Molecular events related to oxidative and nitrosative stress during brain aging, neurodegeneration and neurotrauma

Table of Contents

1 Introduction

2 Review of literature
2.1 Brain and Oxidative stress
2.1.1 Free radicals
2.1.2 Mitochondria as a source of ROS
2.1.3 Oxidative stress and cellular damage
2.1.4 Antioxidant Defenses in the brain
2.2 Brain Aging
2.2.1 Theories of Aging
2.2.2 Age related changes in the Brain
2.2.3 Oxidative Stress and mitochondrial damage in Aged Brain
2.2.4 Brain aging as a risk factor for TBI related poor prognosis
2.3 Traumatic Brain Injury (TBI)
2.3.1 Epidemiology
2.3.2 Pathobiology of TBI
2.3.3 Glasgow Coma Scale (GCS) and Glasgow Outcome Score (GOS)
2.3.4 Pathoanatomical typology of TBI
2.3.5 Models of TBI
2.3.6 Biomarkers of TBI
2.3.7 Protein dynamics and rational for Neuroproteomics in TBI
2.3.8 TBI and neurodegeneration
2.4 Parkinson's disease (PD)
2.4.1 Clinical symptoms and diagnosis
2.4.2 Pathobiology
2.4.3 Etiological factors
2.4.4 Mechanisms of neurodegeneration in PD
2.4.5 Experimental models of PD
2.4.6 Current approaches to treatment:

3 Scope of the research

4 Objectives of the Research

5 Materials and Methods
5.1 Materials
5.2 Human tissue samples
5.3 Histology and Immunohistochemistry (IHC)
5.4 Cell culture experiments:
5.4.1 Cell line
5.4.2 Maintenance of cells
5.4.3 Counting of cultured cells
5.4.4 Treatment of cells
5.5 Estimation of cell viability (MTT assay)
5.6 Measurement of reactive oxygen species (ROS)
5.7 Estimation of hydrogen peroxide (H2O2)
5.8 Estimation of lipid peroxidation
5.9 Preparation of whole brain protein extracts
5.9.1 Protein estimation
5.10 Preparation of mitochondria
5.11 Preparation of Neuropil fractions
5.12 Estimation of Total Glutathione (reduced and oxidized; GSH+GSSG)
5.12.1 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB) recycling method
5.12.2 Estimation of GSH/GSSH by O-pthalaldehyde (OPA) method
5.13 Superoxide dismutase (SOD) activity
5.14 Catalase activity
5.15 Glutathione-S-transferase (GST)
5.16 Glutathione Reductase (GR) activity
5.17 Glutathione peroxidase activity (GPx)
5.18 Thioredoxinreductase (TrxR) activity
5.19 Mitochondrial function assays
5.19.1 Complex I (CI) activity assay
5.19.2 Malate Dehydrogenase (MDH) Assay
5.19.3 Citrate Synthase (CS) assay
5.19.4 3-(4,5-Dimethylthiazol-2-yl)-2,5-DiphenyltetrazoliumBromide (MTT) Assay
5.19.5 Succinate Dehydrogenase (SDH)
5.20 Sodium dodecyl sulphate- Poly acrylamide gel electrophoresis (SDS PAGE) and Western Blot
5.20.1 Sample preparation
5.20.2 SDS PAGE
5.20.3 Coomassie Brilliant Blue (CBB) staining
5.20.4 Silver staining
5.20.5 Electrotransfer
5.20.6 Western blotting
5.21 Determination of protein nitration [3-nitrotyrosine (3-NT) slot blot]
5.22 Determination of protein carbonylation
5.23 Proteomics
5.23.1 Mitochondrial Protein extraction and normalization
5.23.2 Isobaric tag for relative and absolute quantitation (iTRAQ) labeling and Strong Cation Exchange Chromatography
5.23.3 LC-MS/MS Analysis
5.23.4 Data Analysis
5.23.5 Biological Network Analysis
5.24 Statistical analysis

6 Results
6.1 Role of pre/postmortem factors on the oxidant/antioxidant markers in the non­traumatized regions of postmortem brains from TBI patients
6.2 Analysis of the distribution of oxidant and antioxidant markers in the sub-cellular
fractions of non-traumatized regions of brains from TBI patients: Assessment of role of pre and postmortem factors
6.3 Anatomical heterogeneity in the distribution of redox markers in the human brain with the persistence of the signature pattern at different ages: Correction for pre/postmortem factors
6.4 Analysis of histopathological parameters and oxidant and antioxidant markers in the tissue and sub-cellular fractions of traumatized regions of human brains from TBI patients: Role of aging
6.5 Proteomic analysis in brain tissue from TBI: Comparison of contusion, pericontusion with controls followed by validation
6.5.1 Down-regulated proteins
6.5.2 Up-regulated proteins included:
6.6 Assessment of antioxidant markers in PD brains: Focus on glutathione
6.7 Potential therapeutic effects of curcumin derivatives in dopaminergic neuronal cell line via elevated GSH: Implications for PD and TBI

7 Discussion
7.1 Glutathione metabolism is modulated by postmortem interval, gender difference and agonal state in postmortem human brains
7.2 Effect of Storage Time, Postmortem Interval, Agonal State, and Gender on the Postmortem Preservation of Glial Fibrillary Acidic Protein and Oxidatively Damaged Proteins in Human Brains
7.3 Effect of Premortem and Postmortem Factors on the Distribution and Preservation of Antioxidant Activities in the Cytosol and Synaptosomes of Human Brains
7.4 Region-specific changes in redox status of human brains: Vulnerability to degeneration and trauma:
7.5 Role of oxidative stress in the injured regions in TBI
7.6 Proteomic analysis in TBI
7.7 Alteration in the glutathione metabolism in synaptic terminals but not in synaptic-free mitochondria in the frontal cortex of Parkinson’s disease brains
7.8 Curcumin derivatives display improved protection against GSH depletion mediated oxidative stress in a dopaminergic neuronal cell line: Implications for PD and TBI

8 Summary

9 Conclusions

10 List of Publications

11 References

DECLARATION

I, Harish G., hereby declare that the study presented in this thesis entitled “Molecular events related to oxidative and nitrosative stress during brain aging, neurodegeneration and neurotrauma” has been carried out by me in the Department of Neurochemistry, National Institute of Mental Health and Neuro Sciences (Deemed University), Bengaluru, under the guidance of Dr. Srinivas Bharath M.M.

I also declare that the subject matter of this thesis has not formed the basis for the award of any other degree, diploma, associateship or fellowship, prior to this date.

Harish G.

Place: Bengaluru

Date:

Dedicated to the kith and kin who donated the brain of their beloved ones for scientific research. Their willingness to help scientific community despite the bereavement is an example of altruism and nothing short of it.

ACKNOWLEDGEMENTS

“A silent gratitude isn't much use to anyone” - G B Stern

It is with extreme gratitude that I must first acknowledge and thank my mentor, Dr. Srinivas Bharath M.M., a wonderful scientist, philosopher and a kind human being who believes in “science against all odds”, for his support and guidance throughout my doctoral program at NIMHANS. From the beginning, he has taught me to be critical and conscientious towards research and exemplified the qualities of strength, passion, and hard work in all he does. I feel incredibly privileged to have trained under his guidance and appreciate the breadth of knowledge I have gained under his supervision.

I earnestly thank Dr. Shankar SK and Dr. Anita Mahadevan for their relentless support and selfless aid they have rendered, even in the midst of their extremely busy schedule. They continually and persuasively conveyed a spirit of adventure in regard to research and scholarship, and an excitement in regard to teaching. Without their supervision and constant help this dissertation would not have been complete. I sincerely acknowledge them for efficient guidance with the histopathology data and their caring and concern about this dissertation.

I am greatly indebted to Dr. B A Chandramouli and Dr Nupur Pruthi for providing cranniotomy samples and helping me to get clinical details of the samples from patients. Special thanks to Dr Nupur Pruthi for his guidannce in obtaining clinical details of the patients and radiology dataeven in the midst of his exceedingly busy schedule.

I would like to express my earnest gratitude to Dr. Rita Christopher at the Dept. of Neurochemistry, for her encouragement, thoughtful discussions and support throughout the PhD program.

I thank Dr. Sharada Subramaniyan and Dr. Nandakumar for enchanting discussions during seminars and guest lectures.

I thank Dr Gayathri and Dr Sagar for the help rendered in carrying out electron microscopy study of mitochondrial and synaptiosomal fractions. I also thank Dr Thennarasu for suggestions and statistical analysis of the data.

I thank all the staff at Human Brain Tissue Repository (HBTR), esp. Mr. Prasanna, Mr. Shivaji and Mrs. Shakthi for their help in collecting and processing samples for immunohistochemistry. I also thank Vinuth at Institute of Bioinformatics for the proteomic analysis of the samples.

“If you want to do something good, the whole universe will support”, is what Dr Ravi said when I went to him asking for permission to use ultracentrifuge, which is placed in P3 biosafety lab. I am grateful to Dr V Ravi, Vijayalakshmi, and Ullas for the timely help rendered by them to continue my work uninterrupted.

When there was HINI breakout and the P3 lab couldn’t be used, help came from Dr. Savithri, Dept. of Biochemistry (IISc). I thank her for permitting me to use their ultracentrifuge facility. I also thank Prabha Hegde, Ambily, Nishad, Rishikumar N and Vasanth for providing necessary assistance during my visits to IISc.

I thank all the staff at academic section specially Mr. Kumar; and many thanks to coordination section for efficient coordination!

I am greatly indebted to the Junior and Senior research fellowships provided by the Indian council of Medical Research (ICMR) for my PhD. Special thanks to Dr. Sandhya Diwakar at ICMR for her timely response to correspondences.

I was helped a lot by my seniors Dr Mythri, Dr Venkateshappa and Dr Renjini, who patiently tought me and guided me through their experience in a number of techniques related to the current dissertation. Special thanks to Mythri ma’am for all the support she has been throughout my PhD, including help with careful construction of this thesis. Working with Venkatesh sir was fun and most productive (8 publications as co-author!), he inspired me as a cool teacher and a best friend, I missed him the most when he left NIMHANS.

Many thanks to my friend Sunil for providing intution into numbercrunching statistical procedures and refreshing perspectives on philosophy of science.

Together with my juniors Raghunath, Sunitha, Anu mary V, Sonam, Vidya, Ranganayaki, Naveen, colleagues Dr Apurva and Dr Aparna I relished some of the most beautiful memories at NIMHANS.

I owe my deepest gratitude to my parents, sister (for pocket money support!), mama and aunt, and friends for their cherishing love, support and encouragement.

Last but not the least it would be blasphemous not to thank the almighty to which we owe everything. So I pay my utmost gratitude to science, logic and technology.

LIST OF ABBREVIATIONS

Abbildung in dieser Leseprobe nicht enthalten

1 Introduction

Even at rest, an estimated 20% of the total oxygen uptake by our body is utilized by the brain, which is the most metabolically active organ in the human body (Owen , et al. 2011). While >95% of the oxygen taken up by the cells is utilized by the mitochondria, <5 % is converted to highly toxic free radicals which damage the cellular components. Brain is enriched with polyunsaturated fatty acids which are easy targets of reactive free radicals. Studies show that mitochondria are the major sites (Chance , et al. 1979) and targets of superoxide generation (Aguilaniu , et al. 2003, Staniek , et al. 2000). To counter this oxidant burden, a competent antioxidant system comprising of small molecules [glutathione (GSH), vitamin E etc.] and antioxidant enzymes has evolved. Interestingly, brain is relatively deficient in certain antioxidants and related enzymes (Nunomura , et al. 2006). Although the red-ox equilibrium is maintained during physiological homeostasis in the brain, during disease, aging and injury the balance is lost resulting in oxidative damage. Oxidative stress could contribute to mitochondrial damage and both these processes are relevant for the mechanistic basis of physiological aging.

Aging is a physiological process associated with morphological and functional changes in cellular and extracellular components, resulting in a progressive imbalance of the regulatory and metabolic mechanisms. Oxidative damage and structural modification of proteins leading to mitochondrial damage is one of the hallmarks of brain aging. Increased oxidation of mitochondrial proteins with age has been demonstrated (Floyd , et al. 2001, Sohal , et al. 1993). Decreased mitochondrial membrane potential and decreased electron transfer activity of respiratory complexes are reported in senescent brains (Navarro , et al. 2010). Age related structural, chemical, and metabolic changes, both at the level of individual neurons and in medium-scale neuronal networks, can significantly affect the ability of the CNS to adapt to internal and environmental changes (Mattson , et al. 2006).

Age can also influence the human brain’s susceptibility to degeneration and neurotrauma. Traumatic brain injury (TBI) involves a primary injury resulting in disruption of brain parenchyma followed by secondary events including biochemical, cellular, and molecular events (Zasler , et al. 2013). Oxidative stress, mitochondrial dysfunction (Lifshitz , et al. 2004) and protein aggregation are associated with TBI (Johnson , et al. 2010). TBI can induce accumulation of several proteins that are key pathologic aggregates found in degenerative pathologies. The most widely studied of these proteins in TBI include P-amyloid precursor protein, amyloid-P(AP) peptides, neurofilament proteins, and synuclein proteins (Smith , et al. 2003). Research evidences demonstrate that TBI can set a stage for neurodegeneration (Freire 2012).

Brain aging is attributed to several neurodegenerative diseases such as Parkinson’s disease (PD), Alzheimer’s disease (AD), mild cognitive impairment (MCI) etc. (Duncan 2011). PD, the second most common neurodegenerative disorder next to AD is caused by the selective loss of dopaminergic neurons in the substantia nigra (SN) of the ventral mid brain leading to the depletion of dopamine in the striatum. Human SN is relatively more susceptible to oxidative mechanisms due to deficient protective mechanisms, increased iron and lipid peroxidation etc. (Dauer , et al. 2003). Oxidative damage in PD is closely associated with mitochondrial dysfunction (Exner , et al. 2012).

Emerging research evidences have implicated brain aging and neurotrauma as important risk factors contributing to neurodegeneration as seen in PD (Hutson , et al. 2011). Interestingly, these three phenomena could be interlinked by common mechanisms such as oxidative/ nitrosative stress and mitochondrial dysfunction. However, the relationship among these mechanisms in the context of human brain physiology and pathology is not clearly understood.

Most of the studies investigating brain aging, PD and TBI have employed animal and fly models. Many markers of oxidative/nitrosative stress such a lipid peroxidation, DNA damage and protein oxidation/nitration and associated mitochondrial dysfunction have been reported from such studies. Though animal experiments have helped in understanding brain pathophysiology, extrapolation to human system is not realistic because of species barrier, diversity in anatomy, physiology, biochemistry and genetics, except for some phenomenological similarities. Further, due to the non-availability of samples, such studies are very limited in human brains. This reason has also restricted the number of studies aimed at investigating the proteomic changes associated with TBI. The current proposal aims at investigating the role of oxidative/ nitrosative stress and mitochondrial damage in aging, neurodegeneration and neurotrauma in postmortem human brain samples. The study also aims to carry out proteomic analysis of the human brains from TBI subjects to understand and differentiate the primary and secondary events in TBI.

2 REVIEW OF LITERATURE

2.1 Brain and Oxidative stress

2.1.1 Free radicals

Chemical species with a single unpaired electron is called a free radical. This unpaired electron is highly reactive as it seeks an electron to pair with, producing another free radical. A newly formed free radical is unstable and contributes to a chain of free radical reactions. These radical species can be classified into primary radical species [e.g., hydroxyl radical (•OH), superoxide anion(O2’-) Carboxyl ion (CO2’-) and nitric oxide ion (NO^)] and non-radical species [e.g., hydrogen peroxide (H2O2), hyponitrite (N2O2), oxygen (O2), nitrogen dioxide (NO2), hydroperoxyl (HO2), and hypochlorous acid (HOCl)](Halliwell 2006). Generally the term reactive oxygen species (ROS) is used synonymously with free radicals, but technically, free radicals formed from ground state O2 are called ROS, while oxidants derived from nitric oxide (NO») are referred to as reactive nitrogen species (RNS) (Turrens 2003). Fig. 1i briefs the biochemistry of ROS formation.

2.1.2 Mitochondria as a source of ROS

Single electron reduction of O2 generatesO2*'.Mitochondria house a highly reducing intra-mitochondrial environment with various single electron carriers including flavoproteins, iron-sulfur (Fe-S) clusters and ubisemiquinone. Hence a mitochondrion is thermodynamically capable of generating Oa*" anion. Electron transfer during oxidative phosphorylation is a tightly coupled process involving iron sulfur (Fe­S) clusters and single electron carriers apart from the complexes. In spite of this tight coupling, a small amount of electrons leak, resulting in single electron reduction of O2 generating O2*- radicals (Reaction 1) (Andreyev , et al. 2005, Cadenas , et al. 2000).

Abbildung in dieser Leseprobe nicht enthalten

Over the years, studies have identified a variety of sites in mitochondria which generate O2*- and most of them reside in respiratory complexes and individual enzymes. In the brain, under normal conditions, mitochondrial complex I appears to be the primary source of O2*-generation (Barja 1999, Barja , et al. 1998). Within complex I, the FMN moiety, Fe-S clusters, and semiquinones have been suggested to be responsible for Ov- production(Kushnareva , et al. 2002).Presence of inhibitors like rotenone, which inhibits complex I, or antimycin, an inhibitor of mitochondrial complex III are known to increase (V- generation. Several pathological scenario ranging from aging to Parkinson's disease(PD) (Trojanowski 2003) indicate complex I as the major source of ROS (Barja and Herrero 1998).

In complex III, autoxidation of ubisemiquinone appears to be the source of most of the O2”. Production of O/- is inhibited once electrons flow between the Rieske Fe-S protein and oxygen is blocked (by myxothiazol, cyanide or cytochrome c depletion), which results in the availability of ubisemiquinone for (V- production (Trumpower 1990, Turrens , et al. 1985).A Schematic Model of ROS Generation in the Mitochondria is shown in Fig. 1ii (Balaban , et al. 2005).

Several flavoproteins in the mitochondrial matrix space, like thea-lipoamide dehydrogenase moiety of the alpha-ketoglutarate dehydrogenase complex in tricarboxylic acid cycle (Starkov , et al. 2004)or the electron transfer flavoprotein of the P-oxidation pathway (St-Pierre , et al. 2002), are the additional candidate sites of (V- production. Other sites in the mitochondria known to generate ROS include aconitase enzyme; pyruvate dehydrogenase; dihydroorotate dehydrogenase; the monoamine oxidases A and B; and cytochrome b5 reductase(Lin , et al. 2006).

Studies on brain homogenates show that nearly 60% of estimated maximal ROS production was of mitochondrial origin (Malinska , et al. 2010). This could be highly relevant in pathological conditions since interruption to the electron flow would considerably increase O/- production from damaged mitochondria (Malinska , et al. 2010, Napankangas , et al. 2012).

Following is a brief description of some of the important ROS and RNS:

2.1.2.1.1 Superoxide radical (O2~)

(V- is a moderately reactive radical considered as the “primary” ROS, whose generation can lead to more reactive “secondary ROS”. It can undergo dismutation, to form H2O2 catalyzed by the enzyme superoxide dismutase (SOD)(Venditti , et al. 2013). Most of the (V- generated in the mitochondria is eliminated within the matrix while a part of the Oo- produced in the inter membrane space is expelled into the cytoplasm via voltage-dependent anion channels (Han , et al. 2003).Activity of enzymes such as xanthine oxidase and NADPH oxidase also produce O2”.

2.1.2.1.2 Hydrogen peroxide (H2O2)

H2O2 is formed by the intracellular detoxification of O/- by Mn or Cu or Zn dependent SOD enzymes (Reaction 2). H2O2 is removed by the enzymes catalase, glutathione peroxidases, and peroxiredoxins (Guerra , et al. 2000). H2O2 is also believed to be involved in signaling pathways and activation of early genes fos and jun(Raghupathi 2004).

Abbildung in dieser Leseprobe nicht enthalten

2.1.2.1.3 Hypochlorous acid (HOCl)

Myeloperoxidase catalyzes the formation of HOCl (Rossi , et al. 1985)(Reaction 3). Phagosomes of neutrophils express this enzyme to produce HOCl as an antimicrobial agent for elimination of bacteria (Winterbourn , et al. 2000). HOCl disrupts bacterial DNA replication by oxidizing the DNA bound to the bacterial membrane (Rossi, Bellavite, Berton, Grzeskowiak and Papini 1985).

Abbildung in dieser Leseprobe nicht enthalten

2.1.2.1.4 Hydroxyl radical (HOT

Free Fe[2]+ or Cu+ can catalyze the decomposition of H2O2 to form -OH by a reaction called Fenton reaction (reaction 4).

Abbildung in dieser Leseprobe nicht enthalten

In the Haber-Weiss reaction H2O2 reacts with Ov- to yield •OH, OH- and O2 (Reaction 5) (Nordberg , et al. 2001):

Abbildung in dieser Leseprobe nicht enthalten

-HO is one of the most toxic ROS because of its high reactivity, short half-life, and irreversible modification of protein, lipid and DNA (Halliwell 2006).

2.1.2.1.5 Nitric oxide (NO)

NO, peroxynitrite (-OONO-), and nitrogen dioxide (NO2) are the most common reactive RNS formed in the cell. NO is produced by the activation of the enzyme nitric oxide synthase (NOS). There are three isoforms of NOS: neuronal (nNOS), inducible (iNOS), and endothelial (eNOS). NOS catalytically oxidize L-arginine to form L- citrulline with NO as a by-product, using an electron from NADPH (Reaction 7) (Bredt 1999). Since, NO has long half-life (1-[10]s) when compared to other ROS [O2-(10-[8]s) and HO^ (10-[9]s)] (Balazy , et al. 2003), whenever it reacts with other free radicals, it stabilizes the reacting free radical thus acting as ROS scavenger(Rubbo , et al. 2000). NO also serves as an intracellular signaling molecule for guanylatecyclase and protein kinase under physiological concentrations(Ignarro 1990).Physiological functions of NO include regulation of various cellular processes that include vasodilatation, modulation of signaling cascades, and immune responses. Toxic effects of NO include tissue injury and the progression of various inflammatory pathways in various neurodegenerative disorders (Halliwell 2006).Conjugation with Glutathione (GSH) takes care of its detoxification. Under depleted GSH conditions, excessive NO can nitrosylate proteins, thus leading to their damage, or react with other ROS to form other RNS thereby exacerbating cellular damage.

2.1.2.1.6 Peroxynitrite (-OONO) (PN)

Reaction of NO with O/- forms peroxynitrite (PN)(reaction 6).

Abbildung in dieser Leseprobe nicht enthalten

Although SOD detoxifies Oo- H2O2, during elevated NO concentration, NO competes with SOD for O/- thus accelerating PN production(Beckman , et al. 1996). Homolysis of -OONO forms HO^, which damages cellular components. PN is also known to cause age-associated oxidative stress (van der Loo et al., 2000; Imam and Ali, 2001) by initiating fatty acids peroxidation, epoxidation and nitration (O'Donnell , et al. 1999).Production of ROS and RNS is not always harmful to normal cellular functions, since they play an important role in defense against infection and cellular signaling (Nordberg and Arner 2001).

2.1.3 Oxidative stress and cellular damage

During normal physiological conditions, the generation of ROS and RNS will be under stringent homeostatic control. However, during conditions when the ROS and RNS level exceed the capacity of the inherent antioxidant defense reserves or when there is depletion of antioxidant reserve or both, such a scenario is termed “oxidative stress”. Unchecked oxidative stress leads to the destruction of cellular components including protein, lipids, DNA, and ultimately leading to cell death by apoptosis or necrosis(Kannan , et al. 2000).The importance of oxidative damage of proteins, lipids and DNA are described below:

2.1.3.1 Protein Oxidation

Protein oxidation involves covalent modification of a protein directly by ROS or indirectly by reaction with secondary by-products of oxidative stress. Although protein oxidation is considered as an important post-translational modification involved in cell signaling(Wall , et al. 2012) under normal physiological conditions, most of the oxidative modifications of proteins lead to diverse adverse functional consequences. For example, oxidative modification is shown to inhibit enzyme activities(Stadtman 2006). Glutamine synthetase, mitochondrial aconitase, adenine nucleotide translocase, and carbonic anhydrase have been identified as being increasingly oxidized during aging (Carney , et al. 1991, Miyoshi , et al. 2006, Yan , et al. 1997). Oxidatively modified proteins are found to be thermodynamically unstable and assume partially unfolded tertiary structures which readily form aggregates. Protein oxidation leads to increased hydrophobicity and altered rate of protein degradation. Also protein oxidation promotes non-specific protein-protein interactions and protein aggregates through covalent and non-covalent linkages (Agarwal , et al. 1994). Studies have indicated oxidized proteins as intermediates in the formation of amyloid fibrils(Squier 2001). Significant increase in the levels of protein oxidation is observed in various neurodegenerative diseases including Alzheimer’s disease (AD)(Aksenov , et al. 2001, Jenner 2003, Sultana , et al. 2006) and PD (Halliwell 2006). Table 1 lists the types of oxidative modifications(Shacter 2000). Some of the commonly occurring oxidative modifications of proteins are as follows:

2.1.3.1.1 Protein Carbonylation

Levels of protein carbonyls are known to be a good marker of oxidative stress (Levine , et al. 1990).There is a significant increase in the levels of protein carbonyls in aging and during pathological conditions such as head injury, stroke and age-related neurodegenerative disorders (Ansari , et al. 2008, Chang , et al. 2007, Martinez , et al. 2010, Opii , et al. 2007, Stadtman 2006).There are three major mechanisms by which proteins undergo carbonylation(Stadtman 2001). These are (a) Oxidation of specific amino acids in the protein; (b) P-scission of peptide backbone and; (c) Covalent modification of amino acids by reactive aldehydes. Fig. 2a shows mechanism of protein carbonylation.

2.1.3.1.2 Protein Nitration

Protein nitration is another form of protein oxidation, involving RNS such as PN. The initial reaction of PN and carbon dioxide produces nitrosoperoxycarbonate, which undergoes rearrangement to form nitrocarbonate. Homolysis of nitrocarbonate generates a carbonate anion and a nitrite radical, which then react with tyrosine moieties on proteins leading to the formation of 3-nitrotyrosine (3-NT) [Fig. 2b; Radi (2013)]. Protein nitration is indexed by the levels of 3-NT. Tyrosine nitration modifies key properties of the amino acids such as its redox potential, phenol group pKa, hydrophobicity, and volume. Thus, addition of a nitro group (-NO2) into protein tyrosines can lead to profound structural and functional changes, which in turn can contribute to altered cellular and tissue homeostasis. Hence 3-NT has been established as a biomarker of cell, tissue, and systemic “nitrosative stress”(Radi 2012).

2.1.3.2 Lipid peroxidation

Unsaturated fatty acids, particularly 0-3 fatty acids such as linoleic acid, and w-6, including arachidonic, docosatetraenoic acid and decosohexaenic acid, are major components of cell membranes and organelles. Brain is rich in w-6 fatty acids and these polyunsaturated fatty acids are highly susceptible to free radical attack. The end products of lipid peroxidation including malondialdehyde (MDA), 4-hydroxynonenal (4- HNE), acrolein, and other aldehydes (Williams , et al. 2003)are the most commonly observed lipid peroxidation products in neurodegenerative diseases(Negre-Salvayre , et al. 2008).MDA is formed by peroxidation of Arachidonic acid and 4-HNE is formed by the peroxidation of linoeic acid . Compared to free radicals, reactive aldehydes formed as a result of lipid peroxidation have longer half-lives. They can be readily added to proteins by Michael addition reaction, leading to the formation of covalent protein­bound adducts with cysteine, histidine and lysine residues (Butterfield , et al. 1997).The chemistry of lipid peroxidation is shown in Fig. 2c (Aitken , et al. 2011).

Lipid peroxidation results in qualitative changes in fatty acid composition changing the ratio of poly-unsaturated fatty acids (PUFA) and other fatty acids in phospholipids. Lipid peroxidation causes structural disorganization of the cellular membranes and deterioration of pores crossing the double phospholipid layers. Effective changes include lowered membrane fluidity and altered active ion transport, which in turn could alter the intracellular concentration of ions and other compounds (Valko , et al. 2007).

2.1.3.3 DNA oxidation

ROS and RNS can oxidize DNA by modifying bases. Most of the nucleic acid oxidations happen by •OH radical (Castellani , et al. 2004). One of the markers of oxidative DNA damage is 8-hydroxyguanine (8-OH-G), which is formed as a result of oxidation of guanine residue by •OH radical. This results in 8-OH-Gpairing more favorably with adenine rather than with cytosine, such that the mispairing produces GC to TA transversions (Shibutani , et al. 1991). Oxidation of thymine similarly gives rise to 5-hydroxymethyluracil. 8-Hydroxyadenine, an oxidized product of adenine, is also found to mispair with guanine(Bannister , et al. 1985).ROS can cause single strand DNA nick (ssDNA nick) or double strand DNA break (dsDNA break) through abstraction of a single hydrogen atom (Cui , et al. 2000).

RNS generally causes deamination of bases replacing NH2 groups with OH groups thereby causing AT to GC transversions. Adenine, cytosine and guanine are transformed into hypoxanthine, uracil, and xanthine, respectively. Uracil mispairs with adenine and Hypoxanthine mispairs with cytosine(Bannister, Halliwell and O'Neill 1985).ROS mediated DNA damage is illustrated in the Fig. 2d (Cooke , et al. 2003).

2.1.4 Antioxidant Defenses in the brain

Living organisms have evolved a variety of antioxidant defense mechanisms to protect themselves from free radicals. These include: (a) scavenging of free radicals and their precursors (b) maintaining metal ions in a protein-bound form to evade ROS production and (c) inducible antioxidant defenses.

Cellular antioxidants in the brain could be classified into enzymatic and non- enzymatic antioxidants. Non enzyme antioxidants include ascorbic acid (Vitamin C), a- tocopherol (Vitamin E), GSH, lipoic acid, ubiquinones, carotenoids, flavonoids and others. Antioxidant enzymes such as SOD (Cu/ZnSOD and MnSOD), catalase and glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S transferase (GST) constitute the enzymatic antioxidant defense machinery (Gilgun-Sherki , et al. 2001).Major antioxidant enzymes in the brain are shown in Fig. 2e.

2.1.4.1 Antioxidant enzymes

One of the key discoveries to establish free radical theory of aging was SOD. It is a key antioxidant enzyme discovered by McCord and Fridovich(McCord , et al. 1969). It is a metalloenzyme which catalyzes the dismutation of toxic (V- to H2O2. MnSOD is located in mitochondrial matrix, whereas Cu/ZnSOD is found in cytosol, mitochondrial intermembrane space and extracellular fluid. Activity of Cu/ZnSOD activity is found to be decreased in stroke and a variety of neurodegenerative diseases, including Arterio- lateral sclerosis, AD, PD, spinal and brain injury (Demirkaya , et al. 2001, Vaziri , et al. 2004). Transgenic animals with increased SOD expression show increased life span and less damage following ischemic injury (Saito , et al. 2003).

To efficiently remove H2O2, two antioxidant enzymes are available: catalase and GPx, which transform H2O2 into H2O. Catalase functions when H2O2 concentrations are significantly higher than physiological levels. However, GPx is more sensitive and functions even on low H2O2 concentrations (Drevet, 2006). GPx also neutralizes lipid peroxides and requires GSH as a substrate, in which reduced glutathione (GSH) is converted to oxidized form (GSSG).Studies have linked GPx for its potential role in Lewy body degradation in human PD and dementia brain tissues (Power , et al. 2009). Decreased activity of GPx was found to cause senescence and brain dysfunction in aging model of mice (Kishido , et al. 2007). Protective role of catalase and SOD are observed in in vivo toxic PD model using 6-hydroxydopamine (6-OHDA)(Iglesias-Gonzalez , et al. 2012). In another study, mimetics of SOD and catalase were found to decrease oxidative stress and restore cognitive function in aged mice (Clausen , et al. 2010).

GR reduces GSSG to GSH thereby ensuring that all protein thiols are maintained in their native reduced state(Ganea , et al. 2006). Decreased GR activity was associated with increased susceptibility to oxidative stress in rats (Barker , et al. 1996) and ischemic vulnerability in carotid occlusion model of gerbils (Stanimirovic , et al. 1988). GST belongs to a family of detoxification enzymes which catalyze the conjugation of GSH to a wide variety of xenobiotics (McIlwain , et al. 2006). Human GSTs occur in three forms: cytosolic, mitochondrial and membrane-bound microsomal. Polymorphic GST Omega genes were previously reported to associate with risk and age-at-onset of AD and PD. Regional variation in GST expression is linked to the differential regional susceptibility to degeneration after toxic insults (Johnson , et al. 1993).

2.1.4.2 Non enzyme antioxidants

a-tocopherol prevents propagation of lipid peroxidation by donating an electron to peroxyl radicals (Reiter 1995).Humans and primates do not synthesize Vitamin C, which is considered a powerful hydrophilic scavenger (Frei , et al. 1989). It is distributed throughout the brain and its concentration in the CSF is about 10 fold than in the plasma. It serves as a strong reducing agent by donating electron(s) and directly neutralizing ROS(Halliwell 2006).

Histidine dipeptides such as carnosine, homocarnosine, and anserine are important anti-oxidants, unique to brain and muscle. They chelate transition metals and prevent their involvement in the Haber-Weiss reaction. They can also scavenge a variety of ROS, mimic SOD, and exhibit peroxidase-like activity (Kohen , et al. 1991). a-lipoate and its reduced product dihydrolipoate act as potent antioxidants in both intra- and extracellular environments and were reported to show neuroprotective properties in a variety of brain and neuronal tissue pathologies (Astiz , et al. 2012, Packer , et al. 1995).

2.1.4.2.1 Glutathione

GSH, a tripeptide is the most abundant non-protein thiol in animal cells. It is a co-factor for many enzymes and also plays a major role in the post-translational modification of proteins. GSH is synthesized in the cytosol by the ATP-dependent enzymes Y-glutamylcysteine ligase (GCL) and GSH synthetase.

GSH exists in either reduced (GSH) or oxidized (GSSG) form and participates in cellular redox reactions by reversible oxidation of its thiol functional group. It functions as a coenzyme of several cellular enzymes. GSH forms the major cellular antioxidant defense and is present in high concentrations in both the mitochondria and cytosol. Mitochondria contain nearly 15% of the total cellular GSH (Shan , et al. 1993). Under normal and unstressed conditions, 99% of Glutathione exists in a reduced (GSH) form with intracellular concentrations ranging from 0.5 to 10mM depending on the cell type(Butterfield , et al. 2002).GSH in its reduced form is essential for the maintenance of thiol redox status, detoxification of endogenous and exogenous reactive species, protection against oxidative damage, storage and transport of cysteine, protein and DNA synthesis, cell differentiation and cell cycle regulation(Butterfield , et al. 2002).Depletion of GSH in oxidative stress conditions is associated with increased susceptibility to cell damage (Cakatay , et al. 2001, Calabrese , et al. 2000). GSH and GSH-related enzymes play an important role in detoxifying ROS. The thiol group of GSH reduces ROS in the presence of GPx, while GR reduces GSSG back to GSH using NADPH. This thiol-disulfide redox cycle helps in maintaining the thiol groups of proteins in a reduced state. Fig. 2f depicts metabolism and functions of GSH in the brain(Dringen , et al. 2000).

GSH is an excellent scavenger of the lipid peroxidation products HNE and acrolein, both of which are found to bind proteins, thereby inhibiting their functions(Pocernich , et al. 2000). GST catalyzes the conjugation of GSH to a variety of xenobiotics for detoxification (McIlwain, Townsend and Tew 2006). GSH also detoxifies saturated epoxides, quinones, esters, and aryl nitro compounds by neutralizing the nucleophilic attack from these compounds (Hammond , et al. 2001). Via non-enzymatic reactions, GSH binds to metal to form complexes to terminate the Fenton reaction (Hammond, Lee and Ballatori 2001). GSH deficiency or a decrease in the GSH/GSSG ratio could result in an increased susceptibility to oxidative stress, and the resulting damage is thought to be involved in several diseases such as cancer, PD and AD (Ballatori , et al. 2009, Smeyne , et al. 2013). Several studies have associated dysregulation of GSH homeostasis to neurodegeneration (Johnson , et al. 2012).

2.2 Brain Aging

Similar to other countries worldwide, India houses a large geriatric population. Increasing geriatric population with increasing age related ailments has necessitated research in the field of Aging. Thus, the study of Biological mechanism of aging is not merely a topic of scientific curiosity, but also a crucial area of research and healthcare challenge.

“Aging” is one of the most fascinating topics that have interested philosophers and scientists for centuries. Over the years, the researchers have postulated several theories to explain the aging phenomena. Denham Harman postulated that aging is a deleterious, progressive, intrinsic, and universal process, which is a progressive accumulation of alteration as a function of time associated with or responsible for the ever-increasing susceptibility to age-related disease and death (Harman 1991). Aging is associated with (a) progressive loss of physiologic functions (b) atrophy to most of the organs (c) increased susceptibility to infections, trauma and neurodegeneration (d) susceptibility to malignancy and (e) decreased gaseous exchange during respiration (Vina , et al. 2007).

2.2.1 Theories of Aging

More than 300 theories have been proposed to explain the aging phenomenon (Ashok , et al. 1999, Bindon 1989, Medvedev 1990) (Table 2), but no single theory can explain all the mechanisms of aging. A credent theory of aging should be able to explain the loss of homeostasis in aged individuals, explain the variation in life-span among cohort genetic strains and species, pinpoint crucial factor(s) responsible for life-span extension (either by genetic mutation or experimental regimens such as caloric restriction) and demonstrate that any variation of senescent factors could manipulate the rate of aging (Sohal 2002).Most acceptable explanation for the mechanistic basis of aging was proposed by Harman (1956) and is called the “free radical theory of aging”.

2.2.1.1 Free Radical theory of Aging

Harman suggested that aging, as well as the associated degenerative diseases, could be attributed to the deleterious effects of free radicals on various cell components. The antioxidant systems are unable to counterbalance the free radicals continuously generated during the life of the cell. This results in oxidative damage in the cell and thus in tissues. Old animals show higher index of oxidation of biological molecules DNA, proteins and lipids than the young ones owing to increased free radical production (Spiteller 2001, Stadtman 2006, Wei , et al. 2002).

The discovery of SOD whose function is to remove (V- provided strong evidence that free radicals are involved in the process of aging. Free radical theory of aging lacked the precision of the sub-cellular location of the oxidative reactions mediated by ROS. Harman revised the free radical theory of aging to implicate mitochondria as they generated significant amounts of cellular energy through consumption of most of the oxygen in the oxidative phosphorylation(Harman 2006). Several studies emerged in support of this theory and it has expanded to the mitochondrial free radical theory of aging. Mitochondrial free radical theory of aging proposes that free radicals produced by mitochondria as by-products during normal metabolism result in oxidative damage, and accumulation of oxidative damage is the main driving force for aging.

2.2.2 Age related changes in the Brain

With advancing age, human brain shows anatomical, molecular and functional changes making it susceptible to the incidence of neurological disorders(Shetty , et al. 2011) and traumatic brain injury (TBI)(Sivanandam , et al. 2012). This includes the loss of structural integrity, alteration in levels of enzymes, hormones, genetic and epigenetic modulation, dysregulated metabolism, oxidative stress, altered protein processing and synaptic function, ultimately leading to lowered physiological and cognitive functions(Ketonen 1998, Lee , et al. 2000, Vanguilder , et al. 2011).

2.2.2.1 Molecular changes:

Brain aging involves altered levels of neurotransmitters, enzymes, hormones and metabolites (Smith , et al. 2005). From early adulthood, the levels of dopamine decline by 10% due to loss of dopaminergic neurons between frontal cortex and striatum, or decreased number of dopamine receptors and their reduced binding affinity (Ota , et al. 2006). Reduction in dopamine is associated with age-related decline in cognitive and motor performance. Loss of synaptic plasticity in the old brain is attributed to reduced serotonin and glutamate levels (Chang , et al. 2009, Yamamoto 2001). Activities of enzymes which regulate monoamine neurotransmitters increase with age and may liberate free radicals from the reactions that exceed the inherent antioxidant activity (Esiri 2007).

Aging affects the expression of neurotransmitter receptors. It has been shown that the number of neurons expressing certain ionotropic glutamate receptors and N- methyl-D-aspartate (NMDA) receptor subunits is significantly reduced during aging (Hof , et al. 2002). Quantitative analysis of the distributions of Glu R2 and NMDA R1 in long and short cortico-cortical connections in young and old macaque and patas monkeys revealed a down-regulation of the expression of both receptors with aging. Glu R2 expression was decreased to a greater extent in the prefrontal cortex compared to other areas, such as the temporal cortex, whereas significant reductions in NMDA R1 occurred mainly in the long cortico-cortical projections from the superior temporal cortex (Hof , et al. 2002).

Endocrine changes in the brain also influence aging brain and its cognitive performance. While hormones like growth hormone, thyroxine, melatonin and sex hormones including testosterone, di-dehydroepiandrosterone, estrogen and progesterone decrease during aging (Schumacher , et al. 2003, Veiga , et al. 2004), stress hormone cortisol shows significant increase (Lupien , et al. 2009). Structural and functional changes related to aging are attributed largely to the modulation in the level of estrogen and its receptors(Thakur , et al. 2006).

2.2.2.2 Changes in Calcium homeostasis

In the aged neurons, there is increased Ca[2]+ release from the endoplasmic reticulum through both the InsP3 and ryanodine-receptors (Thibault , et al. 2007). Hence release of ryanodine could be a biomarker of functional aging (Gant , et al. 2006). Aged neurons contain more depolarized mitochondria and this affects both the energy balance and mitochondrial Ca[2]+ uptake (Toescu 2005). Decreased Ca[2]+ buffering or delayed uptake results in significant increase in the time taken for the relaxation of Ca[2]+ signals following stimulation. Rise in intracellular Ca[2]+ is countered by rapid Ca[2]+ sequestering, by cytosolic Ca[2]+ binding proteins (Brewer , et al. 2006). Age-related alteration in calcium homeostasis probably due to declined function of these proteins and transport systems could contribute to age-related neurodegenerative diseases (Gomez-Villafuertes , et al. 2007). Age-related increase in L-type Ca[2]+ channels specific for hippocampal pyramidal cells and decrease in NMDA receptor function in hippocampus and frontal cortex, suggest that certain neuroanatomical sites and specific cell populations are involved in Ca[2]+ dysregulation. Cell-specific susceptibility to Ca[2]+ dysregulation and oxidative stress could explain region specific neurodegeneration (Kumar , et al. 2009).

2.2.2.3 Changes in gene expression

Gene expression studies in brain across the lifespan reveal alterations in molecules related to stress, inflammation, immune response, mitochondrial functions, growth factors, neuronal survival, synaptic plasticity and calcium homeostasis (Lu , et al. 2004). Transcriptional profiling of the aging human frontal cortex brains showed that ~4% of the genes expressed in the brain are age regulated (Lu , et al. 2004). Gene expression changes during aging become apparent in middle age and most pronounced after 70 years of age. Several genes involved in synaptic functions that mediate memory and learning were significantly down-regulated with age, including synaptic vesicle proteins, glutamate receptor subunits, and members of the signal transduction systems that mediate long term potentiation (LTP). Especially, the synaptic Ca[2]+ signaling system appears to be affected with reduced expression of calmodulin 1 and 3, calcineurin B' a, CAM kinase II' aand IV and multiple protein kinase C isoforms(Lu , et al. 2004). Genes involved in vesicle-mediated protein transport and mitochondrial function was prominently down-regulated with aging. Genes involved in stress response including antioxidant defense, DNA repair, and immune function are up-regulated during aging (Erraji-Benchekroun , et al. 2005). Cellular and molecular changes in the aging brain adversely affects the functions such as attention, speech, sleep, decision making, working and long term memory.

2.2.2.4 Age related changes in GSH and GSH -related enzyme activities in the brain

GSH levels are not similar in different brain areas (Abbott , et al. 1990). Kang , et al. (1999) reported that the levels of GSH in 4-month-old mouse brain varied from region to region with cortex > cerebellum > hippocampus > striatum > substantia nigra. Zhu , et al. (2006) showed that GSH level was similar in all brain tissues examined (cortex, striatum, midbrain, and cerebellum) regardless of genders. This is in agreement with another study (Liu 2002), in which cortex, cerebellum, and hippocampus showed no significant variations of GSH levels in 3-month-old rats. GSH levels were shown to decline with age in the brain tissue of various species (Kim , et al. 2003). The decrease in GSH level and increase in GSSG level in aged rats produces a significant decrease in the GSH/GSSG ratio, this ratio is often used to indicate the redox state within the tissue (Dringen, Gutterer and Hirrlinger 2000). Liu (2002) reported decrease in GSH/GSSG ratio in aged rats. It was showed that GCL protein was decreased in all the observed regions of rat brain (Liu 2002). GR was found increased in midbrain particularly in the midbrain of older animals. GR was found to be increased in midbrain particularly in older animals (Zhu, Carvey and Ling 2006). During aging, dopamine turnover becomes more prominent which depletes more GSH to cause a greater increase in GSSG, thus stimulating an increase in GR to remove excess GSSG (Ling , et al. 2000).

Lipid peroxidation products are disposed from cells through conversion of GSH to GSSG by GPx. GPx activity was observed to be increased in aged brain (Carrillo , et al. 1992). Significant increase in GPx activity was observed in all four brain regions (cortex, striatum, midbrain, and cerebellum) of aged rats compared to the young rats regardless of gender, demonstrating an adaptive enzymatic alteration in the aged rats secondary to the increase in oxidative stress (Zhu, Carvey and Ling 2006). GST activity is assumed to increase with age as an adaptive mechanism secondary to increase in oxidative stress. GST activity which detoxifies various toxins was found to be increased in old rats; activity was 1.7-fold higher in the brains of 9-month-old rats compared to those of 5- week-old rats (Kim , et al. 2003). Gamma-glutamyl transferase (y-GT) initiates the degradation of extracellular GSH. Zhu, Carvey and Ling (2006) reported 30 to 40% higher y-GT activity in the aged animal brains relative to the young animals, suggesting an excessive degradative GSH metabolism in the aged brains.

2.2.3 Oxidative Stress and mitochondrial damage in Aged Brain

Oxidative stress caused by the imbalance between the generation and detoxification of ROS/RNS, plays an important role in brain aging. Several studies have indicated increase in oxidative damage and decrease in antioxidant enzyme activities in aging brains. Elevated protein oxidation, nitration and decreased antioxidant enzyme activities (GR, GST, thioredoxin reductase, SOD and catalase) were observed with increasing age in the hippocampus and frontal cortex of human postmortem brain samples (Venkateshappa , et al. 2012). Decrease in complex I activity and other antioxidant enzyme activities were observed through aging in SN of human postmortem brains (Venkateshappa , et al. 2012). In a canine model of human brain aging, the brains of aged dogs accumulated increased levels of protein carbonyl and it was associated with reduced inherent antioxidant enzymes such as glutamine synthetase activity or superoxide dismutase(Head , et al. 2002)

Free radicals generated by mitochondria, cause damage to cellular and mitochondrial components (Vina , et al. 2003). Damaged mitochondria progressively become less efficient, lose their functional integrity and release more free radicals, increasing oxidative damage and culminating in an accumulation of dysfunctional mitochondria with age. Accumulation of damaged molecules contribute significantly to the aging process (Cadenas and Davies 2000). Decreased respiratory activity of mitochondria was observed with age in liver, muscle, and brain (Vina, Borras and Miquel 2007). Transcription of mitochondrial genes and also mitochondrial membrane potential was decreased with age in rats and Drosophila (Calleja , et al. 1993, Vina, Borras and Miquel 2007).

Age-related mitochondrial DNA (mtDNA) changes including multiple mutations were detected and their frequency increased with age. Levels of a 5-kb deletion that particularly affects mtDNA COX genes were increased with age (Corral-Debrinski , et al. 1992). Low abundance heteroplasmic mutations were detected in COX1, a COX subunit gene located on the mtDNA(Lin , et al. 2002).

There is decreased membrane potential in cortical and striatal mitochondria (LaFrance , et al. 2005) and in whole brain mitochondria in aged rats (Sastre , et al. 1998). Decreased ATP synthesis in aging rat brain due to decreased activity of F1- ATPase was observed in brain mitochondria isolated from aged rats (Lam , et al. 2009).Age-related mitochondrial changes include decline in complex I and complex IV activity, with preserved complex II activity. This suggests that mtDNA changes may mediate the observed complex I and IV functional changes since, Complex I and IV are partly mtDNA-encoded, while complex II is encoded by nuclear DNA(Navarro , et al. 2007).

Around 40-65% decreased activities in complex I, complex IV and mtNOS was noted in senescent brains(Navarro , et al. 2005). Isolated mitochondria from the brain of aged rats and mice show increased oxidation products of biomolecules, decreased membrane potential, and increased size and fragility (Navarro and Boveris 2007, Navarro , et al. 2004). Mitochondria of hippocampus and cerebral cortex appear to have increased oxidative stress than in whole brain during rat aging (Navarro , et al. 2008). Decreased complex I activity in aged rats relative to young ones could be probably due to decreased cardiolipin (phospholipid required for complex I function) with significant increase in peroxidized cardiolipin (Petrosillo , et al. 2008).Mitochondria from aged mammalian brain showed decrease in complex IV activity (Navarro , et al. 2005).Decreased cytochrome oxidase was observed in human substantia nigra (Itoh , et al. 1996) and rat hippocampus (Bertoni-Freddari , et al. 2004).

Mitochondria from aged animals show structural alterations in mitochondrial membrane potential, reduced mitochondrial buffering capacity, chronically depolarized state of the membrane (due to increased proton leak) and reduced ATP synthesis (Toescu 2005). With age there is a decrease in the number of mitochondria, with large bioenergetically inefficient ones replacing the functionally efficient, small-sized forms(Sastre , et al. 2003).However, the total mitochondrial volume remains roughly the same (Bertoni-Freddari , et al. 1996). Studies in aging mice show that, the threshold for Ca[2]+-induced, cyclosporin-sensitive, Ca[2]+ release was significantly lower in isolated brain and liver mitochondria. Aging mice exhibited enhanced permeability transition pore activation in lymphocytes, brain, and liver, suggesting increased susceptibility to Ca[2]+-dependent cell death(Mather , et al. 2000).

2.2.3.1 Lipid peroxidation

Significant changes in the composition of membrane lipid are reported in brain aging. Decreased PUFA and increase in monounsaturated fatty acidsin cerebral cortex and cerebellum are the major changes in aged rats (Giusto , et al. 2002). Arachidonic acid(AA), along with n-6 and n-3 fatty acids, also decrease in brains of aged rats accompanied with cognitive deficit (Ulmann , et al. 2001). Hence lower AA could contribute to the cognitive deficit since AA concentrations are correlated with LTP. HNE forms covalent adducts of histidine, lysine and cysteine residues in proteins to modify their functions (Esterbauer 1993). Also, protein bound acrolein could be a potential marker of oxidative stress in aging (Uchida , et al. 1998). The amount of HNE-modified protein staining increased logarithmically with aging in human oculomotor neurons (Yoritaka , et al. 1996). Also, the HNE-modified proteins, along with neurofibrillary tangles, were observed in the senile plaques in aged dogs (Papaioannou , et al. 2001). MDA can react with amino acids to form adducts. In aged human brain, MDA was increased in inferior temporal cortices and in cytoplasm of neurons and astrocytes compared to the young controls (Dei , et al. 2002).In canine model of aging, MDA were increased in the prefrontal cortex of aged brains (Head , et al. 2002). The basal MDA level was significantly raised by 19% in hippocampus of old rats (Cini , et al. 1995).

Numerous studies suggest that the generation of MDA in brain increases with age. However, immunohistochemical studies did not detect significant increase of MDA- modified proteins in aged rat brains, suggesting that MDA-modified proteins might react with lipid peroxidation products and form crosslinks with each other(Grune , et al. 2001),

2.2.3.2 Protein oxidation

Many studies have demonstrated an age-related increase in the expression of neurofilament protein, which makes neurons more prone to the formation of neurofibrillary tangles ultimately leading to neurodegeneration and dementia(Hof , et al. 1990, Vickers , et al. 1994). During normal aging, there is a widespread increase in protein oxidation, which significantly contributes to cellular aging (Stadtman 2006, Stadtman 2001).

Increased protein carbonyls were detected in frontal and occipital cortex of aged humans, cortex of rats (Smith , et al. 1991) and Mongolian gerbils (Dubey , et al. 1995) in the forebrain of Wistar rats (Cakatay , et al. 2001), and in the brain homogenates of aged dogs (Head , et al. 2002). High level of protein carbonyls was observed in substantia nigra (SNc) from aged human brains compared to other regions. Further, the carbonyl content of the major proteins in each region was linearly dependent on molecular weight (Floor , et al. 1998). Protein carbonyl levels depend not only on its formation, but also the degradation of the oxidized protein (Agarwal and Sohal 1994). Proteasome dysfunction was observed during the aging model of human fibroblasts(Sitte , et al. 2000), as well as in the tissues of aging animals (Keller , et al. 2000). Oxidative modification of the proteasome could probably underlie proteasome dysfunction in the central nervous system (CNS). HNE, NO, and related oxidants inhibit proteasome function, suggesting that oxidative stress is responsible for the increase in protein carbonyl levels in aging brain, regardless of activity of proteasomes(Grune , et al. 1998).

Increased protein 3-NT levels were found in the hippocampus and the cerebral cortex of aged rats (Shin , et al. 2002), the CSF of aged human (Tohgi , et al. 1999), and the sub-cortical white matter of aged monkeys (Sloane , et al. 1999). Immunohistochemical studies observed most prominent labeling of 3-NT in purkinje cells, cerebellar cortex, as well as in the surroundings of neuropil in cerebellar nuclei of aged rats (Chung , et al. 2002). Increased protein 3-NT level in a region-specific fashion could be attributed to the level of NOS(Tohgi , et al. 1999). Aging study in monkeys revealed nitrated a-synuclein in dopaminergic neurons of SN with implications for synucleinopathies (McCormack , et al. 2012).

Protein abnormality in aged brain is manifested by neurofibrillary tangles (NFT) or senile plaques (SP). These structures accumulate with age in the neuropil of frontal cortex and hippocampus (Peterson , et al. 1989) in the enlarged axonal and dendritic processes that sprout and degenerate. The degenerating neurites are surrounded by extracellular proteinaceous filaments called P-amyloid (AP). SP are present in brain of primates, dogs and polar bears, but not rats. NFT are composed of primary microtubule-associated protein tau (tau) and other insoluble proteins (Ishihara , et al. 2001). NFTs are found in normal aged brain, and the number of NFT increased during the ninth and tenth decades of life. NFTs are more abundant in the medial temporal lobe of postmortem brains and may constitute a pathological substrate for memory loss not only in AD but also in normal aging and mild cognitive impairment (Guillozet , et al. 2003). NFTs and SP occur within the same anatomical regions, both in normal aging and AD and the severity of the lesions increases with age and disease (Arriagada , et al. 1992, Giannakopoulos , et al. 1994, Sparks , et al. 1993). Similar to western developing countries, the brains of aged people from India also reveal NFT and SP with a similar frequency and phosphorylated cytoskeletal protein profile (Yasha , et al. 1997). In an aging study on human brains, tau aggregates in neurons and astrocytes were observed in both frontal and entorrhinal cortices in an age-dependent manner. Age-dependent pattern of neuronal, extraneuronal and glial tau protein accumulation in non- neurodegenerative sections were also observed (Yang , et al. 2005).

2.2.4 Brain aging as a risk factor for TBI related poor prognosis

As reviewed in the previous section, cells in the brain experience increased amounts of oxidative stress, altered energy homeostasis, accumulation of oxidized proteins and increased DNA damage. These altered structural, chemical, and metabolic changes, both at the level of individual neurons and in medium-scale neuronal networks, can significantly affect the ability of the CNS to adapt to internal and environmental changes (Mattson and Magnus 2006). The cellular changes during normal aging make neurons increasingly susceptible to excitotoxic damage through the impairment of ion pumps, dysregulation of Ca[2]+homeostasis, and decreased mitochondrial function(Dickstein , et al. 2007, Mattson and Magnus 2006). Evidence suggests that aged animals respond to stimuli with amplified inflammatory response as demonstrated by the presence of activated microglia (Godbout , et al. 2005). Continued presence of reactive microglia in the aged brain creates an environment permissive to a prolonged and amplified neuroinflammatory response that can lead to subsequent complications, especially after TBI (Godbout , et al. 2006).Given that all these processes are demonstrated in the evolution of the injury after traumatic insult, the process of aging itself could significantly increase the vulnerability and impair the recovery after TBI in aged individuals. Hence aging is increasingly considered a major risk factor for TBI related poor prognosis.

2.3 Traumatic Brain Injury (TBI)

TBI involves a primary injury that includes direct impact to the head resulting in disruption of brain parenchyma and secondary injury characterized by a series of biochemical, cellular and molecular events involved in the evolution of secondary damage (Zasler, Katz and Zafonte 2013) which might have long-lasting neurological outcome.

2.3.1 Epidemiology

TBI is a global health-care crisis. It is the leading cause of morbidity, mortality, disability and socio-economic losses among various injuries in India. Socio­demographic epidemiologic and economic transition with unprecedented motorization, urbanization, rapid industrialization, changing life styles and values of people along with the absence of safety policies has resulted in increased number of deaths, hospitalizations and disabilities due to TBI (Gururaj 2002).

It is estimated that nearly 1 million people are injured and 20,000 people die of TBI in India every year. In Bangalore alone, 10,000 people sustain brain injuries and more than 1000 succumb to these injuries every year (Table 3).

Abbildung in dieser Leseprobe nicht enthalten

Table 3: Epidemiological indicators of TBI in India and Bangalore (estimates for 2001)(Gururaj 2002)

Studies revealed that the highest occurrence of TBI was found in the age group of 20-29 years, followed by 30-39 years. The male to female ratio among the TBI patients was 1:0.3. In young adults TBI was most often the result of motor vehicle accidents and in the children and elderly, TBI was most often the result of falls (Gururaj 2002, Hyder , et al. 2006, Masson , et al. 2001). Several Indian studies have shown that not only road traffic accidents, but also increasing role of violence in the etiology of neurotrauma. The other causes of injuries like industrial accidents, sports and recreational injuries were found to be very scant from the Indian subcontinent. When it comes to recovery after TBI, a number of factors affected the outcome viz. age, physiological status, pre-hospital care, severity and locale of brain injury, hospital interventional strategies and availability of rehabilitation services. Severity of TBI was a direct determinant of recovery status with significant statistical correlation (p<0.001). The recovery rate was highest among the mild TBI patients (65%) compared to moderate and severe TBI patients (55% and 38%) respectively (Gururaj 2002).

2.3.2 Pathobiology of TBI

TBI is a dynamic and complex pathophysiological event resulting in structural and functional deficits (Masel , et al. 2010). It is classified based on pathoanatomy (Vascular and Parenchymal injuries), severity (Mild, Moderate and Severe), and Outcome (Glasgow outcome scale) (Fig. 3a). Other types includes primary and secondary brain injuries; where primary brain injury constitutes trauma to the head (focal or diffuse) and secondary brain injury includes the pathophysiological processes that follow mechanical insult(Ragaisis 2002).

Primary injury events include immediate mechanical disruption of the brain tissue at the time of injury resulting in contusion, damage to the blood vessels and axonal shearing (Davis 2000). During the primary injury, mechanical compression and penetration caused by the impact induces tissue deformation, tearing, and hemorrhage which invoke rapid oncotic injury and necrotic cell death. The impact force causes contusions that injure distal gray matter regions and the interconnecting axons of the white mater. Secondary injury events that evolve over a few minutes to months after the primary injury include cascades of metabolic, cellular and molecular events which ultimately lead to cell death, tissue damage and atrophy in different regions of the brain (Bramlett , et al. 2007, Marklund , et al. 2006, Thompson , et al. 2005).Secondary injury is triggered by the excessive release of glutamate from damaged nerve terminals that activate ionotropic glutamate receptors (Palmer , et al. 1993) and lower brain pH to neurotoxic levels (Bullock , et al. 1998). Glutamate driven excitotoxicity leads to uncontrolled shifts in sodium, potassium and calcium ion levels which in turn disrupt ionic homeostasis. This may lead to severe swelling of the neurons and subsequent cellular death (Bullock , et al. 1998). Increased oxidative stress within 60 min following TBI causes oxidative damage to neurovascular structures. Altered ionic flux triggers a second wave of cell death, which culminates through activation of proteases and associated apoptosis for several weeks after injury (Raghupathi 2004). Simultaneously microglial inflammatory response increases intracranial pressure (ICP), astroglial proliferation (astrogliosis) and elevated ROS levels.

Increased oxidative stress causes significant burden on endogenous antioxidant reserves and causes mitochondrial dysfunction, which in turn exacerbates ROS production. This could trigger neutrophil mediated inflammation that can also contribute to secondary damage. Oxidative stress may also induce expression of early genes (C-Fos, C-Jun, and Jun B) which have been associated with programmed cell death, termed apoptotic and heat shock proteins (Awasthi , et al. 2003).

2.3.3 Glasgow Coma Scale (GCS) and Glasgow Outcome Score (GOS)

GCS is a widely used scoring procedure for mental and neurological status following TBI and was introduced by Teasdale and Jennett (Sternbach 2000, Teasdale , et al. 1998). GCS scales the severity of the injury into mild, moderate and severe. The GCS score of 13 to 15 is mild, 9 to 12 is moderate injury, and 3 to 8 corresponds to severe injury [Table 4A;Teasdale, Pettigrew, Wilson, Murray and Jennett (1998)]. The score is based on the sum of three components: eye opening (E), verbal response (V), and best motor response (M). For instance, if an individual at the accident scene opened eyes to voice, used inappropriate words, and demonstrated a flexion response to motor stimulation, the scoring would be E+V+M = 3 + 3 + 4 = 10. Here it produces a graded score in the moderate severity range (Grote , et al. 2011).

The GOS was created in an effort to provide a global assessment of disability post severe TBI. The GOS has 5 categories: good recovery, moderate disability, severe disability, vegetative state, and death [Table 4B, Jennett , et al. (1975)]. Good recovery and moderate disability are generally grouped together as favorable outcomes, with the other 3 categories making up the poor outcomes. It is a fairly reliable measure but lacks sensitivity due to its broad categories.

2.3.4 Pathoanatomical typology of TBI

2.3.4.1 Primary brain injury

The key components of primary injury include cortical disruption, axonal injury, vascular injury, and hemorrhage. Parenchymal injuries are classified as contusions, lacerations or diffuse axonal injuries. The primary injuries are also defined by the type or pattern of injury caused: focal or diffuse. Focal injuries include focal cerebral contusion, traumatic intracerebral haematoma, epidural - and subdural haematoma. On the other hand, the diffuse injuries include traumatic subarachnoid haemorrhage, cerebral concussion, and diffuse axonal injury (DAI).

Haemorrhages : Injuries to the major blood vessels cause vascular injuries in the brain which are further identified by the anatomical localization as epidural-, subdural-, subarachnoidal- or intracerebral haematomas, which can be seen on computed tomography (CT)(Silver , et al. 2011). In Epidural haematoma, blood builds up between the dura mater and the skull. Subdural haematoma involves the blood accumulation between the dura matter, and the arachnoid matter. Subarachinoid haemorrhage happens when bleeding occurs between the arachnoid membrane and the pia matter, and bleeding within the brain parenchyma results in intracerebral haematomas [Fig. 3b, Whitfield , et al. (2009)]. Acute subdural hemorrhages are usually associated with a much poorer prognosis than epidural hemorrhages.

Diffuse axonal injury (DAI) : DAI is characterized by damage to axons, and in most cases, petechial haemorrhages. DAI pattern is more accurately described as multifocal, appearing throughout the deep and subcortical white matter and particularly common in midline structures including the splenium of the corpus callosum and brainstem on post-mortem pathological examination (Adams , et al. 1982). Axonal damage results in swollen, tortuous and transected fibres throughout the white matter, including the corpus callosum, deep grey matter, cerebellar folia and brainstem tracts. However, axonal damage can be detected histologically by the accumulation of P- amyloid precursor protein as early as 35 minutes after head injury (Hortobagyi , et al. 2007). After a period of several days to weeks, there is accumulation of microglia around damaged axons followed by Wallerian degeneration (cut in nerve fibres) of axons. Calpain activation in DAI results in damage to cytoskeletal proteins(Kampfl , et al. 1996) disrupting axonal transport mechanisms and resulting in protein accumulations at the site of injury and eventual axotomy (Maxwell , et al. 1997).

Cerebral Contusions : Contusions are considered to be the hallmark of brain damage. A contusion is a focal brain damage caused by contact between the surface of the brain and the bony protuberance of the base of the skull. Rapid acceleration­deceleration forces also cause contusions (Adelson , et al. 1998).Contusions consist of parenchymal damage and micro haemorrhages around the brain capillaries. They are most commonly found in the frontal and temporal lobes [Fig. 3b (vi)].

Although contusions were previously defined as traumatic necrosis followed by reabsorption, the progressive nature of the injury cannot be explained satisfactorily. Brain contusions involve a dynamic and expansive process leading to the deterioration of the neurological state (Narotam , et al. 1998). Microscopic observations reveal perivascular hemorrhage, astrocytic oedema, infiltration of macrophages, changes in the myelin structure, apoptosis, phagocytosis, and atrophy (Schalen , et al. 1991). The clinical implications of cerebral contusions may be dramatic as some studies indicate poor prognosis with increased contusion volume (Ragaisis 2002, Sutton , et al. 1993).

Initial appearance of contusions evolves over time as demonstrated by pathological studies. Right after the injury, a contusion is visible as microscopic regions of perivascular hemorrhage that follows the tracts of small vessels in the cortex. Blood components seep into the adjacent cortex and neuronal structures in the vicinity resulting in neuronal degeneration subsequently producing a glial scar. As the hemorrhage extends into the white matter, it causes demyelination of axons and loss of neuronal tracts. Necrotic tissue is gradually removed by macrophages, and the contusion looks like a shrunken glial scar often brownish in color as a result of residual hemosiderin filling the macrophages (Granacher 2003).

Many researchers distinguish the injured brain into contusional (forming the core of injury, generally necrotic) and surrounding pericontusional lesion (synonyms: penumbra, ischemic and hypoperfusional region, generally inflammatory and non- necrotic). Histological examination of the pericontusional tissue from surgical samples revealed oedematous astrocytes. Pericontusional zone was found to be highly dynamic and can expand to increase intracranial pressure and subsequent neurological worsening status of the patient (Bullock , et al. 1998, Kroppenstedt , et al. 1999, Minambres , et al. 2008, Shreiber , et al. 1999).

2.3.4.2 Secondary brain injury

The focus of TBI pathophysiology research is largely been in secondary injuries, since primary injuries are essentially irreversible. Clinical and experimental research has identified a multitude of potentially harmful events after TBI that starts with the primary injury and continues for months and years after the incident (Smith , et al. 1997, Williams , et al. 2001). The pathophysiological processes of secondary brain injury are complex and not completely understood. Key processes include excitotoxic damage, altered inflammatory response, altered calcium homeostasis and oxidative stress. Fig. 4a gives a schematic representation of secondary damage associated with TBI (Zasler , et al. 2007).

2.3.4.2.1 Excitotoxicity

Excessive release of excitatory amino acid neurotransmitters, particularly glutamate result in excitotoxic death of neurons (Bullock , et al. 1998, Robertson , et al. 2001). Excessive availability of glutamate results in over-stimulation of ionotropic and metabotropic glutamate receptors with consecutive Ca[2]+, Na+, and K+-fluxes (Floyd , et al. 2005, Yi , et al. 2006). Activation of constitutive NO synthase leads to NO production, PN formation and DNA damage. Activation of poly (ADP-ribose) polymerase (PARP) [DNA repair enzyme] leads to ATP depletion, metabolic failure and cell death (Zhang , et al. 1994).

Excitotoxicity results in increased intracellular Ca[2]+, which can result in irreversible damage to the mitochondria (Robertson 2004). Mitochondria is a Ca[2]+ sink and abnormal intramitochondrial Ca[2]+ levels disturb the electron transport chain, severely affecting the energy production (Sullivan , et al. 2005). This results in a deficiency of ATP at a time when the cell desperately is in need of protection, restoration of ion homeostasis and repair. Injured mitochondria could signal apoptotic proteins and also generators of ROS (Cornelius , et al. 2013, Lewen , et al. 2001). Fig 4b describes the main events of excitotoxic damage.

2.3.4.2.2 Inflammatory response

TBI induces a complex array of inflammatory response which acts as a double edged sword; some mechanisms attenuate and others exacerbate the deleterious effects of injury (Morganti-Kossmann , et al. 2002). The immediate response to an insult is characterized by activation of macrophages/microglia in the brain parenchyma accompanied by infiltration of activated leucocytes from the periphery (Bellander , et al. 1996). Leukocytes also enter the brain through disrupted BBB or via trans-endothelial migration and diapedesis.

Apart from the cellular response during TBI-induced inflammatory cascade, there is increased release of proinflammatory cytokines and activation of the complement system (Lin , et al. 2013).Increased release of inflammatory cytokines such as the IL-1 family, IL-6, IL-10,TNF-a, adhesion molecules such as ICAM-1 and complement activation play an important role in inflammatory reactions following TBI (Holmin , et al. 1997, Kumar , et al. 2012, Schmidt , et al. 2005, Venkatesan , et al. 2010). Increased cytokines act as chemoattractants for the leukocytes (Smith , et al. 2013). Studies have shown upregulation of IL-1 and TNF-a in the injured hemisphere up to three months after TBI in rats, suggesting their role in chronic degeneration (Holmin , et al. 1999). TNF-a together with IL-1P secreted from glial cells after injury act in an autocrine manner, stimulating proliferation and activation, leading to astrogliosis (Wang , et al. 2002). TNF-a mediates BBB-breakdown and increases leukocyte adhesion (Shohami , et al. 1999). In anothr study, it was demonstrated that inhibition of TNF-a reduced oedema and protected hippocampal neurones (Shohami , et al. 1996). Cellular immune reponse includes activation of glia, neurons, and cerebral accumulation of blood leukocytes (Czigner , et al. 2007). TBI also induces activation of complement system in humans and its components have been found in serum, in ventricular CSF and in brain tissue after TBI (Bellander , et al. 2011). The effects of its activation includes increased vascular permeability, cytokine production and facilitation of phagocytosis (Bellander , et al. 2004, Bellander , et al. 2001).

2.3.4.2.3 Necrosis and apoptosis in TBI

Both Apoptotic and necrotic neurons have been identified within contusions, and in regions remote from the site of impact in the days and weeks after injury. Degenerating oligodendrocytes and astrocytes have been observed within injured white matter tracts in TBI (Raghupathi 2004). Physical or ischemic insult, results in necrotic death of cells and is characterized by cellular swelling and disruption cell membrane in conjunction with lysis of nuclear chromatin. Cellular contents are released within the damage tissues evoking chemical mediators and inflammatory responses within local areas which contribute to the demise of cells, originally spared in the primary injury to the brain (Raghupathi , et al. 2000). Apoptosis is morphologically characterized by cell shrinkage, formation of an apoptotic nucleus, chromatin degradation and the formation of apoptotic bodies. Apoptotic signal pathways are observed to contribute to the death ofCNS cells following TBI (Eldadah , et al. 2000, Raghupathi , et al. 2002, Raghupathi, Graham and McIntosh 2000).The nature and/or severity of the injury and the bioenergetic status of the cell regulate the mechanism of cell death resulting in apoptosis or necrosis (Raghupathi, Graham and McIntosh 2000). Apoptotic cell death is energy dependent while the necrotic pathway is independent of energy. Morphological features of both apoptosis and necrosis were observed in in the same neural cell, (Raghupathi, Graham and McIntosh 2000, Stoica , et al. 2010)

Intracellular ATP level probably determines the kind of death signal for cells in TBI. As long as ATP is present within the injured neuron, apoptotic pathways are initiated, while its depletion (due to mitochondrial and/or PARP-activation) causes shift toward necrotic pathway (Raghupathi, Graham and McIntosh 2000, Stoica and Faden 2010). In this direction PARP-inhibitors are considered as drugs which have reduced brain damage following TBI in animal models (LaPlaca , et al. 2001).

2.3.4.2.4 Oxidative stress

Oxidative stress in TBI is due to excitotoxicity and exhaustion of the inherent antioxidant reserves (e.g. SOD, GPx, and catalase). Deleterious effects include peroxidation of cellular and vascular structures, cleavage of DNA, protein oxidation, and inhibition of the mitochondrial electron transport system (Bayir , et al. 2005, Chong , et al. 2005).

The sources for free radicals following TBI include activated microglia, infiltrating inflammatory cells (enriched with xanthine oxidase that generates O2”), dysfunctional mitochondria and increased free iron due to catabolism of extravasated haemoglobin (Halliwell , et al. 1986). Free iron ions accelerates the formation of hydroxyl radical (.OH, OH-), from superoxide and hydrogen peroxide, through the Fenton and Haber Weiss reactions (Halliwell , et al. 1992), which exacerbates the oxidative damage. Studies of experimental TBI have documented increase in hydroxyl radicals OH . and O2- immediately after injury (Fabian , et al. 1998).

Depletion of low molecular weight antioxidants vitamin E, ascorbate (vitamin C), uric acid, melatonin, and histidine-related compounds are observed in damaged tissue (Atlante , et al. 2001). The antioxidant reserves are depleted for at least seven days in TBI (Bayir , et al. 2002). Temporal decrease in antioxidant enzymes has been observed. For example, SOD activity decreases at 24 h and during 7 days after severe TBI (Cernak , et al. 2000).

Elevated lipid peroxidation was documented in severe TBI (Bayir , et al. 2004). Lipid peroxidation results in disintegration of membrane and increased microvascular permeability (Mathew et al., 1996). In humans, TBI increased plasma and CSF levels of malondialdehyde (MDA), as early as 2-3 h which persisted until 7 days post injury (Bayir , et al. 2002). Hydrogen peroxide has been recognized as a second messenger in intracellular signaling which might trigger activation of immediate early genes such as c­fos and c-jun, heat shock proteins, adhesion molecules, cytokines, growth factors, apoptosis-related proteins and proteases (Awasthi , et al. 2003). Fos protein forms a complex with Jun and regulates the expression of amyloid precursor protein, nerve growth factor, and opioid precursor protein among others. These genes appear to be up- regulated after TBI and their expression has been associated with apoptosis (Clemens 2000, Dragunow , et al. 1993, Hensley , et al. 2000).

Several studies have showed the implication of PN in post-TBI pathophysiology. Isoforms of NOS are known to be up-regulated during the first 24 h after TBI in rodents (Gahm , et al. 2000). The acute treatment of injured mice or rats with NOS inhibitors found to have a neuroprotective effect, establishing the neurotoxic role of PN after TBI (Wada , et al. 1998). PN-mediated damage has been documented in rodent TBI models including an increase in 3-NT levels and ADP ribosylation (Mesenge , et al. 1998). Fig 4c describes oxidative stress pathways activated by TBI and antioxidant protective systems.

2.3.4.2.5 Mitochondrial Dysfunction in TBI

During excitotoxicity, when the intracellular Ca[2]+ reaches a concentration of 500 nM, mitochondria acts a Ca[2]+ sink to modulate Ca[2]+ homeostasis in the cytosol (Schinder , et al. 1996). But excessive mitochondrial Ca[2]+accumulation results in uncoupling of electron transfer from ATP synthesis leading to mitochondrial dysfunction (Vergun , et al. 2003). Disturbed electron transport in the mitochondria due to Ca[2]+ overload produces electron leak resulting in free radical generation which can overwhelm antioxidant mechanisms and contribute to cell death (Lang-Rollin , et al. 2003)

Mitochondrial isoform of NOS is believed to be activated upon Ca[2]+ uptake, channelizing electron leak into generation of PN (Giulivi 1998; Tatoyan and Giulivi 1998). PN can oxidize a number of proteins in its vicinity and may cause loss of protein function by conformational changes as evidenced by its direct activation of membrane permeability transition (Bernardi , et al. 2006). Post-traumatic mitochondrial excessive Ca[2]+ uptake can lead to the formation of mitochondrial permeability transition pore (MPT). The MPT results in mitochondrial swelling and metabolic failure, release of death factors and ultimately cell death (Cheng , et al. 2012, Crompton 1999, Zoratti , et al. 2005). Some studies indicated that oxidation of thiol groups of proteins found in inner mitochondrial membrane (which leads to conformational changes) resulted in the MPT formation(Bernardi , et al. 2006).

Excessive Ca[2]+ uptake can also stimulate the production of ROS through activation of membrane permeability transition, release of cyt c, respiratory inhibition, release of pyridine nucleotides, and depletion of mitochondrial glutathione (Starkov , et al. 2004). ROS are known triggers of the mitochondrial apoptotic cascade via interactions with proteins of the MPT complex (Tsujimoto , et al. 2007). In TBI pathology, mitochondrial dysfunction serves as signaling platform and its membrane connects different death programs (Cheng, Kong, Zhang and Zhang 2012, Galluzzi , et al. 2008).

Several studies have documented perturbations in mitochondrial respiration and Ca[2]+homeostasis. Mitochondria isolated from the hemisphere ipsilateral to injury demonstrated reduced ability to sequester Ca[2]+(Xiong , et al. 1997). These alterations in mitochondrial respiration and Ca[2]+ transport were reversed by calcium channel blockers (Verweij , et al. 1997). Another study in isolated mitochondria and synaptosomes from injured cortex showed reduced membrane potential providing evidence of mitochondrial inner membrane permeability changes (Sullivan , et al. 1999).

Mitochondrial dysfunction in TBI has been proved by identification of oxidized and nitrated proteins in mitochondria from rodent models of TBI(Opii , et al. 2007, Reed , et al. 2009). Opii , et al. (2007) demonstrated several oxidized mitochondrial proteins in cortex and hippocampus of experimental TBI model. The list of identified proteins from cortex included- pyruvate dehydrogenase (PDH), voltage-dependent anion channel, fumaratehydratase 1, ATP synthase, and prohibitin. Cytochrome C oxidase Va, isovaleryl coenzyme A dehydrogenase, enolase-1, and glyceraldehyde-3- phosphate dehydrogenase had undergone oxidative modification in hippocampal mitochondria following TBI. Consistent with the proteomic data authors also found a reduction in the activities of PDH, complex I, and complex IV possibly due to oxidation of these proteins.

2.3.4.3 Secondary injury and neuroprotection

Secondary injury events of TBI may be pharmacologically modulated as shown by many studies (Luo , et al. 2011). These observations have led to the evaluation of a number of pharmacological strategies, including Ca[2]+ channel blockers, corticosteroids and other antioxidants, glutamate receptor antagonists, thyrotropin-releasing hormone analogs, opioid receptor antagonists, and magnesium administration, as well as various anti-inflammatory and immune modulatory treatments (Faden 1996). Some of these approaches, such as the use of NMDA, have strong experimental support (Faden , et al. 1989, Luo, Fei, Zhang, Qu and Fei 2011). It has been shown in rat model that modulation of apoptotic cell death by inhibiting caspases also improves outcome after TBI (Yakovlev , et al. 1997). Another strategy that has gained increasing experimental support but not yet been translated into well-designed clinical trials is the use of either combination therapies that block different components of the secondary injury cascade or administration of multipotential drugs (Faden 1993). Multipotential drugs are the therapeutic agents that modulate multiple secondary injury mechanisms to improve histological and functional outcomes after TBI. Table 6 shows the list of Multipotential drugs for TBI under clinical trial (Loane , et al. 2010).

2.3.5 Models of TBI

TBI models are essential for studying the biomechanical, cellular and molecular aspects of human TBI that cannot be addressed in the clinical setting. On a therapeutic note, TBI models could be extremely useful for developing and characterizing novel therapeutic interventions. A single model of TBI that represents all the clinical, neuropathological, inflammatory and biochemical features is currently not available. Over the years, a number of TBI models are evolved to produce a relatively homogeneous type of injury.

2.3.5.1 In vitro models of TBI

In vitro models of injury provide a platform for performing repeatable, well- controlled, environmentally isolated experiments. They can be used to examine the response of the brain parenchyma to mechanical stimuli without systemic confounds, such as systemic inflammation or hypoxia/ischemia.

In vitro transection models re-create axotomy on multiple cells (macroscopic) using a plastic stylet, a rotating scribe, or blades; alternatively, the axotomy can be performed at the single-cell (microscopic) level (Mukhin , et al. 1998, Mukhin , et al. 1997, Tecoma , et al. 1989). Compression model mimics focal injuries, which cause laceration of brain tissue in in-vitro using impactor or weight drop methods. The severity of the injury can be controlled by adjusting the force, depth, shape of impactor, and duration. In slices of tissue, this model generates a region of primary injury directly below the impactor as well as surrounding regions thus leading to secondary injury events (Church , et al. 2005, Sieg , et al. 1999). Events of closed head injury could be modeled in vitro using hydrostatic pressure models, which reproduce the time course of pressure changes thought to occur during closed-head TBI(Murphy , et al. 1993, Shepard , et al. 1991). In vitro stretch model utilizes cultures on deformable silicone membranes, which generates the injury through stretching the silicone substrate. Cultures remain adhered to the membrane during deformation, such that the tissue strain is 93 and 86% of the membrane strain in the x- and y-axis, respectively. In this model, the damage to the cell following injury is positively correlated with strain (Morrison , et al. 2006).

2.3.5.2 Animal models of TBI

Extrapolation of the in vitro models to the in vivo scenario has met with two major limitations associated with their preparation. First, tissue and cells may behave differently ex vivo in response to injurious stimuli. Second, the process of dissection and slicing prior to the experimental injury may affect tissue response (Morrison , et al. 2011). To address this issue, numerous animal models of TBI have been developed in view of heterogenous nature of clinical status, mode of injury and its severity. Although larger animals are closer in physiology to humans, rodents are mostly used owing to their modest cost and standardized outcome measurements (Xiong , et al. 2013).

One category of models produces a cortical contusion, while the others produce diffuse injury by acceleration and deceleration of the brain. However, none of the models completely simulate all the facets of human TBI and the mechanisms behind the human pathology. A table showing commonly used animal models of TBI is shown in the table 5.

Feeney , et al. (1981) introduced a model for cortical contusion injury in the rat called the weight drop model. In this model, the skull is exposed (with or without craniotomy) to a free falling, guided weight. The severity of the injury in these models dropped. Injuries in this model progress from white matter haemorrhages directly under the contused cortex in first few hours after injury to the development of a necrotic cavity by 24 hours (Feeney, Boyeson, Linn, Murray and Dail 1981).

To mimic diffuse axonal injury (DAI)an impact acceleration model (a kind of weight drop model) which is typically caused by falls or motor vehicle accidents was developed(Marmarou , et al. 1994). Marmarou’s model is characterized by widespread and bilateral damage of neurons, axons, dendrites and microvasculature as well as extensive DAI, particularly in the corpus callosum, internal capsule, optic tracts, cerebral and cerebellar peduncules, and the long tracts in the brainstem(Foda , et al. 1994). It is also characterized by motor and cognitive deficits such as difficulties with beam walking and memory (Schmidt , et al. 2000),

Controlled cortical impact (CCI) is another injury model based on cortical compression originally characterized by Dixon et al (1991) (Dixon , et al. 1991). The CCI model uses a pneumatic impact device to drive an impactor onto the exposed, intact dura and mimics cortical tissue loss, acute subdural haematoma, axonal injury, concussion, BBB dysfunction and even coma (Lifshitz , et al. 2007). It has been observed that higher piston velocity results in a more forceful impact and more compression because more energy is delivered to the brain. CCI is most often delivered between bregma and lambda, which causes deformation of the underlying cortex (Dixon, Clifton, Lighthall, Yaghmai and Hayes 1991).

In the Fluid percussion injury (FPI) models, an injury is produced by a fluid pressure pulse to the intact dura through a craniotomy, which is made either centrally around the midline between bregma and lambda (McIntosh , et al. 1987) or laterally over the parietal bone between bregma and lambda (McIntosh, Noble, Andrews and Faden 1987). The percussion injury produces brief displacement and deformation of brain tissue, and the severity of injury depends on the strength of the pressure pulse (McIntosh, Noble, Andrews and Faden 1987). FPI replicates clinical TBI without skull fracture and also replicates some of the key features of TBI such as intracranial haemorrhage, brain swelling and progressive grey matter damage (Graham , et al. 2000)

2.3.5.3 Limitations of current TBI models

In vitro and in vivo models cannot substitute for the human system, due to species barrier and significant differences in molecular mechanisms and genetic/epigenetic diversity. Physiological differences exist between humans and rodents in terms of brain structure and function. Differences in structural characteristics may lead to substantially different responses to trauma of comparable severity or type, from species to species (Povlishock , et al. 1994). To fill some of these lacunae, some studies have utilized human post mortem brain samples to study the molecular events (Frugier , et al. 2010).

2.3.6 Biomarkers of TBI

At present, the primary clinical indicators for the presence of brain injury are the GCS, pupil reactivity, and head CT. While these indices are useful for stratifying the magnitude and extent of brain damage, they have limited utility for predicting adverse secondary injury events or detecting subtle damage. Biomarkers, reflecting a biological response to injury or disease, have proven useful for the diagnosis of many pathological conditions. Biomarkers of TBI are essential to provide useful information about type, severity of the injury and outcome. Both CSF and blood markers are explored for potential markers for their clinical application.

CSF is considered as an optimal source of biomarkers of TBI since it bathes the brain its composition reflects biochemical changes that occur in this organ (Blennow , et al. 2010). Several established CSF biomarkers include proteins that indicate integrity of BBB and neuroinflammation, as well as axonal, neuronal and astroglial damage. The standard biomarker of BBB function is the CSF: Serum albumin ratio (Tibbling , et al. 1977) since albumin in the CSF is mainly from peripheral blood. In human TBI, elevated ratio of CSF: serum was associated with inflammatory response (Csuka , et al. 1999).

Inflammatory cytokines, such as IL-6, IL-8 and IL-10, were elevated in CSF in response to severe TBI. The magnitude of the rise correlated with the patients outcome and with the extent of BBB damage (Csuka , et al. 1999). Total tau and neurofilament light polypeptide (NFL) are the two well established CSF markers indicating axonal injury. Increased Tau was correlated with lesion size, clinical outcome and severity of injury (Franz , et al. 2003, Zemlan , et al. 2002). Similarly, NFL levels were higher in patients who suffered mild TBI (Neselius , et al. 2012). Amyloid precursor protein (APP) is considered to be another marker of axonal damage, following TBI since it accumulates in neurons and axons causing axonal damage (Ahlgren , et al. 1996, Gentleman , et al. 1995). Increased levels of neuron specific enolase (NSE), a glycolytic enzyme enriched in neurons was found to correlate with mortality after TBI both in adults and children CSF (levels are higher in no survivors than in survivors) (Chiaretti , et al. 2009).

In routine clinical practice, collection of peripheral blood samples is considerably easier than obtaining of CSF, which makes blood a standard material to test for a marker. Over the years, proteins like S100P, GFAP and Neuron specific enolase (NSE) have been evaluated for their potential clinical application as markers in the serum of TBI patients. One of the most promising markers of TBI is S100P, a 10-12 kDa calcium binding protein found in the cytoplasm of astrocytes and Schwann cells of the nervous system. It is involved in intracellular signal transduction processes through the inhibition of protein phosphorylation, and calcium homeostasis (Rustandi , et al. 1998, Wilder , et al. 1998). In vitro and in vivo studies have found neurotrophic and neuroprotective role for S100P (Barger , et al. 1995, Iwasaki , et al. 1997). In vitro studies show a significant increase in the S100P level in the culture medium until at least 48 hours after the injury (Willoughby , et al. 2004). Most of the clinical studies correlated S100P levels with the prognosis of patients. A large number of them indicate a correlation between increased levels of S100P and a poor outcome in severe head injuries. Serum S100P level >2pg/l within one to six hours of a severe head injury is a sensitive predictor of an unfavorable outcome (Woertgen , et al. 1999). Temporal study with 6 months of follow up shows a cut-off value of 2.5 pg/l to be 97% specific and 44% sensitive for the prediction of a poor prognosis in TBI patients (Raabe , et al. 1999).

GFAP is another promising marker for TBI. It is expressed in astrocytes in the CNS, and hence considered to be specific for TBI. Like S100P, increased GFAP in serum was also found to predict poor outcome in TBI patients. After severe TBI: high levels of GFAP (>1.5pg/l) were strongly predictive of death or a poor outcome (Vos , et al. 2004). A correlation between serum GFAP levels and severity of both injury and outcome has been documented (Nylen , et al. 2006, Pelinka , et al. 2004).

In an another study involving 79 TBI patients (moderate to severe on GCS scale), the researchers measured GFAP and S100P levels on hospital admission and the patient outcomes were then measured 6 months after injury. Those who died within the follow­up period exhibited 2.1-fold and 33.4-fold higher median levels of S100P and GFAP respectively (Vos , et al. 2010). This study along with several others strongly suggests the role of S100P and GFAP as potential markers of severity and outcome in TBI patients (Goyal , et al. 2013, Metting , et al. 2012, Okonkwo , et al. 2013, Ondruschka , et al. 2013).

Other candidate biomarkers of TBI are neuronal specific enolase (NSE), myelin basic protein (MBP) and hyper-phosphorylated neurofilament heavy chain (NFH). Initially NSE appeared to be a promising marker due to a its specificity to the brain (Johnsson 1996). Serum NSE levels of >10-pg/l was considered to have pathological correlation (Nygaard , et al. 1998). While some studies found a correlation between NSE levels and clinical outcome in severe TBI, (Herrmann , et al. 1999, McKeating , et al. 1998, Yamazaki , et al. 1995) others found no such correlation (Raabe , et al. 1998, Ross , et al. 1996). With several conflicting results, NSE does not appear to have a significant predictive value of the severity or subsequent outcome. MBP could be a more specific marker of TBI than NSE (specificity 96% versus 64%, respectively), only limitation being its suboptimal sensitivity (44% for MBP versus 71% for NSE) (Berger , et al. 2005). Elevated NFH in serum was correlated with severity for over 6 consecutive days in patients who eventually died from their injury (Zurek , et al. 2012).

Other potential markers are aII spectrin breakdown products. aII Spectrin is primarily found in neurons, expressed in axons and presynaptic terminals(Riederer , et al. 1986). It is broken down by proteases and the breakdown products are up regulated in TBI during neuronal necrosis and apoptosis, respectively (Pike , et al. 2001). Spectrin breakdown products from ventricular CSF were associated with clinical correlates of the severity of the trauma (Farkas , et al. 2005, Mondello , et al. 2010).

A large number of possible molecules in the CSF and serum have been analyzed to investigate their role as potential biomarkers in TBI, but many of them failed as a result of their lack of specificity and sensitivity. This has been the case for lactate dehydrogenase, creatine kinase isoenzyme BB, glutamic pyruvic transaminase, alpha­hydroxybutyric acid dehydrogenase, fructose 1,6-diphosphate aldolase, and malate transaminase (Bazarian , et al. 2006, Gabbita , et al. 2005, Kavalci , et al. 2007).

2.3.7 Protein dynamics and rational for Neuroproteomics in TBI

TBI induces several transitions in transcriptome and proteome, involving several modification steps that include the transcription cues and post-translational processes (Lisacek , et al. 2004). Since TBI causes a number of modifications to the transcriptome, including alternative splicing, polyadenylation, and methylation, changes are observed in their products as well (Dongre , et al. 2001). Newly modified mRNA transcripts translate into different sets of proteins, which are subject to further modification (Dongre, Opiteck, Cosand and Hefta 2001, Phizicky , et al. 2003).

Studies indicate immense alteration to the neuroproteome generated from post- translational activity as the first cellular response to injury. Proteins can undergo nearly 400 different types of PTMs (Morrison , et al. 2002) which include phosphorylation, dephosphorylation by kinases and phosphatases, proteolytic processing, acetylation, glycosylation, farnesylation, S-nitrosylation, lipidation, among many others. Proteins can also be conjugated to small protein tags such as ubiquitin or SUMO or cross-linked by transglutaminases. Proteins acquire new functions or conformational states due to modifications. Some of the deleterious modifications like protein oxidation and nitration may cause loss of function and aggregation following brain injury (Morrison , et al. 2002, Opii , et al. 2007).

Identification of PTMs can play an integral role in studying TBI as it can elucidate the functional significance on the protein identified (Husi , et al. 2000). For instance a study demonstrated the dephosphorylation of neurofilament-68 following TBI (Posmantur , et al. 2000). This data could reflect the dendritic and axonal damage due to NF proteolysis, as dephosphorylation of NF signals its calpain-mediated proteolysis. Two-dimensional gel blots were used to detect altered phosphorylation states of many proteins at 24 hours post-TBI (Jenkins , et al. 2002).

A number of proteomic approaches have examined TBI for biomarker discovery and differential proteome responses in patients and models (Ekegren , et al. 2008, Zetterberg , et al. 2008). Proteomics studies have demonstrated the practicality of identifying injured proteome from brain injury, and have provided insight into individual protein responses across clinical brain injury and in different models. Furthermore, high throughput data provided by mass spectrometric analysis provides an ample reserve of biomarker candidates for subsequent evaluation.

2.3.8 TBI and neurodegeneration

It has been observed that TBI patients display many of the same pathologies associated with several neurodegenerative diseases (Smith, Uryu, Saatman, Trojanowski and McIntosh 2003). Particularly TBI can induce accumulation of several proteins that are key pathologic aggregates found in AD and PD. The most widely studied of these proteins in TBI include P-amyloid precursor protein, amyloid-P(AP) peptides, neurofilament proteins, and synuclein proteins (Smith, Uryu, Saatman, Trojanowski and McIntosh 2003).

Some studies have explored a strong association of TBI with neurodegeneration including Parkinson’s disease. Goldman , et al. (2006) linked TBI with an increased risk for PD in twins and suggested that mild-to-moderate closed head injury may increase PD risk decades later. Johnson , et al. (2012) demonstrated that widespread neurofibrillary tangles and AP plaque pathologies are present in up to a third of patients following survival of a year or more from a single TBI. Bower , et al. (2003) also correlated TBI and PD risk in TBI patients. TBI is also shown to increase the neuronal vulnerability to a subsequent insult (Jenkins , et al. 1989).These observations suggest that the link between TBI and PD may be contingent upon exposure to other risk factors, with TBI being one element sensitizing neurons to other risk factors (Sulzer 2007).

2.4 Parkinson's disease (PD)

PD is the second most common neurodegenerative disorder after AD, with prevalence of approximately 0.3% of the population in industrialized nations. In the western population it affects approximately 1% of the population aged above 60 y (Tanner , et al. 1996). However, similar nationwide epidemiological data on the incidence and prevalence of PD from India is not available. Available data from three different regions of India suggest a low prevalence of PD among Indians. Razdan et al. reported a crude prevalence rate of 14.1 per 100,000 of a population of 63,645 from rural Kashmir (Razdan , et al. 1994). The prevalence for people over the age of 60 years was 247/100,000. Similar low, prevalence rates were reported from Bangalore (27/100,000), and rural Bengal (16.1/100,000) (Singhal , et al. 2003). In contrast, a high crude prevalence rate of 328.3/100,000 was reported among a population of 14,010 Parsis living in colonies in Mumbai (Bharucha , et al. 1988). PD is more prevalent in men than in women, with ratios of 1.1:1 to almost 3:1 being reported (Schrag , et al. 2000). With an increase in life expectancy and increasing geriatric population, demographic projections predict a corresponding increase in the number of PD patients.

2.4.1 Clinical symptoms and diagnosis

PD is a chronic, progressive neurodegenerative disease. It has recently been recognized as a complex illness, encompassing both motor and non-motor symptoms. It presents with four cardinal motor manifestations: bradykinesia (refers to a slowness and paucity of movement), rigidity, tremor, and postural instability with an asymmetric onset spreading to become bilateral with time. Non motor symptoms include depression, sleep disturbance, sensory abnormalities, autonomic dysfunction, and cognitive decline (Langston 2006).

Diagnosis of PD is generally based on the presence of cardinal motor symptoms associated with exclusionary symptoms and response to the drug levodopa (L-DOPA) (Rao et al. 2003). Several rating scales have been used for the diagnosis of PD, but have not been sufficiently validated. Hoehn and Yahr scale is commonly used to compare groups of patients and to provide gross assessment of disease progression, ranging from stage 0 (no signs of disease) to stage 5 (wheelchair bound or bedridden) (Hoehn , et al. 1967). The Unified PD Rating Scale (UPDRS) is the most established scale for assessment of disability and impairment (Post , et al. 2005).

2.4.2 Pathobiology

Neuronal loss in the substantia nigra pars compacta (SNc) and the subsequent depletion of striatal dopamine content are accepted as being responsible for the classical motor dysfunctions of PD (Dexter , et al. 2013). Neuropathological diagnosis of PD includes detection of marked dopaminergic neuronal loss in the SNc, presence of Lewy bodies (LB), and eosinophilic inclusions with dense core surrounded by a pale-staining halo of radiating filaments (Fig. 5) (Dauer and Przedborski 2003). LBs are aggregates of proteins commonly found in PD and other neurological disorders. Major protein constituents of LBs are a-synuclein, ubiquitin, neurofilament protein, tau and a-B crystallin. Other oxidatively modified and damaged proteins, which cannot be cleared by the cellular proteasomal system have been detected within LBs (Shults 2006). They are eosinophilic spherical bodies which can be stained by hematoxylin and immunologically stained for ubiquitin and a-synuclein. Over the years, studies have observed LB pathology in many non-dopaminergic nuclei including locus coeruleus, reticular formation of the brain stem, raphe nucleus, dorsal motor nucleus of the vagus, basal nucleus of the Meynert, amygdala, and hippocampus. Importantly, all of these nuclei degenerate with LB pathology, suggesting a common pathogenic process with that occurring in the SNc. It is the LB pathology in these non-dopaminergic areas in the brain that is probably responsible for many of the non-motor symptoms observed in PD patients (Jellinger 2012).

PD pathology does not start in the SNc, but rather LB pathology and the deposition of a-synuclein are proposed to originate in the olfactory bulb and lower brain stem, from where they spread in stages to involve the midbrain, and eventually spreading to cortical regions. Braak , et al. (2003) proposed caudorostral spread of PD pathology such that a-synuclein deposition begins in the dorsal motor nucleus of the vagus, from where it probably proceeds in an upward direction via the pons to the midbrain and then to the basal prosencephalon and mesocortex, finally reaching the neocortex. It is now widely accepted that “spreading” of the degenerative pathology does occur in PD, although not all the PD cases follow the same pattern (Kalaitzakis , et al. 2008). This has raised an important concept that PD pathology may be propagated from one neuron to another through prion-like mechanism (Goedert , et al. 2010). Post­mortem studies on PD patients who had received intrastriatal grafts of embryonic dopaminergic neurons over 10 years prior to death showed that the grafted neurons exhibit LBs and Lewy neuritis (Kordower , et al. 2008). These findings stimulated the hypothesis that a-synuclein transferred directly from host brain cells to the grafted neurons. Experiments involving cell culture, animal models, and fetal mesencephalic cells transplanted into the PD striatum have demonstrated that a-synuclein indeed can transfer between cells in a prion-like fashion (Luk , et al. 2009, Mougenot , et al. 2012). Such findings will have a major impact on the molecular understanding of pathogenesis in PD.

2.4.3 Etiological factors

2.4.3.1 Aging

Age represents the biggest predisposing factor for PD. The accumulation of oxidatively modified proteins has been shown to increase with age, which correlates with the late-onset of neurodegenerative pathology. Studies on cultured human fibroblasts and human brain tissue have shown that in elderly individuals, approximately one third of proteins have been oxidatively modified (Grune, Shringarpure, Sitte and Davies 2001). Increase in protein oxidation is not linear, but instead occurs as an initial gradual rise that magnifies several fold during late age (Smith , et al. 1991). Increased production of reactive species, decreased antioxidant function, and impaired ability to repair or remove the modified proteins could be the reason for accumulation of oxidative modifications. Dysfunctional clearance of proteins has been supported by findings that the activities of the Ubiquitin proteasome system (UPS), macro-autophagy and Chaperone Mediated Autophagy (CMA) decline with age, consequently diminishing the ability of the cell to clear modified proteins (Sitte, Merker, Von Zglinicki, Davies and Grune 2000). Impaired degradation of proteins with aging causes oxidatively modified proteins to accumulate in the cell, increasing their propensity for aggregation. Additionally, once the activity of these degradation pathways is diminished, a feed-forward effect on oxidative damage may result. Inhibition of proteasomal system increased mitochondrial reactive oxygen system (ROS) and decreased complex I and II activity (Sullivan , et al. 2004). Therefore, age related decline in proteasome and autophagy pathways may be further contributing to oxidative damage.

2.4.3.2 Dopamine (DA) Oxidation

It has been postulated that the oxidative environment of SNc, particularly in dopaminergic neurons might be a key component in the pathogenesis of PD. Normally, vesicular monoamine transporters rapidly sequesters DA within vesicles delaying its oxidation. Once DA remains in the cytosol, it can be rapidly oxidized at physiological pH to generate reactive ortho-quinones, aminochromes, as well as ROS. Neurotoxin induced cell culture and rodent models have shown excessive cytosolic oxidation of catecholamine (LaVoie , et al. 1999). Studies suggest that without the ability to sequester intracellular DA and maintain low cytoplasmic levels, reactive metabolites of DA will cause toxicity in vivo. Accumulation of the DA oxidation product, neuromelanin, has been shown in human SNc. Increased DA oxidation in postmortem PD brain SNc as compared to age-matched controls was indicated by increased levels of cysteinyl- catechol derivatives (Zecca , et al. 2003).

2.4.3.3 Environmental factors

Previous studies provided evidence that the pathogenesis of PD could involve complex interactions between environmental and genetic factors. The contribution of environmental factors was established by the identification of cases of parkinsonism in patients exposed to acute doses of the neurotoxin 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) (Langston , et al. 1983). Other environmental toxins, such as the herbicide paraquat and the pesticide rotenone are also shown to cause dopaminergic neuronal cell loss and Parkinsonism. MPTP is highly lipophilic and can easily cross blood brain barrier (BBB). It is converted via monoamine oxidase B within glial cells and serotonergic neurons to its active form 1-methyl-4-phenyl-2, 3-dihydropyridinium ion (MPP+), which is the active toxic compound. MPP+ is transported into dopamine neurons by the dopamine transporter, and causes selective toxicity to dopaminergic neurons. Within the mitochondria, MPP+ inhibits complex I (Przedborski , et al. 2003). In the mitochondria MPP+ also inhibited the a-ketoglutarate dehydrogenase complex of the mitochondrial tricarboxylic acid cycle. Epidemiological studies suggested the association of pesticides such as rotenone and paraquat in PD pathogenesis. Rotenone is a potent and specific inhibitor of complex I. Treatment with Rotenone and MPTP in rodents induced dopaminergic cell death and inclusion bodies similar to LBs (Betarbet , et al. 2000). Paraquat is one of the most commonly used pesticides. The chemical structure of paraquat is similar to MPP+. Epidemiological studies associate the exposure of this chemical to the development of PD. Recent studies revealed that exposure to a combination of paraquat and maneb (an agricultural pesticide), exacerbate dopaminergic cell loss in the rodent model and cause an increased incidence of PD in humans (Costello , et al. 2009).

2.4.3.4 Genetic aspects of PD

Familial forms of PD and the associated gene mutations currently account for approximately 10% of cases and have distinct clinical and pathological phenotypes. Studies have found at least 17 autosomal dominant and autosomal recessive gene mutations in familial PD (Houlden , et al. 2012). These include a-synuclein mutations and triplication, parkin, leucine-rich repeat kinase 2 (LRRK2), ubiquitin carboxyl- terminal hydrolase L1 (UCH-L1), DJ-1, phosphatase and tensin homolog-inducible kinase 1 (PINK1), and glucocerebrosidase (GBA) (Table 7)(Dexter and Jenner 2013). Of these, parkin and LRRK2 are the most common genetic links to young and late-onset PD, respectively, while GBA mutations may be the most common risk factor. Mutations or triplications of the SNCA gene (encodes a-synuclein) have been associated with familial PD (Singleton , et al. 2003). Fibrillary forms of the a-synuclein protein are identified as major structural components of LBs in PD and other synucleinopathies. a- synuclein mutations cause PD through toxic gain-of-function mechanism consistent with the dominant inheritance pattern of mutations. Missense mutation in a-synuclein, A30P and A53T cause mutant proteins to self-aggregate to form oligomeric species and LB-like fibrils in vitro compared to wild-type a-synuclein (Conway , et al. 1998). a- synuclein oligomers are formed as higher-order aggregates, such as amyloid-like fibrils, which precipitate as the filamentous structures as observed in LBs and Lewy neurites (Fig. 6)

LRRK2 mutations were identified as the causative gene for PARK8-linked familial PD. LRRK2 interacts with parkin (a component of E3 ubiquitin ligase), and mutant LRRK2 induces apoptotic cell death in cultured neurons. Mutations in parkin (a 465 amino acid E3 ubiquitin ligase) account for the majority of early-onset familial PD cases. Parkin transfers ubiquitin to target proteins for degradative (i.e., via the proteasome system) or non-degradative (i.e., signaling) purposes. Loss-of-function of parkin can contribute to UPS dysfunction (Horowitz , et al. 2010). PINK1 encodes a 581 amino acid serine/threonine kinase that is localized to the mitochondria. Mutations in PINK1 are the second most common cause of autosomal recessive early-onset familial PD after parkin. Loss-of-function of PINK1 leads to enhanced increased oxidative stress and mitochondrial dysfunction (Moore , et al. 2005). Mutations in DJ-1 represent a rare cause of early-onset familial PD. DJ-1 could be involved alongside parkin and PINK1 in protecting the mitochondria against oxidative stress. Reduced DJ-1 expression is found to be associated with proteasome inhibition (Horowitz and Greenamyre 2010).

2.4.4 Mechanisms of neurodegeneration in PD

2.4.4.1 Oxidative stress in PD

Although several factors have been proposed for the pathogenesis of PD, oxidative stress has been hypothesized to be linked to both the initiation and the progression of PD. Initial evidence for the existence of oxidative stress in PD came from post-mortem analyses of brain tissues obtained from PD patients. These studies demonstrated increased levels of oxidized proteins, lipids, and nucleic acids. Autopsy PD brains showed, decreased concentration of PUFAs in the SNc, while MDA levels were increased (Dexter , et al. 1989). Additional evidence of lipid oxidation in PD is provided by the demonstration of an increase in 4-HNE with ~58% of nigral neurons positively stained for HNE-modified proteins in PD brain tissues. In contrast only 9% of nigral neurons were positive in the control subjects (Yoritaka , et al. 1996).

Levels of protein carbonyls, markers of oxidative damage to proteins, were also significantly increased in postmortem samples of SNc in PD brains compared to controls (Alam , et al. 1997). Nitration and nitrosylation of a-synuclein and parkin in PD have been documented (Giasson , et al. 2000). 3-NT immunoreactivity was also demonstrated in LBs within melanized neurons and in amorphous deposits associated with intact and degenerating neurons (Good , et al. 1998). Nitration is observed in all tyrosine residues (Tyr39, Tyr125, Tyr133 andTyr136) of a-synuclein, and this nitration contributes to PN-induced aggregation of a-synuclein (Takahashi , et al. 2002). Nitrated a-synuclein is resistant to proteolysis and prone to aggregation, and has reduced lipid binding tendency and solubility in the cells. Nitration confers, a-synulein more immunogenicity, which could explain the increased inflammatory response observed in PD patients (Benner , et al. 2008). Like other markers of oxidative damage, 8-OHdG is markedly increased in SNc of postmortem PD brains (Alam , et al. 1997). Increased levels of oxidative stress related deletions in mitochondrial DNA were reported in few spared dopaminergic neurons in the PD SNc and high levels of these mutations were associated with respiratory chain deficiency (Bender , et al. 2006).

Postmortem studies have also revealed changes in neurochemicals that may predispose the PD brain to oxidative damage. GSH is the earliest known indicator of nigral cell loss, and the magnitude of GSH depletion is correlated with the severity of the disease. Moreover, a significant increase in GSSG turnover occurs in PD (Pearce , et al. 1997). Reduced GSH levels in the brain renders it more susceptible to oxidative insult and neurodegeneration, with increasing age GSH levels has been observed to decline. GSH level in the brain is highest in cortex followed by cerebellum, hippocampus, striatum and the least in SN, which explains the increased susceptibility of SN for oxidative damage during PD (Abbott, Nejad, Bottje and Hassan 1990). GSH depletion in PD is observed in the SNc but not in other regions (Sofic , et al. 1992). During PD pathogenesis, GSH depletion is indeed one of the earliest events observed, which precedes both CI inhibition and dopamine depletion (Bharath , et al. 2002). GSH depletion models exhibit oxidative damage and mitochondrial dysfunction and selective inhibition of dopaminergic neurons (Andersen , et al. 1996). Several postmortem studies have associated neurodegeneration in PD with abnormalities of iron metabolism. Iron is found in high concentrations in SN, and is capable of catalyzing free radical formation hence its metabolism is of particular interest in relation to oxidative stress. Increased levels of iron and decreased levels of ferritin has been observed in the SNc of PD patients (Dexter , et al. 1989).

2.4.4.2 Mitochondrial dysfunction

The first link between parkinsonism and mitochondria became evident in the early 1980s, with the discovery of a neurotoxin MPTP mediated neurodegeneration via complex I inhibition and ensuing mitochondrial dysfunction (Langston , et al. 1983). In support of a direct or indirect role of complex I, complex I activity has been reported to be reduced by about 30% in the SNc and frontal cortex of PD brains (Parker , et al. 2008). Complex I subunits derived from both mitochondrial and nuclear genomes were found to be oxidatively damaged in isolated mitochondria prepared from frontal cortex of autopsy PD brains, as suggested by an increase in protein carbonyls (Keeney , et al. 2006). In another study, SNc neurons from PD and age matched controls was compared, and high levels of deleted mtDNA were detected, suggesting the importance of somatic mtDNA deletions in the selective neuronal loss observed in PD and aging. SNc dopaminergic neurons in PD patients and aged individuals harbor high levels of mtDNA deletions (up to 60% mtDNA deletion), which are associated with cytochrome c oxidase (complex IV) dysfunction (Bender , et al. 2006). Construction of cybrids using mitochondrial DNA (mtDNA) from PD patients has clearly demonstrated the encoding of complex I defect and deletions in mtDNA associated with complex IV with concomitant oxidative damage (Gu , et al. 1998). Alterations in mitochondrial complex I are a major source of ROS generation in patients with PD. Inhibition of complex I hampers the mitochondrial respiratory chain, which causes incomplete oxygen reduction, thereby generating (V- and subsequent generation of ROS. Therefore, dysfunctional mitochondria are the primary intracellular source of ROS and contribute to oxidative stress-mediated neurodegeneration during PD.

Recently, interest in the mitochondrial role has stemmed from genetic investigations in familial PD. Notable mutations associated with altered mitochondrial function were found in a-synuclein, parkin, PINK1, DJ-1 and LRRK2. These mutations can lead to altered protein localization in mitochondria in PD, structural and functional abnormalities in mitochondria, and a decrease in complex I assembly and activity (Schapira 2008). Loss of function of, DJ-1, parkin and PINK1, decreases mitochondrial protection against oxidative stress, which in turn increases mitochondrial dysfunction. Parkin and PINK1 act in tandem to regulate the process of mitochondrial turnover by autophagy, specifically mitophagy. This is particularly important in PD, in which autophagy seems impaired, reducing the cells’ ability to remove damaged mitochondria. Genome-wide association studies, laser-captured human dopaminergic neuron studies, and SNc transcriptome studies have documented evidence pointing to the key role of deficits in the mitochondrial electron transport chain in PD (Elstner , et al. 2011).

2.4.5 Experimental models of PD

Current animal models of PD can be broadly divided into two categories: genetic and neurotoxic models. A common feature of neurotoxic models is their ability to produce oxidative stress and death of DA neuronal populations as seen in PD. Neurotoxic models produced by 6-hydroxydopamine (6-OHDA), MPTP and paraquat and rotenone are the most widely used toxic models of PD. Animal models of PD based on genetic mutations are important as they represent potential therapeutic targets. Table 8 summarizes the advantages and disadvantages of these models and their potential roles in elucidating the mechanisms for PD pathogenesis and in testing experimental therapeutics (Tieu 2011).

2.4.6 Current approaches to treatment:

Standard treatment of PD includes DA replacement employing L-dopa and DA agonists. A series of enzyme inhibitors, namely peripheral decarboxylase inhibitors, catechol-O-methyl transferase inhibitors, and monoamine oxidase-B (MAO-B) inhibitors are also used supportively. Many other are symptomatic approaches treating motor deficits include anti-cholinergics and the weak NMDA receptor antagonist amantadine. L-dopa is considered as a potential reason for both slowing the disease progression and acceleration of the cell death in PD. For many years, the ability of L- dopa to generate free radicals and to kill dopaminergic cells in culture tainted the drug with a neurotoxic label. But, several studies, based on a range of in vivo studies, postmortem studies in humans, and clinical experience refute this claim, but it still remains controversial (Gerlach , et al. 2005). MAO inhibitors selegiline and rasagiline have attracted most attention as potential disease modifiers. These drugs are found to prevent MPTP toxicity in dopaminergic neurons through inhibition of MPP+ formation by blocking MAO-B activity. Both of these drugs are known to alter the rate of loss of dopaminergic neurons through their anti-apoptotic actions, antioxidant effects, anti- glutamatergic effects, and neurotrophic actions (Jenner 2004). But these are all symptomatic approaches to treating the motor deficits of PD with little effect on non­motor symptoms and no proven effect on disease progression. Consequently PD does not have a permanent cure. Therefore, academic institutions and pharmaceutical companies worldwide have committed enormous funds and research efforts in identifying and testing potential drugs that could be used to alleviate such debilitating disorders and retard mental deterioration.

The challenge faced by scientists is to come up with a drug(s) that could simultaneously target multiple disease pathways without significant side-effects, be non-toxic at higher concentrations in humans and have the ability to cross the BBB. Consequently, scientists are envisaging a paradigm shift from monotherapy involving isolated compounds to target a specific cellular pathway to multi-therapy based on various targets with a greater probability for success. Towards this, natural substances from plant origin such as phytochemicals which possess highly beneficial medicinal properties are being exploited (Kumar 2006). Most of the information regarding phytochemicals has originated from long-established knowledge of natural herbs and traditional medicine that has existed for thousands of years in developing countries such as India. This has in recent years dramatically increased the interest in the use of herbal products in the western countries (Pal , et al. 2011, Tsao 2010). Consequently, there is increasing support for combination of modern drugs with traditional medicine to evolve better therapies. One such approach involves the exploitation of nutraceuticals and improved dietary compositions as an independent or supplement therapy to treat or prevent PD.

Several experimental evidences have demonstrated the therapeutic potential of phytochemicals in human diseases including neurodegenerative disorders. It is interesting to note that although phytochemicals normally function as toxins and protect plant source against damage due to pests and harmful organisms, at the lower concentrations consumed by humans, these compounds activate adaptive cellular stress responses in vivo (“hormetic mechanisms”) thereby providing cytoprotection against exogenous toxins. Such hormetic mechanisms relevant to neuronal survival and improved brain function ultimately elevate the levels of antioxidant enzymes, protein chaperones and neurotrophic factors (Mattson , et al. 2007). Polyphenols form a major group of such phytochemicals with potential therapeutic and curative properties.

Polyphenols are the most widely distributed natural compounds in the plant kingdom (Fig. 8). They are the secondary metabolites which are synthesized to protect the plants against microbial attack, pests and ultraviolet radiation. Polyphenols are present in fruits, vegetables; oils etc. and provide plants with brilliant colours and fragrance. Polyphenols might possess many biologically significant functions with implications for human degenerative diseases (Han , et al. 2007).

More recently, curcumin has found its role as a neuroprotective agent in models of neurological disorders such as stroke and AD (Lim , et al. 2001, Thiyagarajan , et al. 2004, Yang , et al. 2005). Lim et al. have shown that dietary curcumin reduces amyloid pathology, oxidative stress and pro-inflammatory markers in transgenic mice; in addition curcumin reduces the levels of GFAP (Lim , et al. 2001). Further, curcumin not only prevented formation of aP aggregates but could also disintegrate preformed aggregates by directly binding to plaques (Yang , et al. 2005). Curcumin can cross the BBB and is found to be least toxic in human subjects at high doses (Lao , et al. 2006, Qureshi , et al. 1992, Shankar , et al. 1980, Shoba , et al. 1998). It can detoxify ROS, prevent protein aggregation and induce neurogenesis in vivo (Kim , et al. 2008, Priyadarsini , et al. 2003, Xu , et al. 2007).

Since PD is a neurological condition involving oxidative and nitrosative stress pathways, curcumin can serve as a potential therapeutic molecule for PD. Although thiol reducing agents such as GSH have been evaluated for their anti-PD activity, their efficiency is limited due to poor ability to cross the BBB (Bharath, Hsu, Kaur, Rajagopalan and Andersen 2002). Curcumin on the other hand can cross the BBB and can be administered at higher doses without the risk of toxicity. Curcumin treatment protected SNc neurons, improved striatal dopamine levels and chelated Fe[2]+, following administration of 6-OHDA in rats (Zbarsky , et al. 2005). Curcumin treatment abrogated dopamine induced striatal neuron cell death (Luo , et al. 1999). Curcumin treatment could further increase the density of dopaminergic neurons in the SNc (Vajragupta , et al. 2003). Interestingly, chronic dietary consumption of turmeric caused an increase in the TH positive neurons in the SNc (Mythri , et al. 2011). This is consistent with earlier reports suggesting that neuroprotection by curcumin was probably due to neurogenesis (Kim , et al. 2008, Xu , et al. 2007). Similarly it was demonstrated that curcumin derivatives, with improved bioavailability offered a better neuroprotection against MPP+ in dopaminergic neurons (Mythri, Harish, Dubey, Misra and Bharath 2011).

Curcumin treatment attenuated 3-nitropropionic acid mediated oxidative stress in vivo (Kumar , et al. 2007). However, PN mediated nitrosative stress and mitochondrial dysfunction were attenuated by pretreatment with curcumin in vitro (Mythri, Harish, Dubey, Misra and Bharath 2011). Curcumin mediated protection from PN toxicity is probably due to its direct interaction with and inactivation of PN in vitro (Iwunze , et al. 2004, Mythri , et al. 2007) or indirectly by enhancing GSH levels in vivo (Jagatha , et al. 2008, Mythri, Jagatha, Pradhan, Andersen and Bharath 2007). Curcumin down regulates inducible-nitric oxide synthase (iNOS) which is probably mediated by the cJun/AP-1 pathway (Brouet , et al. 1995, Chan , et al. 1998, Chen , et al. 2006). It has been suggested that curcumin binding to the AP-1 binding site on the promoter region of iNOS, resulted in its inactivation (Brouet and Ohshima 1995). Curcumin reduces iNOS mRNA levels and blocks induction of NFkB (Pan , et al. 2000). Rajeswari and colleagues have reported an increase in striatal dopamine and dihydroxy phenyl acetic acid (DOPAC) levels following curcumin injection in MPTP injected mice (Rajeswari , et al. 2008). In addition curcumin restored mitochondrial membrane potential, caused elevation in Cu-Zn SOD and modulates NFkB nuclear translocation (Wang , et al. 2009) by inhibition of IL6 and TNFa (Wang , et al. 2009). Curcumin treatment inhibits AP-1 pathway by prevention of c-Jun phosphorylation (Luo, Hattori, Munoz, Qin and Roth 1999) in addition to inhibition of JNK phosphorylation (Sawada , et al. 2002, Yu , et al. 2010) and caspase 3 activation (Yu , et al. 2010). It has been reported that curcumin induces anti apoptotic genes such as Bcl-2 and inhibits iNOS resulting in reduction of ROS, abrogates oxidative stress mediated cytochrome c release or caspase 3 activation, significantly increased levels of anti-apoptotic proteins Bcl-2 and Bcl-xL and decreased levels of pro-apoptotic proteins Bax and Bad (Chen , et al. 2006, Chen , et al. 1998). Curcumin induced cytoprotection was probably mediated by the Bcl-2 - mitochondria - ROS - iNOS pathway (ref).

Interestingly, not only is curcumin an antioxidant molecule but can also increase the levels of GSH, probably by enhancing astrocytic efflux of GSH(Stridh , et al. 2010). Jagatha, Mythri, Vali and Bharath (2008) have shown that curcumin enhances GSH levels in vitro and in vivo, in addition to enhancing the antioxidant enzyme activities of SOD and catalase in the striatum and midbrain of MPTP injected mice (Rajeswari 2006). In addition curcumin treatment results in a 3-7 fold increase in the modifier subunit of the GSH synthesizing enzyme, gamma-glytamyl cysteine ligase (GCL), in both the neurons and astrocytes (Dickinson , et al. 2003, LaVoie , et al. 2005). Curcumin mediated increase in the transcription of GCL genes is probably via regulation of the binding of transcription factors to the 12-tetradecanoate 13-acetate (TPA)-responsive elements (TRE) and electrophilic response element (EpRE) elements (Dickinson, Iles, Zhang, Blank and Forman 2003). It further activates the Nrf2/ARE pathway which in turn induces synthesis of phase II antioxidant enzymes such as GCL (Jeyapaul , et al. 2000, Wild , et al. 1999), GST (Chanas , et al. 2002, Ye , et al. 2007), NQO1 (NADPH: quinone acceptor oxidoreductase) (Ye, Hou, Zhong and Zhang 2007) and HO-1( Hemeoxygenease 1).

Curcumin further inhibited aggregation of a-synuclein in vitro (Pandey , et al. 2008, Wang , et al. 2010) and aggregation of A53T mutant a-synuclein in SH-SY5Y cells in a dose dependent manner. Curcumin, thus enhanced a-synuclein solubility rendering them non-toxic (Pandey, Strider, Nolan, Yan and Galvin 2008). It also exhibited neuro­restorative properties as shown by its ability to disintegrate preformed a-synuclein fibrils (Ono , et al. 2006). Curcumin pre-treatment resulted in a reduction in aggregation of synphilin-1, a component of Lewy neuritis seen during PD, as a function of rotenone induced nitrostive stress, in SH-SY5Y dopaminergic cells suggesting its efficacy in ameliorating nitrosative stress induced damage (Pal, Miranda and Narayan 2011).

Curcumin has been suggested to regulate proteins involved in iron metabolism. It has been reported that curcumin induces activation of iron regulatory protein and repression of ferritin and a reduction in levels of hepcidin, suggesting a role as an iron chelator (Jiao , et al. 2006, Jiao , et al. 2009). Harish , et al. (2010) and Mythri , et al. (2011) have used diester derivatives of curcumin (di-piperoyl, di-valinoyl and di- glutamoyl) with a resultant enhanced protection against GSH depletion mediated oxidative stress and toxin induced cell death in a dopaminergic neuronal cell line.

Particularly the di-glutamoyl derivative showed maximum protection due to its de­esterification following absorption thus providing glutamate as a precursor for GSH synthesis. Thus one could use such pro drugs with improved uptake and better radical scavenging properties to combat oxidative and nitrosative stress in various neurodegenerative disorders such as PD.

Mythri, Veena, Harish, Shankaranarayana Rao and Srinivas Bharath (2011) have demonstrated that chronic dietary consumption of turmeric offers neuroprotection in MPTP induced mouse model of PD. Although curcumin is the most active ingredient of turmeric with implications in neuroprotection, it could be more effective in their natural milieu. Also the biological properties of natural extracts would probably be mediated by their various constituents rather than a single compound. Therefore it would be interesting to study the pharmacokinetics and pharmacodynamics of curcumin or the other biologically active components of turmeric in order to completely understand the mechanism of neuroprotection.

3 SCOPE OF THE RESEARCH

The human brain is subjected to several physiological and pathological processes. Analysis of such processes would be beneficial in understanding the brain function and to delineate the mechanisms underlying brain pathologies. This would also help in evolving novel therapeutic strategies applicable to human diseases. In this regard, it is pertinent to investigate the biochemical processes associated with brain aging, neurotrauma (TBI) and neurodegeneration (PD). Research studies have indicated that aging and TBI could be linked to the susceptibility of the brain to neurodegeneration. Oxidative/nitrosative stress and mitochondrial dysfunction are common mechanisms underlying these phenomena as demonstrated from animal models. However, all the oxidant-antioxidant mechanisms and mitochondrial markers involved in these brain phenomena have not been completely delineated. Further, such studies are limited in human brains due to the non-availability of samples.

Though our understanding of brain function and pathology has advanced with some progress in biomarker discovery and pharmacotherapy, many endeavours have not reached their logical end since the experimental results are not validated in the human condition. This is clinically pertinent since in vitro and in vivo models cannot substitute for the human system, due to species barrier and significant differences in molecular mechanisms and genetic/ epigenetic diversity.

The current proposal aims at investigating the status of oxidant/antioxidant markers and mitochondrial function in human brains during TBI, PD and aging. The current study would also attempt to analyze the proteomic changes associated with TBI compared to age-matched controls which could help in delineating the primary and secondary events in neurotrauma. On a clinical note, natural antioxidants and derivatives would be tested as therapeutic molecules applicable to neurodegenerative diseases and neurotrauma.

4 Objectives of the Research

Objective 1: To analyze the effect of pre and postmortem factors including age on oxidant and antioxidant markers in the non-traumatized regions of human brains from TBI subjects: This includes assay of oxidant markers [protein carbonyls, protein nitration by 3-nitrotyrosine (3-NT) and lipid peroxidation], antioxidant markers (antioxidant enzymes, GSH and associated metabolic enzymes) in whole cell extracts and neuropil fractions (mitochondria, synaptosomes and cytosol) of non-traumatized regions of TBI postmortem brains compared to controls. This would be done to ascertain the influence of premortem (age, gender and agonal state) and postmortem factors [postmortem interval (PMI), storage time] in human brains.

Objective 2: To analyze the markers of oxidative/ nitrosative stress and mitochondrial function in TBI- Role of aging: This includes (a) biochemical analysis (assays for antioxidant/oxidant and mitochondrial markers) histopathological analysis (histology and immunohistochemistry for degenerative, inflammatory and aggregation markers) in young and old brains and (b) proteomic analysis (quantitative mass spectrometric analysis of differentially expressed proteins) in contusion, pericontusion (penumbra) regions vs. non traumatized region including validation studies in fresh and postmortem human brains from TBI subjects.

Objective 3: To understand the role of oxidative stress in human samples and cell culture model of PD: This includes (i) analysis of antioxidant/oxidant markers in the neuropil fractions of PD frontal cortex tissue compared to age-matched controls (ii) assessment of the neuroprotective ability of curcumin bioconjugates in the cell model of early PD.

5 Materials and Methods

5.1 Materials

Chemicals, solvents and reagents used were of analytic grade. Bulk chemicals and solvents were obtained from Merck & Co. Inc. (Whitehouse station, NJ, USA) or Sisco Research Laboratories (SRL) Pvt. Ltd. (Mumbai, Maharashtra, India). Fine chemicals, cell culture materials, Anti-3NT, Anti-Synaptophysin, Anti-beta actin and anti- dinitrophenol (a-DNP) antibody were obtained from Sigma (St. Louis, MO, USA). Proteomic grade trypsin was procured from Promega (Wisconsin, USA). Anti- CHIT 1 antibody was obtained from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). Antibodies against GFAP [GA-5], Neurofilament [NE-14], CD68 [KP1] (BioGenx, Fremont, CA, USA), Iba1 (abcam, Cambridge Science Park Cambridge, UK), HLA DR (DAKO, Denmark) were kindly provided by the Human Brain Tissue Repository (HBTR), Department of Neuropathology, NIMHANS. Anti-horseradish peroxidase conjugated secondary antibodies (anti-rabbit, anti-mouse and anti-goat) were obtained from Bangalore Genei (Bangalore, Karnataka, India). Coomassie Brilliant Blue G-250, was obtained from Bio-Rad Laboratories (Hercules, CA, USA). Nitrocellulose and PVDF membranes were obtained from Millipore (Billerica, MA, USA). N27 cell line was a kind gift from Dr. Curt Freed, University of Colorado (Colorado, USA). Curcumin bioconjugates (viz., D1- Curcumin di piperoyl, D2- Curcumin di-valinoyl and D3- Curcumin di-glytamoyl esters) were gifted by Dr. Krishna Misra (Indian Institute for Information Technology, Allahabad, India).

5.2 Human tissue samples

Postmortem brain tissues were sourced from the Human brain tissue repository (HBTR), Department of Neuropathology, NIMHANS, Bangalore, India. The brain tissues were collected, stored and used for research following approval from the Institutional Ethics Committee and written informed consent from close legal relatives of the subjects.

Craniotomy samples of brain tissue of TBI patients were obtained from NIMHANS emergency OT after obtaining informed consent from the relatives of the patients. A 3cm[3] piece of tissue was also collected at autopsy from TBI patients (who succumbed to head injuries) from the impact site and tissues from contralateral cortex/ different lobe anatomically distant from the contusion that did not show any evidence of injury was also collected. Tissues were divided arbitrarily for the purpose of study into “contusion” zone and “pericontusion” zone based on naked eye examination. Tissues were labeled “contusion” in presence of hemorrhages and immediate adjacent cortical tissue devoid of hemorrhage was considered “pericontusion” (Fig.32-33). Relatively normal appearing parenchyma away from the pericontusion sites were considered as “away from contusion”. In cases obtained from autopsy, tissues could be obtained from a different lobe, distinctly distant from the contusion e.g., for temporal contusions, tissues were obtained from ipsilateral/contralateral frontal lobe.

Age matched control brain samples for all the experiments were obtained from subjects who succumbed to road traffic accidents (non-alcoholics, non-diabetics, not on any medication and with no known neurological or psychiatric disorders). These control brain samples used in the study were anatomically farthest from the site of injury and without distinct oedema or grossly apparent pathology. These tissues were subjected to histopathological assessment and the samples that maintained tissue integrity were utilized for the study. While the major portion of the tissue was frozen for biochemical studies, a limited portion corresponding to the mirror image of the stored tissue bits was fixed in buffered formalin. The protocol of autopsy, tissue handling and other procedures were uniform for all the samples.

The clinical details (including GCS at time of admission, course and terminal event), the details of injury including interval between time of injury and surgery, radiology findings (basal cisterns and mid line shift) and post mortem interval in autopsied cases were obtained from medical records. The GCS score was based on (i) best eye response (E; range: 1-4) (ii) best verbal response (V; range: 1-5) and (iii) best motor response (M; range: 1-6) with a total range of 3 to 15 (noted at the time of hospital admission), with the score of 3 corresponding to the most severe agonal state. Details of cases included for the study is provided in Table 11).

5.3 Histology and Immunohistochemistry (IHC)

The formalin-fixed tissues from different brain samples were processed for routine paraffin embedding. Four micron thick serial sections were collected and stained with haematoxylin-Eosin (H&E), Nissl stain and Luxol fast blue for myelin (Govindan , et al. 2011, Yasha, Shankar, Santosh, Das and Shankar 1997). IHC was carried out by indirect immunoperoxidase technique using antibodies to GFAP (dilutions-1:200), phosphorylated neurofilament directed against medium and high molecular weight chain (dilutions-1:200). Microglial response was detected using Iba-1 (dilutions-1:200) and HLA-DR (dilutions-1:100) for detected activation status of microglia. Infiltration by macrophages was characterized using CD68 (dilutions-1:200).

IHC (Govindan , et al. 2011):

Sectioning: Tissue sections of 4 pm thickness were cut from paraffin-embedded blocks on a microtome and mounted from warm water (40°C) onto silane coated microscope slides. Sections were allowed to dry overnight at room temperature.

Deparaffinization: Fixed tissues were deparaffinized in xylene (2 changes, 10 min each). Rehydration: The sections were rinsed in absolute alcohol (2 changes, 2 min each) and, placed in 3% H2O2 (v/v) prepared in methanol. This was followed by 2 min washes each in absolute alcohol and distilled water.

Antigen retrieval: The sections were rinsed in 0.05M Tris Buffer, pH 7.66 and pressure cooked in 0.01M citrate buffer (pH 6.01) for heat induced epitope retrieval. Slides were allowed to cool for 20 min in room temperature and washed in 0.05M Tris buffer pH 7.6 (2min).

Immunostaining: Sections were blocked using skimmed milk solution or Power block (Biogenex) for 30-45 min. After draining the blocking solution, the sections were incubated with primary antibody for 1-2h. Sections were washed in Tris buffer pH 7.6 (thrice, 2min each) and incubated in secondary antibody for 30min to 1hr. The sections were then washed (3 washes, 2min each) and the color reaction was developed with3,3- diamino benzidine(DAB) solution and Hydrogen peroxide) (Dako, Denmark). Finally, the sections were rinsed in water, counter stained with Haematoxylin, allowed to dry and mounted in Distrinedibutyl phthalate xylene (DPX).

5.4 Cell culture experiments:

5.4.1 Cell line

1RB3AN27 (N27) dopaminergic neuronal cells were used for all cell culture experiments. N27 cells were generated by SV40 LT-antigen immortalization of rat mesencephalic cultures (Adams , et al. 1996, Prasad , et al. 1994). Since N27 cell line possesses all the physiological and biochemical properties of dopaminergic neurons, it serves as a good model for early PD. The cell line expresses both TH and DAT and produce measurable amounts of dopamine (Adams , et al. 1996).

Preparation of medium: Roswell Park Memorial Institute (RPMI) medium 1640 was prepared by dissolving the entire contents of a pack in ~ 800 ml of autoclaved milli Q water. The pH was adjusted to 4.0 with 2N HCl under constant stirring to facilitate complete dissolution of the medium. To this 2 g of NaHCO3 was added and the pH was adjusted to 7.1. The volume was made up to 1000 ml and filtered in the cell culture hood by ultra-filtration via cellulose acetate filters of pore size 0.2pm (Sartorius stedim Biotech GmbH, Goettingen, Germany). Following filtration, fetal bovine serum (FBS) (final concentration: 10 %) and antibiotic mix (final concentration: 1X; 100 U/ml penicillin, 100 pg/ml streptomycin and 0.25 pg/ml amphotericin) were added and the media was stored in sterile bottles at 4 OC until use.

5.4.2 Maintenance of cells

N27 cells were grown as adherent culture in RPMI 1640 medium in a CO2 incubator under standard conditions (95 % CO2 and 5% air, 95% humidity). Cells were sub-cultured once a week via trypsin treatment. Briefly, spent medium was aspirated out using a sterile pipette and the cells were washed with 1X phosphate buffered saline (PBS). Approximately 1 ml of trypsin-EDTA (0.75 % w/v) solution was added to completely cover the cells and placed in the CO2 incubator at 37 OC for 1-2 min. Once cells were detached from the surface of the culture dish (as observed under the microscope) ~ 500 pl of fresh medium containing serum was added (to inactivate trypsin). The cells were then transferred to sterile microfuge tubes, centrifuged at low speed (<2000 rpm for 2 min) and the supernatant was discarded. The cell pellet was resuspended in fresh medium and an aliquot of the cell suspension with known volume was used for counting using a haemocytometer. The required numbers of cells were then transferred to fresh medium taken in a new, labeled culture dish and incubated in the CO2 incubator as above.

5.4.3 Counting of cultured cells

Routine counting of N27 cells in suspension was carried out by haemocytometry­based method as described previously (Sambrook , et al. 2001). An aliquot of the harvested cells was diluted 10-fold in culture medium and 10 pl of the diluted cell suspension was placed on the graduated surface of the haemocytometer, covered with a glass cover slip and observed under the microscope. Counting was manually performed in the 4 quadrants (including all 16 sub-quadrants) of the haemocytometer. The average cell count per ml was calculated as follows: Average number of cells in all quadrants X 10[4] X dilution factor.

5.4.4 Treatment of cells

N27 cultures at ~80-85% confluence were incubated with either BSO (1.5pM)or curcumin (0.5pM and 1pM) or curcumin bioconjugates (0.5pM and 1pM) both (during pre and post-treatment experiments). After the stipulated time period (24 h or more) cells were harvested and used for GSH estimation and measuring antioxidant and oxidant markers.

5.5 Estimation of cell viability (MTT assay)

Principle: 3-(4,5-dimethylthiazol-2- yl)-2,5- diphenyltetrazolium bromide (MTT) is reduced by mitochondrial oxido-reductases in live cells, to purple colored water insoluble formazan precipitate, which can be solubilized in dimethyl sulfoxide. The intensity of the purple color is directly proportional to the number of viable cells.

Procedure: After various treatments, 20 pl of 5 mg/ml MTT was added to cells in each well of a 96-well plate and incubated at 37oC in the CO2 incubator for 2 h. The spent medium was discarded, the dark blue formazan crystalline product was dissolved in dimethyl sulfoxide, and the absorbance was measured in a multi-well plate reader (Tecan, Austria) at 570 nm(van Meerloo , et al. 2011). The results were expressed in percentage with respect to untreated controls.

5.6 Measurement of reactive oxygen species (ROS)

Principle: 2’,7’ -dichlorofluoresceindiacetate (DCFDA) is a cell permeable fluorogenic dye. After diffusion into the cell, DCFDA is de-acetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by hydroxyl, peroxyl and other ROS in the cell into 2’, 7’ -dichlorofluorescein (DCF). DCF is a highly fluorescent compound which can be detected by fluorescence spectroscopy with excitation and emission wavelength of 480nm and 530nm respectively.

Procedure: Total ROS in N27 cells were measured by a modified method of Ohashi , et al. (2002). Briefly, the spent medium from treated or untreated N27 cells was replaced with 1 ml Locke’s solution (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 5 mM HEPES, 2 mM CaCl2 and 10 mM Glucose, pH 7.4). Ten microliters of DCFDA (1 mM) was then added and the cells were incubated at 37 [0]C (10 min) in the CO2 incubator. The Locke’s solution was then aspirated and the cells were harvested. A small aliquot of cell suspension (10 pl) was kept aside for counting and the rest of the suspension was centrifuged (5,000 X g, 10 min). The cell pellet was washed with PBS twice and reconstituted in lysis buffer (10 mM Tris-HCl containing 0.5% Tween-20). The lysate was centrifuged at 10000Xg (10 min), the fluorescence of the supernatant was measured (Excitation @ 480 nm; Emission @ 530 nm) and the value was expressed as Arbitrary fluorescence units/number of cells.

5.7 Estimation of hydrogen peroxide (H2O2)

Principle: The H2O2 assay was based on the oxidation of ferrous ions (Fe[2]+) to ferric ions (Fe[3]+) by peroxides wherein the ferric ions (Fe[3]+) react with the indicator dye xylenol orange to produce a purple colored complex measureable at 560 nm (Sorbitol was included in the assay to amplify the color intensity).

Procedure: Water soluble hydroperoxide level in N27 cells/tissue extracts was determined as previously described (Deiana , et al. 1999, Jiang , et al. 1990). N27 cells/minced brain tissue were sonicated (5 sec x 4 on ice; pulsed setting) in 200pl of PBS and centrifuged at 13,500Xg (10 min) at 4 [0]C. Thirty microliters of the supernatant (corresponding to ~0.2 mg protein) was added to 0.95 ml of FOX1 (Ferrous ion oxidation xylenol orange reagent-100pM xylenol orange, 250pM Ammonium ferrous sulphate, 100mM sorbitol and 25 mM H2SO4) reagent and incubated at room temperature for 30 min. The reaction mixture was centrifuged at 800Xg (10 min) and the absorbance of the supernatant was measured at 560 nm. The amount of H2O2was calculated using its molar extinction coefficient (1.5 x 10[4]mol-[1]cm-[1]) and expressed as pM/mg of protein.

5.8 Estimation of lipid peroxidation

Principle: Lipid peroxidation was measured by estimation of malondialdehyde (MDA) by thiobarbituric acid (TBA) reaction method modified from Ohkawa , et al. (1979). MDA, a product of lipid peroxidation, reacts with TBAto form 1:2 adduct (MDA- TBA2). This adduct is extracted into n-butanol, and its absorbance is recorded spectrophotometrically at 532 nm.

Procedure: N27 cell extracts/tissue homogenates were sonicated on ice (5 sec X 3 pulse). One hundred microliters of the sonicate was added to a mixture containing 0.75 ml of acetic acid (pH 3.5, 20% v/v), 0.1 ml SDS (8% w/v) and 0.75 ml TBA (0.8%, w/v) (The acidic pH facilitates the hydrolysis of MDA-protein adducts resulting in better recovery of MDA). The reaction mixture was heated in boiling water bath for 45 min. The adducts formed were extracted into 1.5 ml of 1-butanol by vortexing and centrifugation at 2500 rpm (10 min) and their absorbance was measured at 532 nm. The amount of MDA formed was calculated using its molar extinction coefficient (1.56 x 105 M-[1]cm-[1]).

5.9 Preparation of whole brain protein extracts

Whole brain extracts were prepared by homogenization in PBS containing 1X protease inhibitor cocktail using either a Wheaton dounce type glass homogenizer or a Potter-Elvehjem motorized Teflon homogenizer. The homogenate was then sonicated on ice (10 s X 3). The extract was centrifuged (15000 Xg, 5 min) and the total protein in the supernatant was estimated (Chandana et al, 2009).

5.9.1 Protein estimation

Principle: The Bradford assay (Bradford 1976) is a colorimetric assay for protein determination based on an absorbance shift in the dye Coomassie brilliant blue-G250. Coomassiebrilliant blue (red in unbound form) on binding to protein changes to stable blue form with absorbance shift from 465nm to 595nm. As the increase of absorbance at 595 nm is proportional to the amount of bound dye, and thus to the amount (concentration) of protein present in the sample, this can be used as a measure for the protein concentration of the unknown sample.

Procedure: Briefly, 10pl of the suitably diluted protein sample was dispensed in a 96-well plate to which 200 pl of 1X Bradford reagent [100mg Coomassie Brilliant Blue G-250, 50 ml ethanol (95%) and 100 ml orthophosphoric acid (85%) in 200 ml distilled water] was added, thoroughly mixed and incubated for 10 min. The absorbance was read at 595 nm in multi-well plate reader (TECAN, GmbH, Austria). The concentration of protein in the unknown sample was calculated by comparing with Bovine serum albumin (BSA) standards (50 - 500 pg/ml). All estimations were performed in triplicate.

Alternatively, proteins estimation was carried out using a spectrophotometer (Model: UV-1601PC, Shimadzu Co. Japan), in a 1 ml reaction volume containing 100 pl of diluted sample and 900 pl of 1X Bradford reagent. The absorbance at 595 nm was recorded and the protein concentration in the sample was calculated against BSA standards.

5.10 Preparation of mitochondria

Mitochondria were prepared from frozen brain tissue, based on the principle of differential centrifugation, as described earlier (Trounce , et al. 1996). Brain tissue weighing ~200-500 mg, was washed, minced thoroughly with a clean pair of scissors and homogenized manually (18 gentle strokes), in ~1.5 ml isolation buffer [320 mM sucrose, 5 mM TES [tris (hydroxymethyl) methyl aminoethane sulfonic acid, 1 mM EGTA, pH 7.2] using a Wheaton glass homogenizer. The homogenate was then transferred to microfuge tubes and centrifuged at 1,000 g for 5 min at 4°C to facilitate separation of the nuclear fraction, unbroken cells and other cell debris. The supernatant was collected and the pellet was resuspended in isolation buffer followed by a second round of homogenization and centrifugation as mentioned earlier. The supernatants from both steps were then pooled and centrifuged at 8,500 g for 10 min at 4°C. The crude mitochondrial fraction obtained in the pellet was resuspended in minimum volume of isolation buffer (~150 pl), overlaid on 6% (w/v) Ficoll solution, and centrifuged at 75,000Xg for 30 min at 4°C, to remove the myelin content. The pellet was then resuspended in ~150pl reconstitution buffer (250 mM sucrose, 10 mM TES, pH 7.2) and stored as aliquots at -80°C.

5.11 Preparation of Neuropil fractions

Neuropil constitutes three different fraction of the brain tissue viz. Post mitochondrial supernatant, Mitochondria and Synaptosomes. Post mitochondrial supernatant constitutes the cytosolic fraction without any cellular organelles. Synaptosomal fraction is used as model of functional synapse for the study of neuronal terminal processes. They are usually “pinched-off” from homogenized neurons and could be isolated through the use of ultracentrifugation and a discontinuous sucrose density gradient. Synaptosomes are composed of a continuous plasma membrane that have functional pumps and channels capable of ion exchange, and respond to depolarization. They also contain some mitochondria and hence are capable of carrying out respiratory activities. Synaptosomes provide a complete model of a functional synapse, since they are shown to be capable of protein synthesis.

Neuropil fractions were prepared as described above with slight modifications. The supernatant obtained after 8500xg separation (which constitutes the post mitochondrial supernatant or cytosol) is centrifuged (15000 Xg, 10 min). The pellet obtained in this step is resuspendend in isolation buffer (200pl) and overlaid on a discontinuous ficoll gradient consisting of 6% (w/v) ficoll (density = 1.065 g/ml, 4 ml), 9% (w/v) ficoll (density = 1.075 g/ml, 1 ml) and 12% (w/v) ficoll (density= 1.085 g/ml, 4 ml). The gradient was centrifuged at 75000 Xg for 1 h at 4°C. The myelin layer at the top of the gradient was removed, the middle synaptosomal fraction was separated and the pure mitochondrial pellet present at the bottom was resuspended in reconstitution buffer (250mM sucrose and 10mM TES, pH 7.2) and utilized for biochemical experiments.

5.12 Estimation of Total Glutathione (reduced and oxidized; GSH+GSSG)

5.12.1 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB) recycling method

Principle: Total glutathione in human brain or N27 cell extracts was estimated by the 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB) recycling method as described earlier (Tietze 1969). In principle, the GSH present in the sample reduces the dye DTNB, to 5- thio 2-nitro benzoic acid (TNB) and in turn gets converted to GSSG. However, in the presence of NADPH, the enzyme glutathione reductase (GR), converts GSSG back to GSH, which can again react with DTNB, resulting in a cyclic reaction. The rate of formation of TNB is monitored kinetically at 412 nm as an increase in absorbance and compared with GSSG standards. The rate of the reaction is used to estimate the total GSH.

Procedure: Tissue/N27 cells were homogenized in PE buffer (100 mM potassium phosphate buffer, pH 7.4, containing 1 mM EDTA). The sample was then sonicated (10s X 3) and the total protein was estimated. The sonicate was treated with equal volume of 5% sulfosalicylic acid (SSA) (w/v) to precipitated proteins and centrifuged at 16,000 Xg for 15 min. The supernatant obtained was used for GSH estimation. Ten microliters of the supernatant was incubated with assay buffer (PE buffer containing 0.8 mM DTNB and 0.32 U/ml GR) in a final reaction volume of 250pl. The reaction was initiated by addition of 0.6 mM NADPH. The reaction kinetics of DTNB recycling was monitored at 412 nm for 3 min, in a multi-well plate reader (Tecan infinite M200, Austria). The absolute GSH level in each sample was calculated based on GSSG standards (0 to 100 ng) and normalized per mg protein.

5.12.2 Estimation of GSH/GSSH by O-pthalaldehyde (OPA) method

Principle: GSH reacts with OPA to form a stable, highly fluorescent tricyclic derivate at pH 8.0, while GSSG reacts with OPA at pH 12.0. This fluorescent compound can be activated at 350nm and has emission peak at 420nm. During measurement of GSSG, GSH is complexed to N-ethylmaleimide (NEM) to prevent oxidation of GSH to GSSG upon exposure to air.

Procedure: GSSG was estimated by the method as previously described (Hissin , et al. 1976), with slight modifications. The sonicated tissue extract was acid precipitated using 5% sulphosalicylic acid (SSA). 25pl of supernatant was incubated with 0.05 M NEM for 20 min to interact with GSH present in the sample. From this reaction mixture 10pl of sample is mixed with 180pl of 0.1 N NaOH, and 10 pl OPA (20 mg/ml) and incubated at room temperature for 15 min. Fluorescence reading was recorded (excitation: 350 nm, emission: 420 nm). The absolute GSSG value was calculated from the linear standard plot using standard GSSG.

For GSH (reduced) estimation 50pl of SSA supernatant was mixed with phosphate buffer pH 8. To 10 pl of the above, 10 pl OPA (20 mg/ml) and 180 pl of phosphate Buffer (pH 8) were added and incubated for 15 min at room temperature. Fluorescence reading was recorded (excitation: 350 nm, emission: 420 nm). The absolute GSH value was calculated from the linear standard plot using standard GSH.

5.13 Superoxide dismutase (SOD) activity

Principle: SOD catalyzes the dismutation of the superoxide anion (O2-.) into H2O2 and molecular oxygen. SOD activity was assayed using its inhibitory action on quercetin oxidation (Bagnyukova 2003). Quercetin is oxidized by O2-.produced by TEMED, which is effectively inhibited by SOD in the sample. The rate of inhibition of quercetin oxidation is monitored at 406 nm for 10 min.

Procedure: The reaction mixture contained 30 mM Tris-HCl buffer (pH 9.1), 0.5 mM EDTA, 50 mM TEMED, 0.05 mM quercetin and 10pl of soluble extract (containing 10pg of protein). The reaction was monitored at 406 nm for 10min. One unit of SOD activity was defined as the amount of enzyme (per mg protein) that inhibits quercetin oxidation reaction by 50% of maximal value.

5.14 Catalase activity

Principle: Catalase is involved in the detoxification of H2O2, a toxic product of both normal aerobic metabolism and pathogenic ROS production. This enzyme catalyzes the conversion of two molecules of H2O2 to molecular oxygen and two molecules of water and the rate of decomposition of H2O2 is measured at 240 nm.

Procedure: Catalase activity was assayed by the method of Aebi (1984). Briefly, 1ml of the reaction mixture containing 50 pl of sample was mixed with 900 pl of phosphate buffer (0.1M, pH 7.0) and 50 pL of H2O2 (8.8mM). The decrease in absorbance (at 240nm) was followed for 5 min at room temperature using a UV-Visible spectrophotometer. The enzyme activity was expressed as pmol H2O2 consumed /min/mg protein (MEC = 43.6 mM-1 cm-1).

5.15 Glutathione-S-transferase (GST)

Principle: GST represents a family of enzymes that play an important role in detoxification of xenobiotics. GST catalyzes the conjugation of the thiol group of GSH to electrophilic xenobiotics to protect cells against toxicants. It utilizes GSH to scavenge toxic compounds including those produced due to oxidative stress and is part of the defense mechanism against the mutagenic, carcinogenic and toxic compounds (Boyland , et al. 1969). GST was assayed by measuring the rate of enzyme catalyzed conjugation of GSH with 1-chloro -2,4-dinitrobenzene (CDNB) according to the method of Guthenberg , et al. (1985).

Procedure: To 236pl reaction mixture (0.1 M Phosphate buffer, pH 6.5; 0.5 mM EDTA, and 1 mM GSH), 4pl of 1.5 mM CDNB and 10 pl sample were added and the increase in absorbance at 340 nm was monitored for 5 min. The enzyme activity was expressed as nmoles of S-2,4- dinitrophenyl glutathione formed /min/mg protein (M EC=9.6 mM_[1] cm_[1]).

5.16 Glutathione Reductase (GR) activity

Principle: GR is a flavoprotein that is required for the conversion of oxidized GSSG to GSH. At the same time, it oxidizes NADPH. This universally present enzyme is essential for the maintenance of GSH level in vivo (Carlberg , et al. 1985). GR therefore plays a major role in glutathione peroxidase (GPx) and GST reactions as an adjunct enzyme in the control of ROS (Bompart , et al. 1990). GR catalyzes the NADPH- dependent reduction of GSSG to GSH. The oxidation of NADPH to NADP+ is accompanied by a decrease in absorbance at 340nm (Rigobello , et al. 2005), which is directly proportional to the GR activity in the sample.

Procedure: To 180 pl reaction mixture containing Tris-HCl buffer (0.1 M, pH 8.8; 0.1 mM EDTA), NADPH (0.2mM) and GSSG (2 mM), 10 pl sample was added and the decrease in absorbance at 340 nm was monitored for 5 min. The enzyme activity was expressed as nmoles NADPH oxidized/min/mg protein (MEC=6.2x10[3] M_[1] cm_[1]).

5.17 Glutathione peroxidase activity (GPx)

Principle: GPx converts GSH to GSSG while reducing lipid hydroperoxides to their corresponding alcohols or free H2O2 to water. The assay measures GPx activity indirectly by a coupled reaction with GR. The GSSG, produced in the reaction, is recycled by GR and NADPH:

Abbildung in dieser Leseprobe nicht enthalten

The oxidation of NADPH to NADP+ is accompanied by a decrease in absorbance at 340 nm which is directly proportional to the GPx activity in the sample.

Procedure: GPx activity was determined using the substrate t-butyl hydroperoxide (tbHP) as described earlier (Flohe , et al. 1984). The reaction mixture containing 50 pl sample, 0.1 M phosphate buffer, 0.5 mM EDTA, 100 pl GR (0.24 U), 100 pl GSH (1 mM), 100 pl NADPH (0.15 mM) and incubated at 37°C for 3 min. The reaction was initiated by the addition of 100 pl tbHP (0.12 mM). Change in absorbance at 340 nm was monitored for 5 min spectrophotometrically and the activity was expressed as nmoles of NADPH oxidized /min/mg protein (MEC= 6.22 x 10[3] M-[1]cm -[1])

5.18 Thioredoxinreductase (TrxR) activity

Principle: The thioredoxin (Trx) system [TrxR and Trx] together with GSH system regulate cellular redox processes. TrxRs are selenium-containing pyridine nucleotide-disulfide oxidoreductases (Mustacich , et al. 2000) that transfer electrons from NADPH to Trx, which in turn reduces Trxperoxidase, methionine sulfoxide reductase, ribonuclotide reductase and other important redox proteins. TrxRs reduce several substrates other than Trx, including selinite, lipid hydroperoxides, vitamin-K and H2O2 (Mustacich and Powis 2000). TrxR activity was assayed based on reduction of DTNB with NADPH to TNB (Hill , et al. 1997), a yellow product that can be measured at 412 nm.

Abbildung in dieser Leseprobe nicht enthalten

Procedure: The reaction mixture contained 0.2M sodium-potassium phosphate buffer pH 7.4, 1 mM EDTA, 0.4mM NADPH and 25pl of sample. The reaction was initiated by the addition of 10pl of 2mM DTNB and the yellow product formed is measured at 412nm for 5min. The activity of TrxR is expressed as nM of DTNB reduced/min/mg of protein.

5.19 Mitochondrial function assays

5.19.1 Complex I (CI) activity assay

Principle: CI is the first enzyme in the electron transport chain, which accepts electrons from NADH and transfers them to the mobile electron carrier ubiquinone via a series of Fe-S clusters. Cyanide, an inhibitor of complex IV that prevents movement of electrons further down the electron transport chain is added to the reaction mixture. Consequently, the electrons from ubiquinone are transferred to Dichloroindophenol (DCIP), a colored, secondary electron acceptor whose reduction can be monitored by a change in the absorbance at 600 nm.

Procedure: CI enzyme assay was carried out as previously described (Trounce, Kim, Jun and Wallace 1996). The assay was initiated by addition of aliquots of mitochondria (5 pg in 25 mM phosphate buffer pH 7.2 + 5 mM MgCl2) to 50 mM Tris- HCl (pH 7.4), 500 pM EDTA, 1% BSA, 200 pM NADH, and 200 pM decylubiquinone with or without 2 pM rotenone, in the presence of 2 mM NaCN with 0.002 % DCIP as a secondary electron acceptor. The decrease in the absorbance at 600 nm was recorded as a measure of the rate of enzyme reaction at 37°C for 10 min and specific activity was calculated.

Specific activity was calculated using the following formula:

Abbildung in dieser Leseprobe nicht enthalten

The reaction was measured spectrophotometrically in a reaction volume of either 500 pl using a spectrophotometer or 200 pl in an ELISA plate reader. The specific activities with and without rotenone were calculated independently. The activity obtained in the absence of rotenone corresponds to total NADH dehydrogenase activity since rotenone is a specific inhibitor of CI. The activity in the presence of rotenone corresponds to non-mitochondrial NADH dehydrogenase activity. Hence the difference between the two gives the activity specific to mitochondrial CI. Thus results were plotted as relative rotenone-sensitive specific activity.

5.19.2 Malate Dehydrogenase (MDH) Assay

Principle: MDH catalyzes the reversible reduction of oxaloacetate to malate using NADH as a co-substrate.

Abbildung in dieser Leseprobe nicht enthalten

Procedure: MDH activity was assayed according to the method described previously (Harish , et al. 2012, Kitto , et al. 1967). The reaction buffer (230 pl) [potassium phosphate buffer (0.1 M, pH 7.5), 0.03 ml NADH (14.3 mM) and 0.05 ml oxaloacetate (20 mM)] was added to 10pg of sample in a 96-well plate in triplicate and the decrease in absorbance at 340 nm was followed spectrophotometrically for 3 min. The enzyme activity was expressed as nmol NADH /min/mg protein (MEC = 6.22 X 10[3] mol-[1] cm-[1]).

5.19.3 Citrate Synthase (CS) assay

Principle: CS is a pace-maker enzyme in the Krebs cycle localized in the mitochondrial matrix and is commonly used as a quantitative marker enzyme for the content of intact mitochondria. CS catalyzes the reaction between acetyl coenzyme A (acetyl CoA) and oxaloacetic acid to form citric acid. The hydrolysis of the thioester of acetyl CoA results in the formation of CoA-SH. This thiol reacts with DTNB in the reaction mixture to form TNB which could be spectrophotometrically measured at 412 nm.

Procedure: CS activity was determined as the rate of DTNB reduction at 412 nm (Harish , et al. 2012, Srere 1969). The reaction mixture (194 pl) contained 100 mM Tris- HCl (pH 8.1), 0.2 mM DTNB, 0.1 % Triton X-100, 0.1 mM Acetyl-CoA and 5pg of total protein. The reaction was initiated by the addition of 20 pl of 10mM oxaloacetate (final concentration 0.2 mM) (Harish , et al. 2012, Srere 1969). The results were expressed as nmol DTNB/min/mg protein (MEC=13.6 mM-[1]cm-[1]).

5.19.4 3-(4,5-Dimethylthiazol-2-yl)-2,5-DiphenyltetrazoliumBromide (MTT) Assay

It is a colorimetric method that measures the reduction of MTT by mitochondrial reductases and was performed as described previously (Cohen , et al. 1997, Harish , et al. 2012). Briefly, to 1 ml of assay buffer (Mannitol Sucrose-HEPES, 20 mM sodium succinate, 1 mM NADH, pH7.4) containing 5 pl of sample (~5 pg protein), 15 pl of MTT (5 mg/ml) was added and incubated at 37[0]C for 2 h. The formazan crystals formed were dissolved in 100 pl SDS-dimethyl formamide (DMF) buffer (45 %DMF in distilled water and 10 % SDS, pH 4.7), and the absorbance was measured at 570 nm.

5.19.5 Succinate Dehydrogenase (SDH)

Principle: SDH is a membrane bound respiratory complex II enzyme, which participates both in electron transport and Kreb’s cycle. SDH catalyzes the oxidation of succinate to fumarate with the reduction of ubiquinone to ubiquinol. SDH oxidizes succinate while reducing p-iodonitrotetrazolium chloride to formazan (A490nm). SDH activity was determined as an end-point assay based on the method described earlier (Pennington 1961) with minor modifications (Harish , et al. 2012).

Procedure: Mitochondrial sample (10 pl) was added to 70 pl of 0.01 mol/l sodium succinate prepared in 0.05 mol/l phosphate buffer pH 7.5 and incubated for 15 min at 37[0]C. Next, 70 pl of 1 pg/pl p-iodonitrotetrazolium violet was added to the reaction and incubated at 37[0]C for an additional 10 min. The reaction was stopped by the addition of 100 pl of a 5:5:1 (v:v:w) solution of ethyl acetate: ethanol: trichloroacetic acid and the absorbance was measured at 490 nm. The specific activity was expressed as absorbance/mg protein.

5.20 Sodium dodecyl sulphate- Poly acrylamide gel electrophoresis (SDS PAGE) and Western Blot

5.20.1 Sample preparation

Fifty pg of the protein sample (mitochondrial or total cell extract) was mixed with equal volume of 2X Laemmli buffer (loading buffer) (100 mM Tris-Cl pH 6.8, 4 % w/v SDS, 20% v/v glycerol, 200 mM DTT or beta mercaptoethanol (ME) and a pinch of Bromophenol blue) and boiled for ~10 min. The samples were then centrifuged to pellet insoluble proteins and the supernatant was loaded onto the gel.

5.20.2 SDS PAGE

The glass plates were properly mounted according to the manufacturer’s instructions (Bangalore Genei, Bangalore, Karnataka, India or Biorad Laboratories Inc., Hercules, CA, USA) and checked for leakage. The resolving gel was poured in-between the assembled glass plates and overlaid with water saturated butanol. Upon polymerization, the butanol overlay was discarded and the stacking gel was poured. The comb was carefully inserted in the staking gel quickly before it polymerized. Care was taken not to introduce air bubbles. The samples were loaded in the corresponding wells (50pg/lane) and separated electrophoretically at 125 V for 2-2.5 h.

Composition of resolving gel -

Abbildung in dieser Leseprobe nicht enthalten

Composition of electrophoresis buffer (1X pH 8.3) was 25 mM Tris-base, 250 mM Glycine and 0.1 %( w/v) SDS. Following electrophoresis, gels were carefully removed and stained with freshly prepared coomassie brilliant blue (CBB) R-250 for 1 hr or by silver staining.

5.20.3 Coomassie Brilliant Blue (CBB) staining

Following electrophoresis, gels were incubated in freshly prepared CBB staining solution (250 mg CBB, 50 ml methanol, 10 ml glacial acetic acid and 40 ml distilled water) for 1 h. Once stained, gels were destained with destaining solution (50 ml methanol, 10 ml glacial acetic acid and 40 ml distilled water) until bands appeared on the gel. Gels were then transferred to distilled water and images captured in the gel documentation system (Biorad Laboratories (Model: 2000TM), Inc., Hercules, CA, USA) attached to a computer and analyzed by Image J program, NIH, USA.

5.20.4 Silver staining

Gels were fixed in fixative (50 ml methanol, 12 ml glacial acetic acid and 0.5 ml formaldehyde made up to 100 ml with distilled water) for 1-2 h or overnight. Gels were washed first with 50% methanol twice for 20 min each,and then once in 30% methanol for 20 min. Gels were then washed with water for 2-5 min followed by sensitization with 0.02% sodium thiosulphate for 1 min. The gels were then washed with water thrice, 20 s each. Gels were then incubated for 30 min in silver nitrate solution [200 mg AgNO3, 75 pl HCHO in 100 ml distilled H2O] in dark. The gels were washed with water thrice (20 s each) and developed in developing solution (6 g Na2CO3, 50 pl HCHO, 0.04% Na2S2O3 [w/v] in 100 ml distilled water) until spots began to appear. Reaction was stopped by the addition of fixative and gels were then washed with distilled water. The images were captured using the gel documentation system as mentioned previously.

5.20.5 Electrotransfer

Following separation by SDS PAGE, proteins were electrophoretically transferred from the gel to nitrocellulose (NC) membranes using a semi-dry transfer apparatus (Sree Maruthi Scientific Works, Bangalore, India). Prior to transfer, the Whatman filter paper (grade no. 3) and NC membrane cut to the size of the gel were pre-soaked in ice cold electrotransfer buffer (pH>8.0, 48 mM Tris-base, 39 mM Glycine, 20% Methanol, 0.0375 % (w/v) SDS). The sandwich for transfer was prepared as follows: Two Whatman filter papers were carefully placed on the anode side of the transfer apparatus and ~ 1 ml of transfer buffer was added. The NC membrane was then carefully placed above the filter papers followed by the SDS gel. Care was taken not to introduce air bubbles in between any two layers. Two more filter papers were placed above the gel and more buffer was added. The cathode plate was then replaced and proteins were transferred electrophoretically for 2 h at 125 mA constant current.

5.20.6 Western blotting

Following transfer, the blot was incubated in blocking solution [1x PBS containing 0.01% Tween-20 (PBST) and 5 % skimmed milk powder for 1 h at room temperature or overnight at 4 ° C to block non-specific binding. The blot was then incubated in primary antibody diluted in PBST containing 5 % BSA for 1 ¥2 h at room temperature (1:1000 for a-3NT, a-GFAP, P-actin). The blot was then washed with PBST (5 min X 5) to remove excess primary antibody and then incubated for 1 ¥2 h at room temperature with Horse Radish Peroxidase (HRP)-conjugated secondary antibody (diluted in PBST containing 5% skimmed milk powder, 1:3000 for a-rabbit or a-goat and 1:1000 for a-mouse). Following incubation, membranes were washed with PBST and the immune reaction was visualized by development in 1x PBS containing 3,3'- diamino benzidine tetrahydrochloride (DAB) [1 mg/ml (w/v)] and 0.1% H2O2.

5.21 Determination of protein nitration [3-nitrotyrosine (3-NT) slot blot]

Principle: Tyrosine nitration of proteins at ortho position is a form of protein oxidation. 3-NT thus formed is detected immunochemically using specific antibody against 3-NT.The level of 3NT modification within a given protein sample can be measured by a semi-quantitative dot blot or slot blot method (Ansari , et al. 2010).

Procedure: 50 pg of protein extracted from different brain samples were spotted in triplicate onto an NC membrane and air dried. The membrane was then washed with PBST and blocked with 5 % skimmed milk solution for 30 min. The blot was then incubation with 1:1000 primary a-3NT antibody for 1 ¥2 h. It was then processed as mentioned previously for western blotting. Spot intensities were quantified by the densitometric scanner (Quantity One 4.2.2., Biorad Laboratories Inc.) and the arbitrary absorbance values were normalized against the respective beta actin values.

5.22 Determination of protein carbonylation

Principle: Protein carbonyls can be measured indirectly by derivatization of the carbonyl groups with 2, 4-dinitrophenylhydrazine (DNPH), which form DNP-Protein adduct. The latter can be immunochemically detected using a specific antibody against dinitrophenol (a-DNP) (Oxyblot).

Abbildung in dieser Leseprobe nicht enthalten

Protein carbonyl DNPH Protein-DNP

Procedure: Oxyblot was carried out based on the protocol published earlier (Subramaniam , et al. 1997). Brain protein extract or N27 cell extract at 4 mg/ml protein concentration was derivatized with 5 pl DNPH (10 mg/ml stock prepared in 50% sulphuric acid) in a final reaction volume of 20 pl in the presence of 10 pl 12% SDS for 20 minutes at room temperature. The reaction was stopped by addition of neutralization buffer (2 M Tris in 30% glycerol). Derivatized sample was spotted in triplicate onto an NC membrane (5 pl/spot). The membrane was washed with PBST, blocked and incubation for 1 ¥2 h with primary a-DNP antibody (1:200) at room temperature. It was then incubated in HRP-conjugated anti-rabbit secondary antibody for 1 ¥2 h followed by color development in DAB solution as mentioned previously. Spot intensities were quantified by a densitometric scanner and the values normalized against the respective anti-beta actin values as above.

5.23 Proteomics

5.23.1 Mitochondrial Protein extraction and normalization

Crude mitochondria (containing non-synaptic mitochondria, synaptosomes and microsomes) were isolated from TBI samples obtained from 8 subjects along with 8 age matched controls. We used Contusion and Pericontusional penumbra from 8 TBI subjects and 8 Fronto-temporal regions from age and sex matched controls. Isolated crude mitochondria were sonicated (once 10 s, 37% Amplitude) with Phosphate buffer saline and 0.05% SDS. Protein estimation was carried out using Bradford method. Twenty microgram of protein from each sample within a group was pooled and protein normalization was confirmed by SDS-PAGE on a 10% gel.

5.23.2 Isobaric tag for relative and absolute quantitation (iTRAQ) labeling and Strong Cation Exchange Chromatography

Protein samples were reduced, cysteines blocked, and digested with trypsin. iTRAQ (Applied Biosystems catalog no 4352135) labeling of peptides was carried out according to the manufacturer’s protocol. Briefly, i00pg of protein from each experimental group was treated with 2pl of reducing agent (tris (2-carboxyethyl) phosphine) at 60[0]C for 1 h and alkylated with 2.5pl of methyl methanosulfonate for 10 min at room temperature. Proteins were digested using sequencing grade trypsin (Promega) (1:20) overnight at 37°C. Peptides from control, Contusion and Pericontusion samples were labeled using iTRAQ reagent containing reporter 115, 116 and 117 respectively. Labeling was carried out for 1 ¥2 h at room temperature. After labeling, the peptides from all the three samples were pooled and fractionated using strong cation exchange chromatography on Polysulfoethyl A column (PolyLC, Columbia, MD) (100x2.1 mm, 5 pm particles with 300 A pores) using an LC Packing HPLC system connected to a Probot fraction collector. SCX fractions were collected in a 96 well plate using a 70-min gradient of KCl from 0 to 350 mM concentration in 5 mM potassium phosphate buffer, 25% acetonitrile (pH 2.85). The fractions collected were pooled based on SCX profile into 20 fractions dried and reconstituted in 20 pl of 0.1% formic acid. Samples were desalted using C-18 stage tip dried and stored in -20[0]C till mass spectrometric analysis.

5.23.3 LC-MS/MS Analysis

LC-MS/MS (Liquid chromatography-tandem mass spectrometry) analysis was carried out on LTQ Orbitrap Velos ETD - mass spectrometer (Thermo Scientific) interfaced with Proxeon Nano LCII. Peptides were analyzed on a reversed phase liquid chromatography. The RP-LC system equipped with a pre-column (2cm, 5p - 100Ao), analytical column (12 cm, 3p - 100Ao) made with magic AQ C18 material (Michrom) was packed in-house. Further, the peptides were sprayed using nano electro spray emitter tip 10p. (New Objective, Woburn, MA) fixed to an NSI source. The peptides were loaded on the pre column using 97% solvent A [0.1% formic acid (aq)] and resolved on the analytical column using a linear gradient of 10-30% solvent B (95% acetonitrile, 0.1% formic acid) for 60 min at a constant flow rate of 0.35 ^l/min. The spray voltage and heated capillary temperature were set to 2.4 kV and 250[0]C, respectively. The data was acquired in a data dependent manner. From each MS survey scan, 20 most intense precursor ions were selected for fragmentation. MS and MS/MS scans were acquired in an Orbitrap mass analyzer and the peptides were fragmented by higher energy collision dissociation with normalized collision energy of 41%. MS scans were acquired at a resolution of 60,000 at 400 m/z while MS/MS scans were acquired at a resolution of 15,000. The automatic gain control (AGC) for full FT MS was set to 1 million ions and for FT MS/MS was set to 0.1 million ions with maximum time of accumulation 200 ms and 500 ms, respectively (Proteomic experiments were carried out in neurochemistry lab of NIMHANS, IOB lab at NRC-NIMHANS and at IOB, Whitefield, Bangalore).

5.23.4 Data Analysis

The MS data was analyzed using Proteome Discoverer (Thermo Fisher Scientific, Version 1.4.0.288). The workflow consisted of spectrum selector and reporter ion quantifier. MS/MS search was carried out using Sequest search algorithm against the NCBI RefSeq database (release 52). Search parameters included trypsin as the enzyme with 1 missed cleavage allowed; oxidation of methionine was set as a dynamic modification while methylthio modification of cysteine and iTRAQ modification at N- terminus of the peptide and lysine were set as static modifications. Precursor and fragment mass tolerance were set to 20 ppm and 0.1.Da, respectively.

5.23.5 Biological Network Analysis

The up-regulated and down-regulated proteins were further analyzed for pathway analysis using the Gene-Spring analysis software version 12.5 (Agilent Biosystems, Santa Clara, CA). Up-regulated and down-regulated proteins obtained after filtering based on fold change cut off (=2.0) were taken as the input list. Biological networks were generated by comparing the input list to the reference list, which contains more than 1.4 million reactions derived from natural language processing­based extraction from literature and from different interaction databases. High confidence networks were further generated by applying filters that included binding, expression, metabolism, transport, promoter binding and regulation category of molecules. The number of molecules per network was restricted to 50. The entities which did not have connections were removed from the network. The constructed network was overlaid on the final input list to visualize the up-regulated and down- regulated genes (Marimuthu , et al. 2013).

5.24 Statistical analysis

All quantitative data were accumulated from at least three independent experiments. Normality of the sample was established by Shapiro-wilk and Kolmogorov- Smirnov test for normal distribution. The final data were expressed as mean ± SD / SEM. Differences between mean values were analyzed by one-way analysis of variance (ANOVA), Mann-whitney’s t test and Kruskal-wallis one way analysis of variance. In order to reveal the differential changes of antioxidant and oxidant parameters across sites (different regions of the brain) general linear model based multivariate site*group analysis was carried out using SPSS v16.0 (statistical software for social sciences). Correlation analysis were done in Microsoft excel 2010 and p value < 0.05 was considered as statistically significant.

6 RESULTS

The current thesis focuses on the role of oxidative stress and mitochondrial function in the human brain as follows:

1. Assessment of the non-traumatized regions of brains from TBI patients: Assessment of the role of pre and postmortem factors including aging.
2. Analysis of the traumatized regions of brains from TBI patients: Role of aging
3. Proteomic analysis in TBI: Comparison of contusion, pericontusion with controls followed by validation.
4. Assessment of antioxidant markers in PD brains with implications for therapeutics: Focus on glutathione.

6.1 Role of pre/postmortem factors on the oxidant/antioxidant markers in the non-traumatized regions of postmortem brains from TBI patients.

We first tested the alteration in the activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase in non-injured anatomical areas FC, CB, MO, SN and HC of postmortem human brain samples with increasing PMI (range: 2.5 to 26 h), gender of the subject and agonal state (GCS score: 3 to 15). Compared to other regions, SN and HC showed lower SOD activity in all samples. Interestingly, none of the regions showed significant alteration in SOD activity with increasing PMI (FC: r= -0.05, p=0.38; CB: r=0.13, p=0.20; MO: r=-0.10, p=0.26; SN: r=-0.38, p=0.13; HC: r=-0.16, p=0.26) (Figure 9A). Except for HC (r=0.41, p=0.05), none of the regions showed significant alteration in SOD activity with increasing GCS rating (FC: r= 0.01, p=0.49; CB: r= 0.11, p=0.23; MO: r=0.04, p=0.40; SN: r=0.04, p=0.45) (Figure 9B). Similarly, none of the regions showed significant alteration in SOD activity between males and females (FC: p=0.08, CB: p=0.28; MO: p=0.98; SN: p=0.29; HC: p=0.79) (Figure 9C). With respect to catalase activity, CB showed a slight decrease in catalase activity compared to FC and MO (Figure 10). However, none of the regions showed significant alteration in the activity with increasing PMI (FC: r=-0.059, p=0.348; CB: r=0.053, p=0.363; MO: r=0.025, p=0.437; SN: r=-0.22, p=0.21 ; HC: r=0.00 , p=0.5) (Figure 10A), GCS rating (FC: r= -0.25, p=0.06; CB: r=0.08, p=0.31; MO: r= 0.06, p=0.37; SN: r=0.28, p=0.16; HC: r=0.08, p=0.39) (Figure 10B) or gender difference (FC: p=0.332, CB: p=0.155; MO: p=0.99; SN: p=0.33; HC: p=0.93) (Figure 10C). These data indicate that the SOD and catalase activities in the human brains show significant stability postmortem.

We then estimated the levels of total glutathione (GSH+GSSG) in these samples and observed that MO, SN and HC showed > 10 fold decrease in total GSH levels compared to FC and CB (Figure 11). While FC showed an increasing trend and CB exhibited decreasing trend, there was no any significant change in GSH levels with increasing PMI (FC: r= 0.205, p=0.085; CB: r=-0.132, p=0.19) (Figure 11A). On the other hand, MO showed significant decrease in GSH with increasing PMI (r=-0.309, p=0.02) (Figure 11B). However, the GSH levels were unaltered with GCS rating in all the regions tested (FC: r=0.15, p=0.17; CB: r=0.08; p=0.31; MO: r=0.03, p=0.44) (Figures 11C and 11D). Similarly in SN and HC, the total GSH content did not change with increasing PMI (SN: r=-0.42, p=0.06; HC: r=-0.02, p=0.46) or GCS rating (SN: r=- 0.06, p=0.06; HC: r=-0.08, p=0.37) (Figures 11E and 11F). While CB and MO showed statistically significant increase in GSH levels in females compared to males, FC only showed an increasing trend (FC: p=0.14, CB: p=0.017; MO: p=0.038) (Figure 11G and 11H). However, SN and HC did not show significant variation in GSH content between males and females (SN: p=0.51; HC: p=0.45) (Figure 11I and 11J). The altered GSH levels in MO and CB indicate the susceptibility of GSH metabolism to pre and post­mortem factors.

The alteration in GSH concentration could be partly in response to altered oxidative markers in theses samples. To test this, we estimated the lipid peroxidation product malondialdehyde (MDA) as a marker of oxidative stress in our samples. We observed that MO showed ~ 3-fold increase in MDA levels compared to FC and CB (Figure 12). MO also showed statistically significant increase in MDA levels with increasing PMI (r=0.25, p=0.047) (Figure 12A) which corroborated with the decreased GSH levels in those samples (Figures 12B). On the other hand, FC, CB, SN and HC did not show any significant change in MDA concentration with increasing PMI (FC: r=0.070, p=0.321; CB: r=0.07, p=0.333; SN: r=-0.09, p=0.44; HC: r=0.22, p=0.11) (Figure 12A). Except HC, none of the regions showed any statistically significant difference in MDA content with GCS rating (FC: r= 0.03, p=0.41; CB: r= 0.23, p=0.07; MO: r=-0.16, p=0.15; SN: r=-0.34, p=0.29; HC: r=-0.35, p=0.03) (Figure 12B). Similarly, none of the regions showed any statistically significant difference in MDA content between males and females (FC: p=0.99, CB: p=0.74, MO: p=0.51; SN: p=0.49, HC: p=0.65) (Figure 12C). These data suggest that GSH elevation in MO (Figure 11E) cannot be explained by the gender dependent MDA levels. To understand whether there is a gender difference in GSH synthesis, we tested the activity of GCL which is the rate limiting enzyme in GSH synthesis and one of the indicators of GSH status. Figure 11D shows that in FC, CB and MO, although there was marginal increase in GCL activity in females compared to males (Figure 11E), the data was statistically insignificant to contribute to GSH synthesis.

Since GSH levels were altered in our samples, we investigated the changes in the activities of three antioxidant enzymes related to GSH dynamics: Glutathione reductase (GR), Glutathione peroxidase (GPx) and Glutathione-S-transferase (GST). Regarding GR, CB showed ~ 40-50 % decrease in GR activity compared to FC and MO (Figure 13). There was no significant alteration in activity with increasing PMI in any of the regions tested (FC: r= -0.101, p=0.252; CB: r=0.017, p=0.455; MO: r=-0.180, p=0.121; SN: p=- 0.57; p=0.07; HC: r=-0.14, p=0.3) (Figure 13A). With agonal state, only CB showed significant increase in GR activity (r= 0.27; p=0.04) while it was unchanged in other regions (FC: r=0.11, p=0.21; MO: r=0.20, p=0.10; SN: r=-0.08, p=0.43; HC: r=0.05, p=0.43) (Figure 13B). However, none of the regions showed any statistically significant difference in activity between males and females (FC: p=0.33, CB: p=0.745; MO: p=0.525; SN: p=0.49; HC: p=0.54) (Figure 13C).

Regarding GPx, MO showed ~2.5 fold increase in GPx activity compared to FC and CB (Figure 14). Although MO showed an increasing trend in GPx activity with increasing PMI, none of the regions showed significant alteration in activity with increasing PMI (FC: r= -0.135, p=0.185; CB: r=-0.044, p=0.385; MO: r= 0.213, p=0.085; SN: r=-0.54, p=0.09; HC: r=0.1, p=0.29) (Figure 14A). Regarding agonal state, GPx activity significantly increased with GCS rating in MO (r= 0.26, p=0.05) but was unaltered in the other regions (FC: r= 0.06, p=0.35; CB: r= 0.19, p=0.11; SN: r=0.05, p=0.46; HC: r=-0.17, p=0.19) (Figure 14B). However, none of the regions showed any statistically significant difference in activity between males and females (FC: p=0.121, CB: p=0.626; MO: p=0.836; SN: p=0.40; HC: p=0.38) (Figure 14C).

We observed that GST was the least altered enzyme in our study (Figure 15) since its activity did not change significantly when compared across different anatomical areas or PMI (FC: r=-0.021, p=0.444; CB: r=0.088, p=0.280; MO: r=-0.167, p=0.139; SN: r=0.03, p=0.47; HC: r=0.17, p=0.25) (Figure 15A), GCS rating (FC: r=0.11, p=0.25; CB: r=0.01, p=0.48; MO: r=0.17, p=0.15; SN: r=0.07, p=0.44; HC: r=0.07, p=0.39) (Figure 15B) or gender (FC: p= 0.22, CB: p=0.56; MO: p=0.69; SN: p=0.07; HC: p=0.58) (Figure 15C).

Histopathological analysis of different neuroanatomical areas of postmortem brain samples (MO, CB and FC) were assessed for alteration in neuronal morphology and changes in axonal tracts in the white matter. The findings in different areas were then compared with GCS score and the PMI (Figures 16a-o). The MO, CB as well as FC revealed well preserved neurons with Nissl substance within soma at higher GCS scores (GCS 9-15) (Figures 16d-g, l-o). At low GCS scores at admission, ischemic changes were seem in neurons in MO (Figure 16 a, h) and FC (Figure 16b) though CB neurons were well preserved (Figures 16c, k). In addition, spongy changes in neuropil was demonstrable in MO with decreasing GCS (Figures 16l) and the white matter of pyramidal tracts maximal at GCS of 3 (Figures 16i) . Similarly with increasing PMI, MO and FC showed well preserved neurons with prominent Nissl substance but increasing neuropil vacuolation was demonstrable in both regions that was maximal at PMI of 19 h suggesting dendritic swelling (Figures 16f, l). The white matter tracts in the medullary pyramids revealed edema and separation of fibres from PMI 15 h onwards which gradually increased (Figures 16e,i). However, no distinct demyelination was observed. The oligodendroglia in white matter revealed pyknosis of the nuclei with perineuronal cytoplasmic vacuolation reflecting anoxic damage, more evident at longer PMI (above 15 h, Figure 16n). The CB however was well preserved with no significant pathological features except for ischemic change in Purkinge neurons (Figure 16o). The maximal pathological changes were observed in MO with increasing PMI and decreasing GCS as reflected by the neuropil vacuolation and ischemic changes in the neurons and this corroborates with the biochemical data which showed significant oxidative changes in the MO. FC and CB in contrast showed relatively milder pathological changes. These data highlight that while SOD and catalase were relatively unaffected by the pre and postmortem factors, GSH and its metabolic enzymes were significantly altered and this was more pronounced in MO of postmortem human brains. These data highlight the influence of pre and postmortem factors on GSH dynamics and the inherent differences in brain regions, with implications for studies on brain pathophysiology employing human samples.

Next, we tested whether the status of oxidatively damaged brain proteins were altered by pre and postmortem factors. This was carried out by western blot based quantitation of protein carbonylation in different samples. Densitometric quantitation of protein carbonyls/P-actin ratio with increasing storage time showed significant decrease in the status of protein carbonyls in CB and MO while it was relatively unaltered in FC (FC: r= -0.14, p=0.19; CB: r= -0.27, p= 0.04; MO: r=-0.42 , p= 0.003) (Figure 17B). Statistical analysis indicated that the stability of the oxidized proteins was unaltered until 91.3 months of storage in CB and until 75.2 months in MO (data not shown) indicating that tissue preservation up to ~6 years at -80 [0]C would still maintain the stability of oxidized proteins. On the other hand, protein oxidation status was unchanged in any of the regions with increasing PMI (FC: r= -0.06, p=0.35; CB: r= - 0.01, p= 0.47; MO: r= 0.02, p= 0.45) (Figure 17C), agonal state (FC: r= 0.16, p=0.15; CB: r= 0.20, p= 0.10; MO: r= 0.07, p= 0.32) (Figure 17D) and gender difference (FC: p=0.134; CB: p=0.28; MO: p=0.054) (Figure 17E).

We then carried out western blot based quantitation of protein 3-NT in different samples using the slot-blot approach. Figure 18A shows a representative slot-blot showing protein nitration and P -actin levels in FC with increasing storage time. Densitometric quantitation of protein 3-NT/P-actin ratio with increasing storage time showed significant decrease in protein nitration level in MO while it was unchanged in FC and CB (FC: r= 0.20, p=0.10; CB: r= 0.13, p= 0.20; MO: r=-0.36 , p= 0.01) (Figure 18B). However, statistical analysis indicated that the stability of the nitrated proteins was unaltered until 75 months of storage in MO (data not shown) indicating that tissue preservation up to ~6.25 years at -80 [0]C would still maintain the stability of nitrated proteins. On the other hand, the protein nitration status was unchanged with increasing PMI (FC: r= -0.02, p=0.44; CB: r= 0.15, p= 0.17; MO: r=-0.03, p= 0.44) (Figure 18C) and agonal state (FC: r= 0.01, p=0.47; CB: r= -0.11, p= 0.25; MO: r= 0.15, p= 0.16) (Figure 18D) in any of the regions. While gender based differences in protein nitration were not significant in FC and MO, protein nitration was lower in females compared to males in CB (FC: p= 0.35; CB: p=0.043; MO: p=0.182) (Figure 18E).

Next we tested the level of GFAP expression (as a marker of astrogliosis) using the slot blot quantitation (along with the corresponding P-actin levels) in different samples. Figure 19A shows a representative slot-blot of GFAP and P-actin levels in FC with increasing storage time. Densitometric quantitation of GFAP/P-actin ratio with increasing storage time showed significant decrease in the status of GFAP in MO while it was unchanged in FC and CB (FC: r= -0.08, p=0.31; CB: r= -0.05, p= 0.39; MO: r= - 0.29, p= 0.03) (Figure 19B). Statistical analysis indicated that the stability of the oxidized proteins was unaltered until 88.5 months of storage in MO (data not shown) indicating that tissue preservation up to ~7.4 years at -80 [0]C would still maintain the stability of GFAP. However, the GFAP status was unchanged in any of the regions with increasing PMI (FC: r= 0.20, p=0.10; CB: r= 0.06, p= 0.34; MO: r= -0.14, p= 0.18) (Figure 19C), agonal state (FC: r= -0.04, p=0.40; CB: r= 0.12, p= 0.23; MO: r= 0.10, p= 0.26) (Figure 19D) and gender difference (FC: p=0.67; CB: p=0.44; MO: p=0.217) (Figure 19E). These data highlight the influence of storage time on preservation of markers of protein damage and astrogliosis and the inherent differences in brain regions, with implications for studies on brain pathology employing stored human samples.

6.2 Analysis of the distribution of oxidant and antioxidant markers in the sub-cellular fractions of non-traumatized regions of brains from TBI patients: Assessment of role of pre and postmortem factors

Next, we investigated the distribution of antioxidant activities in the subcellular fractions of postmortem human brains and tested whether they are influenced by increasing storage time at - 80[0]C (11.8-104.1 months), PMI (range: 2.5-26 h), age of the donors (2 days to 80 y), gender of the subject, and agonal state (GCS score: 3-15). This was carried out by assaying the SOD, GSH, GPx, GR, and GST in cytosol and synaptosomes fractions of FC from human brain samples (n = 45).

The SOD activity was > 5 fold lower in synaptosomes compared to the cytosol. The SOD activity was unaffected by storage time (cytosol: r= - 0.12, p = 0.2; synaptosomes: r= - 0.10, p = 0.29), PMI (cytosol: r = 0.02, p = 0.46; synaptosomes: r= - 0.11, p = 0.24), agonal state (cytosol: r= - 0.12, p = 0.23; synaptosomes: r = 0.04, p = 0.40), or gender (cytosol: p = 0.89; synaptosomes: p = 0.37) (Fig. 20).However, SOD activity increased with age in the synaptosomes, while it remained relatively unaltered in the cytosol fraction (cytosol: r = 0.20, p = 0.10; synaptosomes: r = 0.37, p = 0.01) (Fig. 20D). The total GSH content was ~ 5 fold lower in the synaptosomes compared to cytosol. The total GSH content was unaffected by storage time (cytosol: r = 0.21, p = 0.11; synaptosomes: r= - 0.11, p = 0.27), agonal state (cytosol: r= - 0.12,p = 0.24; synaptosomes: r= - 0.23, p = 0.09), or gender (cytosol: p = 0.35; synaptosomes: p = 0.15) (Fig. 21).However, total GSH decreased in the cytosol with increasing PMI (cytosol: r= - 0.28, p = 0.04; synaptosomes: r = 0.05, p = 0.38) (Fig. 21B) and age, while it was unaltered in synaptosomes (cytosol: r= - 0.28, p = 0.03; synaptosomes: r = 0.11, p = 0.24) (Fig. 21D). On the other hand, GPx activity was ~8 fold lower in synaptosomes compared to cytosol. GPx activity was unaffected by storage time (cytosol: r = 0.05, p = 0.38; synaptosomes: r= - 0.15, p = 0.18), PMI (cytosol: r= - 0.11, p = 0.25; synaptosomes: r= - 0.11, p = 0.24), agonal state (cytosol: r = 0.13, p = 0.23; synaptosomes: r= - 0.13, p = 0.22) or gender (cytosol: p = 0.93; synaptosomes: p = 0.23) (Fig. 22). Interestingly, while the GPx activity decreased with age in cytosol, the activity significantly increased in synaptosomes (cytosol: r= - 0.26, p = 0.05; synaptosomes: r = 0.25, p = 0.05) (Fig. 22D).

The GR activity was ~20 % lower in synaptosomes compared to cytosol. GR activity was unaffected by all factors including storage time (cytosol: r = 0.11, p = 0.27; synaptosomes: r= - 0.18, p = 0.14), PMI (cytosol: r= - 0.13, p = 0.20; synaptosomes: r= - 0.05, p = 0.36), agonal state (cytosol: r = 0.13, p = 0.23; synaptosomes: r = 0.19, p = 0.13), age (cytosol: r= - 0.10, p = 0.26; synaptosomes: r= - 0.08, p = 0.30) and gender (cytosol: p = 0.57; synaptosomes: p = 0.11) (Fig. 23). The total GST activity was > 20 fold lower in synaptosomes compared to cytosol. The GST activity was unaffected by storage time (cytosol: r = 0.07, p = 0.34; synaptosomes: r= - 0.18, p = 0.15), PMI (cytosol: r= - 0.01, p = 0.47; synaptosomes: r = 0.07, p = 0.33), or gender (cytosol: p = 0.72; synaptosomes: p = 0.91) (Fig. 24). However, GST activity decreased in synaptosomes with agonal state (cytosol: r = 0.12, p = 0.23; synaptosomes: r= - 0.27, p = 0.05) (Fig. 24C) and age, while it was unaltered in cytosol (cytosol: r = 0.05, p = 0.38; synaptosomes: r = 0.32, p = 0.02) (Fig. 24D).

Overall, we observed that the antioxidant activities were several fold lower in synaptosomes as compared to cytosol in the postmortem human brains. The activities were affected mainly by the age and to a lesser extent by other premortem and postmortem factors (Table 9). These results should be considered during interpretation of biochemical data in a meaningful manner.

6.3 Anatomical heterogeneity in the distribution of redox markers in the human brain with the persistence of the signature pattern at different ages: Correction for pre/postmortem factors

Next, we analyzed the relative distribution of redox markers in among brains from young subjects and old subjects, followed by correction for different pre and post mortem factors. This was carried out in six anatomical regions: FC, CB, CD, mid brain (MB), MO and HC in young brains (20±5 y; n=ii) and old brains (70±10 y; n=i0) (Table 10).

Initial observation in both young and old brains was that within each group, the antioxidant and oxidant markers showed significant alteration across different brain regions (Figure 25-28). For example, total GSH (Figure 25A) was highest in CB followed by FC, while CD displayed ~ 25 % GSH compared to CB. On the other hand, HC and MO showed only ~5 % total GSH while MB had ~ 2% GSH. Interestingly, the gross pattern of the relative distribution of antioxidant markers in different brain regions was similar between young and old brains for GSH, GPx, SOD and GST (Figures 25, 26). On the other hand, GR and catalase showed variation in the relative distribution of antioxidant markers in different brain regions between young and old brains (Figure 25). Similar analysis of oxidant markers (lipid peroxidation and protein oxidation; Figure 26E-F, 27) and GFAP signal (Figure 28) showed similar pattern in different brain regions between young and old brains.

To analyze the differential changes in redox parameters across different brain regions, general linear model based multivariate site*group analysis was conducted for both age groups to theoretically nullify (factor out) the effects of pre and post-mortem factors (age, gender, PMI and agonal state) thereby representing the absolute value for each parameter (Figure 29-30). Accordingly, the profiles of GSH, GPx, GR, catalase, protein carbonyls, GFAP and MDA did not show significant change in young vs. old brains while SOD (p<0.037), GST (p<0.048) and catalase (p<0.0i) showed significant difference in the intra-anatomical profile between young and old brains. These data indicate that every marker in the brain could have specific pattern that could be maintained uniformly or unevenly across different ages thereby emphasizing the anatomical heterogeneity of these biochemical parameters.

6.4 Analysis of histopathological parameters and oxidant and antioxidant markers in the tissue and sub-cellular fractions of traumatized regions of human brains from TBI patients: Role of aging

Following extensive analysis of redox and mitochondrial markers in the non­traumatized regions of TBI brains, we set out to carry out a comparative analysis of redox markers in traumatized regions of human brains. This was carried out in fresh samples (resected during craniotomy; n=2i) and autopsy samples (n=6) following identification of contusion (FC), pericontusion (FC), away from contusion (Occipital cortex) and age-matched controls (Table 11), based on the analysis of the CT-scan of the patients by a neurosurgeon. All the tissues utilized for biochemical studies were initially subjected to pathological examination and characterization using histochemical staining and immunohistochemistry against GFAP and phosphorylated neurofilament (medium and high molecular weight chain). Microglial response was detected using the markers Iba-1 and HLA-DR while infiltration of the tissue by macrophages was characterized using CD68.

Following histological staining, contusions were seen as zones of variable sized hemorrhages clustered around the crest of the gyri disrupting the surface pia and cortical parenchyma (Fig. 32A). Hemorrhages were produced by the rupture of thin walled parenchymal venules producing coalescing petechial zones of hemorrhage (Fig. 32B). The rupture of vessel walls caused exudation of RBCs and plasma with margination of luminal polymorphonuclear leukocytes along the wall (Fig. 32C). More extensive contusions resulted in tissue destruction and extension into underlying white matter in a wedge shaped fashion. The overlying subarachnoid space revealed variable degree of subarachnoid hemorrhage following rupture of smaller thin walled venules (Fig. 32D). The arterioles and muscular vessels in subarachnoid space displayed features of spasm of muscle coat in the form vacuolation of the smooth muscle coat (Fig. 32E). The intervening parenchyma revealed petechial hemorrhages from the leaky vessels (Fig. 32F) while the neurons revealed pyknotic nuclei and darkly staining shrunken cell bodies, the „dark neurons (Fig. 32F).

The pericontusion zones appearing as penumbra around the zone of contusion displayed oedema in a prominent manner. The neurons in this zone were relatively viable but demonstrated perineuronal vacuolation extending along the dendritic processes (Fig. 33B) suggesting presynaptic pathology probably secondary to mitochondrial alterations. The neuropil and glial cells also revealed vacuolation (Fig. 33C) temporary progressing from fine vacuoles to produce coalescing larger vacuoles with increase in time after trauma. The vessels in the grey and white matter had widened Virchow vacuolation of foot Robin space and vacuolation of astrocytic foot processes (Fig. 33D, F) reflecting breach in blood brain barrier and perivascular edema. The axonal tracts were separated by neuropil edema and demyelination (Fig.33G). Vessels focally had polymorphonuclear leucocyte accumulation in the wall and transuding into parenchyma (Fig. 32E).

Neuronal changes: Neuronal alterations heralded with shrunken and intensely eosinophillic cytoplasm (red neurons) shortly following the injury, reflecting a reparative response of increase in mitochondria within the cytoplasm (Fig. 34A). With increase in time interval following the injury, dark neurons with shrunken irregular dark nucleus (apoptotic cell change) predominated (Fig. 34B). In pericontusional zones, in addition to perineuronal vacuolation (Fig. 34C) dystrophic changes of neuronal soma in the form of accumulation of phosphorylated neurofilament was noted probably secondary to axonal transection (Fig. 34D). The numbers of dystrophic neurons in the grey matter increased with increasing time interval after the injury.

Axonal alterations: In the white matter, axonal alterations were prominent in pericontusional zones compared to relative preservation in the areas of contusion (Fig. 34E). In the pericontusional zones, paralleling the increasing edema, there was separation of axons followed by axoplasmic degeneration and fragmentation (Fig.33F). In cases with more than 72 hours survival following injury, rarefaction of parenchyma with dissolution of myelin was accompanied by marked axonal depletion (Fig.33E, F). The surrounding glial cells demonstrated shrinkage and pyknosis of nuclei (apoptotic change).

Glial alterations: The astrocytic alterations were highlighted by GFAP immunolabeling. The contusion zones were characterized by marked increase in gliosis encircling the zones of hemorrhage (Fig. 35B, C). Beginning in perivascular and perineuronal zones the reactive changes in the protoplasmic astrocytes was seen as numerous ramified branching processes coalescing around neurons forming large plaque like aggregates (Fig. 35C). The intense GFAP accumulation in this plaque like zones reflected increase in synthesis of the glial fibrillary protein and it was confirmed on Western blots. The perivascular astrocytes close to contusion demonstrated vacuolation and separation of the foot processes disrupting the blood brain barrier (Fig. 35D). The astrocytes in pericontusion zone demonstrated dystrophic changes (Fig. 35F, H). In comparison to normal thin fine branching processes of protoplasmic and fibrillary astrocytes in grey and white matter respectively (Fig. 35E, G), the reparative astrocytes demonstrated short irregular processes with beading and fragmentation (Fig. 35F) without prominent hypertrophy of the cell body. In the white matter, the reactive astrocytes demonstrated hypertrophy of the cell body with short truncated cell processes (Fig. 35H). The glial dystrophic changes reflect metabolic disequilibrium in the synaptic neurotransmission enhancing proteolytic and lysosomal activation (reflected in proteomic studies with up-regulation of proteins involved in ubiquitin proteasomal pathway: See subsequent sections)

Inflammatory/microglial alterations: The subarachnoid space and the parenchymal hemorrhages revealed CD68 positive macrophage infiltration (Fig. 36A, B). In the grey matter and white matter, particularly in the pericontusion zones, accumulation of activated microglia was seen (Fig. 36C, D). These had a ramified morphology with cell hypertrophy and branching processes (Fig. 36C, D), tending to aggregate around the neurons, vacuolated glia and perivascular spaces. Some of these were labeled by HLA DR indicating activated state (Fig. 36F) secondary to trauma. Almost all cases examined had hemorrhagic contusions. Ischemic infarcts were not evident.

Characterization of temporal evolution in the neuronal astrocytic and microglial response was not possible due to limited sample size, though a trend the sequential changes were evident. Eosinophilic neurons, basophilic neurons and phosphorylated NF filled neuronal soma was seen with increasing survival times following injury. Similarly the proliferating astrocytes in response to injury displayed dystrophic changes, altering the blood brain barrier, leading to increasing neuropil edema and vacuolation of foot processes. Reactive microglial response changing to ramified forms and expression of HLA DR followed the astrocytic changes closely to scavenge the degenerative neurons. The accumulation of phosphorylated neurofilament in the neuronal soma and rarefaction of the white matter are suggestive of irregular axotomy following injury. A systematic study on a large sample size, at different intervals, on large brain section will provide better insight into the evolution of the pathology.

Following pathological evaluation, the whole tissue extracts from the contused region of different brains were used to determine the status of different redox markers followed by correlation with age and agonal state of the patient (Figure 37-38). Accordingly, catalase (r= -0.50, p=0.01), total GSH (r=-0.36, p=0.03), GPx activity (r= - 0.58, p=0.002) and GST activity (r=-0.40, p=0.04) were significantly lowered in contused samples with increasing age (19 to 70 y) (Figure 37C-E, 37I). On the other hand, activities of GR, TrxR and SOD were unaffected by age (Figure 37F-H). Analysis of oxidant markers in the contused tissue indicated that while lipid peroxidation was significantly elevated with aging (r=0.65, p=0.0004), H2O2 content was unaffected (Figure 37A-B).

Interestingly, correlation of the redox data with agonal state (GCS 15 to 3) showed that only total GSH was significantly decreased with increasing agonal state (r=- 0.44, p=0.03) (Figure 38D), while catalase, TrxR, SOD and GST activities were unchanged with GCS (Figures 38C, 38G-I). On the other hand, the activities of GPx (r=0.41, p=0.04) and GR (r=-0.45, p=0.023) were significantly increased with age (Figure 38E-F). The status of the oxidant markers was relatively unchanged with age (Figure 37). Correlation of the redox markers with mid line shift (MLS in mm: marker of brain injury and structure) did not show any significant change (data not shown). On the other hand, Kruskal-wallis one way ANOVA of the data following grouping samples (severe, n=8; moderate, n=i0; mild, n=5) indicated that GR showed increased activity (Figure 38j) in severe injury cases (p<0.05), while the status of other the markers was unchanged (data not shown). Overall, the data indicate that redox markers are significantly affected by increasing age and not the agonal state and severity of TBI.

Next we compared the status of redox markers in the contused area with the area of pericontusion, away from contusion and age-matched controls. We observed that the activities of GPx (p<0.0i), GST (p<0.0i) and SOD (p<0.0i) and the total GSH content (p<0.05) were significantly decreased in the contused tissue compared age-matched controls while it was unchanged in the pericontused area and area away from contusion (Figure 39). On the other hand, H2O2 was elevated in contusion (p<0.0i) and protein carbonyls were elevated both in contusion (p<0.0i) and pericontusion (p<0.05) (Figure 39A, 39H). Lipid peroxidation was relatively unchanged in all the samples (data not shown).

As observed earlier in the study, the status of a redox marker could be significantly different in its sub-cellular distribution (neuropil fractions: cytosol, non- synaptic mitochondria and synaptosomes) compared to the whole tissue extract. Accordingly, analysis of the redox markers in the neuropil fractions showed that contused area showed significant loss of antioxidant function compared to the control, consistent with the data from whole tissue extract (Figure 40). Further, all the redox markers were significantly decreased mainly in the synaptosomal fraction compared to control (SOD: p<0.05, GSH: p<0.05 in contusion and pericontusion, GPX: p<0.05 in contusion, GST: p<0.0i in contusion, GR: p<0.05 in pericontusion and p<0.0i in contusion) while it was slightly reduced in the non-synaptic mitochondria (Total GSH: p<0.05 both in contusion and pericontusion; GR: p<0.05 in contusion) and predominantly unchanged in the cytosol (except GPx activity in the contusion: p<0.05) (Figure 40). However, the cytosolic and non-synaptic mitochondrial fractions did not display increase in protein carbonyls and 3-NT signal (Figure 41-42) while synaptosomes showed elevated protein oxidation in contusion compared to control (p<0.05) (Figure 43C-D).

These data indicate that the non-synaptic mitochondria and synaptosomes showed significant loss of antioxidant function and elevated protein oxidation which might affect the mitochondrial and synaptic function. To confirm this, we carried out mitochondrial assays in the non-synaptic and synaptic mitochondria which indicated that compared to the controls, contusion and pericontusion showed significant decline in complex I activity (Figure 44A). Although statistically not significant, activities of MDH and SDH (Figure 44B-D) were very much lower compared to normal controls. CS activity was relatively unchanged (Figure 44E). Based on these data, it could be surmised that there is significant loss of antioxidant function and elevated oxidant markers, specifically in the contused region compared to the other regions. Sub-cellular analyses proved that these changes were predominant in the non-synaptic mitochondria and synaptosomes with associated loss of mitochondrial activity thus impinging on synaptic function. To further evaluate this data, we carried out high-throughput analysis of the proteomic changes in the contused area vs. pericontused region compared to age- matched control.

6.5 Proteomic analysis in brain tissue from TBI: Comparison of contusion, pericontusion with controls followed by validation

Proteomic characterization of injured brain tissue using rodent or canine TBI models have been used widely used. In contrast, there are relatively fewer proteomic studies in the brain tissue from TBI subjects (Conti , et al. 2004, Dayon , et al. 2008). In order to analyze the proteomic changes in TBI, we analyzed the contused region and pericontusion (penumbra) region from Frontal/fronto-temporal/temporal cortex compared to age-matched controls (Table 12). Analysis of the soluble fraction of the samples from different tissue samples by iTRAQ labeling followed by LC-MS/MS analysis and identification and quantitation indicates that:

a. In contused regions, 3254 proteins were identified and quantified. Among these, 402 proteins were over expressed (1.5 fold or more) (Table 13) and 119 proteins were under expressed (0.7 fold ore lesser) in contusion compared to age-matched controls (Table 14). In the pericontusion region, among the 3254 identified proteins, 73 proteins were over expressed (1.5 fold or more) (Table 15), while 84 proteins were under expressed (0.7 fold or lesser) compared to age-matched controls (Table 16).
b. Gene ontology (GO) analysis helped in elucidating the localization, different functions and process of identified proteins (Fig. 45). GO analysis localized 61.1% of proteins to intracellular locations, 13.9% were protein complexes, 14.4% were of extracellular, and 6.3% were plasma membrane proteins. Among the total identified proteins, 462 (14%) were mitochondrial and 732 (22.4%) were synaptic proteins (Figure 46A, B).
c. Significantly up-regulated and down-regulated proteins were separately analyzed by GeneSpring software to determine the association of these proteins in biological pathways for better understanding of their biological context, involvement in diverse physiological pathways, and association with injury. Significantly altered proteins (down-regulated or up regulated) were in the following pathways: KEAP1-NRF2 (Fig 47), Spinal cord injury pathway, oxidative stress pathway (Fig 48), Prostaglandin synthesis pathway, ubiquitin proteasome and synaptic vesicle pathway (Fig 49). Among the differentially expressed proteins, those which are implicated in oxidative stress and TBI pathology, and the ones associated with the current thesis are mentioned here.

6.5.1 Down-regulated proteins

- Many subunits of Cytochrome c oxidase (COX) (mitochondrial complex IV) were down-regulated. COX1 (0.59 fold in contusion, 0.8 fold down in pericontusion), COX2 (0.7,0.9), COX7A2L (0.8 in contusion, 0.9 in pericontusion), COX7A1 (0.8 in contusion) and CX6B1 (0.8 in contusion, 0.8 in pericontusion)
- Cytochrome c, a central component of the electron transport chain in mitochondria (0.5 fold less in Contusion and 0.6 fold less in Pericontusion)
- Both SOD1 [Cu-Zn] (0.75 fold in contusion; 0.79 fold in pericontusion) and SOD2 [Mn] (0.49 fold in contusion; 0.39 fold in pericontusion) were down-regulated compared to controls.
- Antioxidant proteins catalase (0.9 fold in pericontusion), Myeloperoxidase (0.7 fold in pericontusion),
- Synaptic vesicle trafficking proteins like Complexin 1 (CPLX1) involved in synaptic vesicle exocytosis (0.61 fold in contusion; 0.75 fold in pericontusion) and synaptosomal-associated protein (SNAP25), implicated in synaptic vesicle membrane docking and fusion (0.77 fold in contusion; 0.76 fold in pericontusion) were significantly down-regulated compared to controls.

6.5.2 Up-regulated proteins included:

- Immune response HLA class protein like HLA-C (7.4 fold in contusion), HLA-G (2.6 fold in contusion), HLA-A (2.1 fold in contusion). Complement protein Complement factor B’ was 2.5 fold more in contusion.
- Antioxidant proteins Peroxiredoxin 2 (1.6 fold in contusion), catalase (3.8 fold in contusion), Myeloperoxidase (3.7 fold in contusion), Hemeoxygenase 1 (HMOX1) (3.6 fold in contusion),
- The expression of chitinase 3-like protein 1 (CHI3L1) and chitinase 1 (Synonyms: chitotriosidase, CHI3, CHIT) was more 2.9 fold higher in contusion and 1.2 times higher in pericontusion compared to controls.
- Astrocytic cytoskeletal protein GFAP was significantly increased in contusion (1.5 fold higher) but not in pericontusion (0.9 fold).
- Proteins associated with ubiquitin proteasome system like E3 Ubiquitin ligase, Ubiquitin like protein (ISG15), TRIM25, Proteasomal ubiquitin receptor (ADRM1), and TMUB2 were 3.9, 2.49, 1.87, 1.52, and 1.52 folds down-regulated in contusion compared to controls.

To check the reliability of the proteomic data we selected proteins for validation based on fold changes of the proteins, possible functional association with TBI pathobiology and availability of the antibodies. The validation data indicated that:

a. The expression of chitinase 1 (Synonyms: chitotriosidase, CHI3, CHIT) was significantly increased in contusion (1.57 fold) and pericontusion (1.6 fold) over age matched controls (Figure 50A, B).
b. The expression of peroxiredoxin-2 was unchanged among contusion (0.9 fold), and pericontusion (0.85 fold) compared to controls (Figure 50C, D).
c. The expression of GFAP was significantly over expressed both in contusion (2.3 fold) and pericontusion (1.53 fold) (p<0.05) compared to controls (Figure 51A, B). This was also evident from the immunohistochemistry data indicated in Figure 35.
d. The expression of synaptophysin was significantly over expressed both in contusion (6.4 fold) (p<0.001) and pericontusion (4.2 fold) (p<0.05) compared to controls (Figure 51C, D).
e. Immunohistochemical analysis for inflammatory markers Iba-1, CD68, and HLA- DR was carried out on contusion, pericontusion and normal controls. Of all the groups we could observe inflammatory response only in contusion and pericontusion, compared to controls. CD68 positive macrophage infiltration was prominently observed in the subarachnoid space and parenchymal hemorrhages (Contusion and Pericontusional zones) (Fig.36A, B). In the grey matter and white matter, particularly on pericontusion zones, accumulation of microglia (Iba 1 positive signal) was observed (Figure 36). These cells had activated ramified morphology with cell hypertrophy and branching processes (Fig. 36C, D) tending to aggregate around neurons, vacuolated glia and perivascular spaces. Some of these cells were labeled for HLA DR indicating activated state while control group did not show HLADR positive cells (Fig.36F).
f. SOD1 was included as a marker of redox balance to estimate the oxidative stress among the two groups. SOD activity is decreased in the synaptosomes and whole tissue extract as indicated in figures 40A and 39C respectively.

6.6 Assessment of antioxidant markers in PD brains: Focus on glutathione

The overall analysis of different redox markers in non-traumatized and injured regions of TBI brains indicated the major role of GSH depletion. This could be relevant to degenerative conditions such as PD. Hence the status of GSH could have a bearing on the integrity of the tissue and brain region. Experimental evidences in PD have demonstrated significant oxidative stress and mitochondrial dysfunction in the SN (Albers , et al. 2000, Banerjee , et al. 2009, Lin and Beal 2006). In our previous study (Mythri , et al. 2011), we observed that compared to SN, non-SN regions such as FC were protected from oxidative and mitochondrial damage due to increased GSH. In the current study, we investigated the distribution of antioxidant markers in different sub- cellular fractions in FC in the brains from cases of PD compared to controls. This is physiologically relevant since the activity in total brain extract might not reflect the status of different markers in the neuropil compartments and different fractions might respond differently to oxidative stimuli. We observed this phenomenon of differential response both in control and PD brains.

In our earlier study, we had reported that the total SOD activity in non-SN regions of the brain was unaltered in PD compared to control (Mythri , et al. 2011). However, the current study showed that the SOD activity in the FC from control brains were ~4 fold lower in the mitochondria (p<0.001) and ~10 fold lower in the synaptosomes (p<0.001) compared to the cytosolic fraction (Figure 52). Further, while the SOD activity in the cytosol and synaptosomes of FC tissue from PD cases was unaltered, mitochondrial SOD activity was found to be increased by ~ 2 fold (p=0.017) compared to control (Figure 52A).

In the control brains, total GSH displayed ~5 fold lower level in mitochondria (p<0.01) and synaptosomes (p<0.05) compared to cytosolic fraction (Figure 52B). On the other hand, we observed distinct elevation in GSH content in the synaptic terminals of the FC from PD brains. Accordingly, synaptosomes from PD brains showed ~5 fold elevation in total GSH compared to control (p=0.01), while the cytosolic GSH was elevated by ~2.5 fold compared to controls (p=0.009). Interestingly, the mitochondrial GSH content was unaltered in PD samples (p=0.23) (Figure 52B).

To investigate whether the elevation in total glutathione was associated with the conversion of GSH to GSSG, we estimated the GSSG in the neuropil fractions. The GSSG content in the control samples showed a trend similar to the total glutathione (Figure 52B and 52C), with the cytosolic GSSG significantly higher that the mitochondrial and synaptosomal GSSG (p<0.05). In the PD samples, the synaptosomes showed the highest percentage increase in GSSG (p<0.01) compared to control followed by cytosol (p<0.001), while it was unchanged in the non-synaptic mitochondria. The ratio of GSSG to total glutathione (GSH+GSSG) in the different fractions did not show any difference between the control and PD samples indicating that the elevated total glutathione was not due to conversion of GSH to GSSG (Figure 52D).

The GPx activity in the control brains was ~3 fold lower in mitochondria (p<0.001) and ~ 5 fold lower in synaptosomes (p<0.001) compared to cytosol (Figure 52C, inset). On the other hand, GPx activity in the PD tissue was elevated by ~2 fold both in the cytosol (p=0.02) and synaptosomes (p=0.02) compared to control, while it was unaltered in mitochondria (p=0.09) (Figure 53A).

GSH reductase activity in the control FC showed similar activity in all neuropil fractions (Figure 53B). However, the GR activity in the FC from PD brains was elevated by ~2.5 fold in synaptosomes (p=0.006) and by ~1.5 fold in the cytosol (p=0.03) compared to the respective controls, while it was unaltered in mitochondria (p=0.54) (Figure 53A). On the other hand, GST in the control brains showed ~6 fold lower activity (p<0.001) and >10 fold lower activity in synaptosomes (p<0.001) compared to cytosol. GST activity in the FC from PD brains was significantly elevated on in the synaptosomes by ~3 fold (p=0.0001) while it was unaltered in cytosolic and mitochondrial fractions (Figure 53C).

These data indicated that (i) the antioxidant markers showed significant alteration in control brains across different sub-cellular fractions of FC with the synaptosomes showing least activity followed by mitochondria and cytosol (ii) synaptosomes from FC of PD brains showed relatively increased GSH content and higher related enzyme activities compared to controls (iii) mitochondria from FC of PD brains showed higher SOD activity compared to controls (Table 17).

Next, we investigated whether markers of oxidative stress in different fractions showed alteration in PD compared to age-matched controls. The mitochondrial fraction showed lowered protein nitration levels (indicated by 3-nityrotyrosine or 3-NT blot) (p<0.01) and lowered protein oxidation (indicated by anti-dinitrophenyl or anti-DNP blot) in PD samples compared to controls (p<0.05) (Figure 54). Similarly, protein nitration was unaltered (p=0.5) and protein oxidation was lowered in PD synaptosomes (p<0.01) compared to controls (Figure 55). The cytosolic fraction from the FC of PD brains showed lowered protein oxidation (p<0.05), while the protein 3-NT level (p=0.13) and GFAP expression (p>0.05) were unaltered (Figure 56). These observations indicate protection against oxidative damage by elevated SOD in the mitochondria and elevated GSH content in the synaptosomes and cytosolic compartments.

Next, we investigated whether the difference in the antioxidant status between synaptosomes and non-synaptic mitochondria is associated with the quantum of mitochondrial content and function. To address this issue, we carried out mitochondrial assays including MTT, CS, SDH and MDH in both fractions (Figure 57). Based on these functional assays, we observed that the synaptosomes showed >5 fold lower mitochondrial content compared to the non-synaptic mitochondria, both in the control and PD samples.

6.7 Potential therapeutic effects of curcumin derivatives in dopaminergic neuronal cell line via elevated GSH: Implications for PD and TBI.

Oxidative stress implicated in PD pathology involves GSH depletion as the earliest event (Bharath, Hsu, Kaur, Rajagopalan and Andersen 2002). In the current study on the non-traumatized and traumatized regions from TBI patients and the FC from PD samples indicated that lowered GSH could significantly affect the neuronal function with implications for degeneration and injury. Hence, therapeutic intervention to decelerate neurodegeneration requires molecules that restore neuronal GSH levels and prevent oxidative damage. It was previously showed that curcumin protects against oxidative and nitrosative stress in vivo either by direct detoxification of toxic species or via induction of GSH synthesis (Jagatha, Mythri, Vali and Bharath 2008, Mythri, Jagatha, Pradhan, Andersen and Bharath 2007). However, the bioavailability of curcumin poses a major obstacle for its therapeutic efficacy. The synthesis and improved cellular uptake of curcumin bioconjugates have been recently reported (Dubey , et al. 2008, Mishra , et al. 2005). In the current study, we have tested the neuroprotective ability of three curcumin derivatives of curcumin (diester with demethylenatedpiperic acid or D1; diester with val or D2; diester with glu or D3) compared to curcumin against GSH depletion mediated oxidative stress in dopaminergic neurons (See figure 58 for structures of the compounds).

We utilized N27 cell line (Adams , et al. 1996), since it represents an excellent cell model to understand GSH depletion mediated oxidative stress associated with PD. N27 neuronal cells treated with BSO (0-25 pM; 24 h) showed a dose dependent decrease in the levels of total cellular GSH (Figure 59A) with 50% depletion obtained at 1.5 pM BSO and maximum decrease at 25 pM. For subsequent experiments, cells were exposed to 1.5 pM BSO for 24h to maintain ~50% depletion of cellular GSH, similar to the decreases observed in the PD SN (Bharath, Hsu, Kaur, Rajagopalan and Andersen 2002).

We confirmed the neuroprotective ability of curcumin and its derivatives against GSH depletion using the BSO-N27 model (Jagatha, Mythri, Vali and Bharath 2008). Treatment of N27 cells with curcumin or the derivatives alone (0.5 uM, 24 h) significantly increased total GSH as shown earlier (Jagatha, Mythri, Vali and Bharath 2008) with D3 showing maximum increase (~64 % compared with untreated control) compared to curcumin (30%), D1 (~25%) and D2 (~25%) (Figure 59B). However, higher concentrations of curcumin and the derivatives did not cause further increase in GSH levels (data not shown) indicating that under these conditions, 0.5 uM curcumin or derivatives is sufficient to induce maximum GSH levels in these neurons.

When N27 cells were treated with BSO (1.5 uM for 24h) followed by BSO withdrawal for 24 h, it completely restored the cellular GSH levels indicating that BSO mediated GSH depletion was specific and reversible (Figure 60). This regimen involving 24 h BSO treatment followed by 24 h withdrawal was applied in all the subsequent experiments. Pre- treatment of BSO treated N27 cells with curcumin and its derivatives (0.5 pM, 24 h) significantly restored the GSH levels compared to BSO alone (1.5 pM, 24 h) (Curcumin= 78 %, D1= 78 %, D2=72 % & D3= 83 % compared to BSO= ~50 % and Control= 100%) (Figure 60). However, post treatment (0.5 pM, 24 h) caused tremendous increase in GSH compared with the BSO alone and much higher compared to untreated control. (Curcumin= 127 %, D1= 124 %, D2= 122 % & D3= 136 % compared to control= 100% and BSO alone= 50%) (Figure 60). We observed that D3 showed higher GSH levels both in the pre and post treatment experiments compared to curcumin.

It is known that GSH depletion causes increased ROS production (Bharath, Hsu, Kaur, Rajagopalan and Andersen 2002). Since curcumin and its derivatives can restore GSH levels in the BSO-N27 model, we tested whether these derivatives could detoxify ROS more efficiently compared to curcumin. Accordingly, we measured the cellular levels of MDA (marker of lipid peroxidation), H2O2 and total ROS in N27 cells treated either with BSO (0.5 pM) alone or in combination with curcumin or its derivatives (both pre and post-treatment) compared to untreated N27 cells. Figure 4A shows that BSO treatment resulted in ~25 % increase in total MDA levels compared to untreated extracts, which was significantly decreased following BSO withdrawal for 24 h (104 %). While curcumin pre- treatment resulted in ~40 % reduction in lipid peroxidation compared to BSO alone, pretreatment with D1 and D3 resulted in ~ 50% reduction and D2 caused ~ 30% reduction in lipid peroxidation. On the other hand, post treatment with these compounds caused higher reduction in MDA levels as shown in figure 61A (65 % reduction with curcumin, 75 % with D1, 80 % with D2, 90 % with D3 compared with BSO alone). These levels were much lower than the MDA levels observed in untreated cell extracts. Such drastic detoxification of lipid peroxidation might be possible due to significantly higher GSH levels during pretreatment (as shown in fig 60). We observed that the exposure to D3 resulted in maximum reduction in lipid peroxidation compared with curcumin and other derivatives.

We surmised that the decrease in MDA levels in cells treated with curcumin and derivatives might be due to the direct involvement of cellular GST activity. Accordingly, GST assay in BSO treated N27 extracts showed ~20 % reduction in activity compared with untreated controls (Figure 61B). Further, withdrawal of BSO or incubation with curcumin and its derivatives (both pre and post treatments) restored GST activity in BSO treated cells comparable to untreated controls. However, a direct correlation between the magnitude of restoration of GST activity and the corresponding MDA values in respective treatment groups was not observed.

Estimation of H2O2 indicated ~40 % increase in BSO treated N27 cells which decreased by ~ 25 % following BSO withdrawal (Figure 61C). The H2O2 levels were further reduced (~ 50 % reduction compared to BSO alone) in cells either pre or post­treated with curcumin or its derivatives. Except for D2 (post-treatment) which showed ~ 80 % decrease in H2O2, the other derivatives showed similar reduction compared with curcumin. Nevertheless, these levels were much lower than the H2O2 levels observed in untreated cell extracts.

Similarly, ROS estimation indicated that compared to untreated controls, there was ~65 % increase in BSO treated N27 cells, which was decreased by ~30 % following BSO withdrawal (Figure 61D). The ROS levels were further decreased in cells pre­treated with curcumin or its derivatives (50 % reduction in ROS with curcumin, 55 % with D1, 70 % with D2 and 80 % with D3 compared with BSO alone). Post treatment was also able to abrogate BSO mediated ROS generation (45 % reduction in ROS with curcumin, 65 % with D1, 60 % with D2 and 90 % with D3 compared with BSO alone). As seen in the MDA experiment, exposure to D3 caused maximum scavenging of ROS compared with curcumin and other derivatives. However, cells treated with either curcumin or the derivatives alone did not show significant increase in ROS levels (data not shown).

Based on the data, we conclude that among the derivatives, D3 displayed significantly enhanced antioxidant potential against oxidative stress in dopaminergic neurons compared with curcumin.

7 DISCUSSION

7.1 Glutathione metabolism is modulated by postmortem interval, gender difference and agonal state in postmortem human brains.

Oxidative stress plays a pivotal role in various physiological and pathological phenomena in the brain. Specific proteins, enzymes and pathways are targeted for oxidative damage in brain aging and neurological disorders (Danielson , et al. 2008). Human samples employed for such analysis utilize tissues stored in brain banks. Although most nucleic acids and proteins are suggested to be reasonably stable post­mortem (Hynd , et al. 2003), any alterations in specific protein molecular markers should be conclusively proven to be caused by specific pathology and not secondary to agonal factors and PMI. Further, inherent biochemical differences in different anatomical areas of the brain could contribute to altered response to pre and postmortem factors. We observed significant variation among the anatomical areas with respect to the status of antioxidant and oxidant markers with MO demonstrating maximum variation. MO demonstrated decreased GSH level with increasing PMI (Figure 11B) which could be attributed to increased binding and detoxification of lipid peroxidation products as demonstrated by increased MDA levels with increasing PMI (Figure 12A). Interestingly, MO showed at least two-fold higher levels of MDA compared to other regions reflecting higher lipid content in that area (Figure 12C). MO also revealed increased GPx activity (Figure 14B) with agonal state compared to FC and CB which might represent a stress response or adaptive mechanism due to neuronal injury. MO also showed more than two-fold higher GPx activity compared to other regions (Figure 14C). MO also showed lower GCL activity compared to other regions (~ 50 % lower compared to CB and >7 fold lower compared to FC) (Figure 12D). Although we do not have supportive data, we speculate that increased GPx activity might contribute to GSH depletion in MO by formation of protein GSH mixed disulfides. The regional differences as displayed by MO might be due to inherent biochemical and neurochemical profile in individual anatomical areas that could be further altered by pre and postmortem factors. The oxidative changes in MO were also reflected in the gross pathological changes in that region compared to FC and CB (Figure 16). As discussed in an earlier study, (Chandana , et al. 2009), before autopsy, when the bodies are maintained at 4 [0]C, the inner regions of the brain cool more slowly than the surface thus probably contributing to regional differences in brain chemistry. It is essential to keep in mind these limitations in interpreting the biochemical parameters in different areas of brain tissue collected at autopsy.

Evaluation of the quality of human postmortem brain tissue is important for biochemical studies. Traditionally, a low PMI (Chandana, Mythri, Mahadevan, Shankar and Srinivas Bharath 2009, Harrison , et al. 1995, Lewis 2002, Trotter , et al. 2002) is characteristic of high tissue quality. Brain tissue pH also reflects the integrity of post mortem tissue (Chevyreva , et al. 2008, Hynd, Lewohl, Scott and Dodd 2003, Kingsbury , et al. 1995, Li , et al. 2004, Monoranu , et al. 2009, Perry , et al. 1982) since it is altered with prolonged agonal state and ischemic brain damage in addition to repeated freeze­thaw cycles (Grunblatt , et al. 2009, Perry, Perry and Tomlinson 1982). RNA quality is also as a marker of tissue quality (Bahn , et al. 2001, Johnston , et al. 1997, Miller , et al. 2004, Preece , et al. 2003, Stan , et al. 2006, Tomita , et al. 2004, Weis , et al. 2007, Yasojima , et al. 2001) which is significantly affected by tissue pH, mode of death (Chevyreva, Faull, Green and Nicholson 2008) and increasing PMI (Birdsill , et al. 2010, Pardue , et al. 1994). Increased tryptophan is also correlated with PMI and storage time (Grunblatt , et al. 2009, Grunblatt , et al. 2010, Perry, Perry and Tomlinson 1982). These data concur with our study which emphasizes that PMI should be short and the body should be stored at 4°C until autopsy in order to preserve brain tissue quality.

Most studies related to the effect of pre and postmortem factors have concentrated on gene expression studies and RNA stability but studies on the postmortem stability of individual proteins/enzymes are limited. One such study comparing the brains between healthy subjects and those dying after a prolonged illness indicated significant reduction in the enzymes glutamate decarboxylase and phosphofructokinase (Perry, Perry and Tomlinson 1982). Previous study from our group showed that SN displayed alterations in the levels of neurofilament and GFAP compared to FC with PMI, while synaptophysin levels were unaltered (Chandana, Mythri, Mahadevan, Shankar and Srinivas Bharath 2009). Activities of the enzymes methyltransferase and acetyltransferase in the brain were relatively preserved with longer PMI and storage duration (Monoranu , et al. 2010). Another study reported degradation of specific proteins related to metabolic, structural, stress response, antioxidants and synaptosomal function induced by artificial PMI and storage temperature (Crecelius , et al. 2008). Vascular endothelial growth factor (VEGF) level in the brain altered with PMI (Thaik-Oo , et al. 2002). It is possible that not all biochemical markers in postmortem brains are altered in the similar fashion in response to PMI.

In a previous study evaluating the effect of PMI on protein stability (Chandana, Mythri, Mahadevan, Shankar and Srinivas Bharath 2009), samples from subjects who had succumbed to spinal cord injury but with relatively normal structure was analyzed. The other samples were from subjects who had sustained head trauma, though the regions used for the study studied were unaffected and anatomically away from the site of injury. Comparison of data in these two sub groups did not indicate any direct influence of tissue injury on the biochemical parameters in the brains sampled. The current study was carried out on brain samples obtained from subjects who succumbed to TBI. Although the regions selected were farthest from the site of primary injury, it is possible that the agonal state represented by GCS score might influence the markers tested in the study. Accordingly, we observed among the antioxidant and oxidant markers, GR activity in CB (Figure 13C) and GPx activity in MO (Figure 14C) were altered with increased severity of brain injury. These data suggest that following primary brain injury, secondary biochemical events in remote anatomical areas might be induced.

The increased GPx activity in MO with severe agonal state (Figure 14B) could be a stress response to increased cellular H2O2 levels due to very low GSH content compared to FC and CB. Increased GR activity in CB with agonal state (Figure 13B) could be explained as follows. Analysis of MDA in different areas with agonal state (Figure 12B) showed maximum (but statistically insignificant) increase in CB compared to the other regions tested. We hypothesize that the slight increase in MDA could be detoxified by reduced GSH. This might alter the GSH/GSSG ratio which could trigger an increase in GR activity such that the ratio and the redox state are maintained to prevent further damage.

With reference to gender based differences, only total GSH showed increasing trend in all the tested anatomical regions in females compared to males with significant elevation in CB and MO (Figure 10D). This might indicate increased antioxidant load with potential role in neuroprotection in the female brain. Increased GSH level could be partly explained by increased trend in the GCL activity in females compared to males in all the regions (Figure 12D). This is supported by Wang et al (2003) who observed that male mice displayed more dramatic age-associated change in GSH content than did female mice in many tissues. On the contrary, Ferris , et al. (1995) proposed that GSH levels are gender-independent. Although gender significantly contributes to aging, lifespan and pathology, the importance of gender in free radical mechanisms is not completely understood. Differences in ROS homeostasis contribute to gender divergence in survival (Ali , et al. 2006). Female mouse brain has lower oxidant and higher antioxidant capacity (Sobocanec , et al. 2003) indicating that gender affects the oxidative protein damage in the brain thereby contributing to its aging (Uzun , et al. 2010). Analysis of oxidative stress parameters in the female rat brain after gonadectomy indicated that protection against oxidative stress is afforded by ovarian sex hormones, thus decreasing the potential for oxidative cell damage in females compared to males (Kume-Kick , et al. 1996). Males may be more susceptible than females to stress induced neurobehavioral changes and free radicals may exert a regulatory influence in such gender dependent responses to stress (Chakraborti , et al. 2007). Consequently, gender difference has a bearing on the susceptibility to disease. Gender affects hypertension- associated oxidative stress, lipid and protein damage, apoptosis in heart and brain tissues (Ren 2007). Gender differences in oxidative stress parameters might be associated with higher prevalence of AD in females (Schuessel , et al. 2004).

7.2 Effect of Storage Time, Postmortem Interval, Agonal State, and Gender on the Postmortem Preservation of Glial Fibrillary Acidic Protein and Oxidatively Damaged Proteins in Human Brains

Since postmortem human tissue is extremely important for neuroscience research, several approaches have been adapted to evaluate the markers of tissue quality. Conventionally, a low PMI (Chandana, Mythri, Mahadevan, Shankar and Srinivas Bharath 2009, Harrison , et al. 1995, Lewis 2002, Trotter, Brill and Bennett 2002) is considered to ensure high tissue quality. Apart from this, brain tissue pH also reflects the integrity of post mortem tissue since it is directly related to the divergent physiological state and resultant metabolic changes ante mortem and mode of death (Chevyreva, Faull, Green and Nicholson 2008, Hynd, Lewohl, Scott and Dodd 2003, Kingsbury , et al. 1995, Li , et al. 2004, Monoranu , et al. 2009, Perry, Perry and Tomlinson 1982). Brain tissue pH value is altered with prolonged agonal state and ischemic brain damage additionally to repeated freeze-thaw cycles (Grunblatt , et al. 2009, Perry, Perry and Tomlinson 1982). Since different anatomical areas of brain have distinct physiology, neuronal connections with varied cellular dynamics and neurotransmitter profiles, we measured pH in FC-cholinergic, CB- GABAergic and MO and found that the pH values did not show significant variance with storage time, GCS rating, PMI and between males and females (data not shown) consistent with recent reports (Grunblatt , et al. 2010). Interestingly, we found that 12 samples showed tissue pH <6.0 which represents significantly acidic pH. When we analyzed the status of the antioxidant and oxidant markers in those samples in the continuous pH range 5.45 to 6.0, we found that the low pH did not significantly affect any of the markers tested in the study (data not shown).

Selective oxidative and nitrative modifications of specific brain proteins might contribute to the progression of neurodegenerative diseases such as PD. For e.g., mitochondrial complex I isolated from the postmortem samples of PD patients shows increased carbonyl levels compared to age-matched controls (Keeney, Xie, Capaldi and Bennett 2006). Similarly, oxidative damage of DJ-1 protein has been linked to sporadic PD and AD based on analysis of human samples (Choi , et al. 2006). However, the role of pre and post-mortem factors on the status of protein oxidation in the human brain samples needs to be delineated for interpretation of the results. In our previous study (Chandana, Mythri, Mahadevan, Shankar and Srinivas Bharath 2009), enhanced protein carbonyl formation was not detected with increasing PMI in all the brain regions studied. Similarly, in the current study, there was no significant effect of PMI and agonal state on protein oxidation in all the three regions tested confirming our previous data (Figure 17). However, with increasing storage time, we observed significant loss of protein oxidation signal in CB and MD (Figure 17). The decreased western signal might be due to the masking of epitopes or degradation/ aggregation of oxidized proteins or other unknown mechanisms that might affect the phenomenon.

Protein oxidation level was significantly lower among female brains in CB compared to males indicating a gender-specific alteration in protein stability and oxidation status (Figure 17). Although gender difference has been implicated in brain aging, lifespan, survival and pathology, the importance role of gender in reactive oxygen species-dependent processes is not well characterized (Ali , et al. 2006). Consistent with our data, other studies using animal brains have recorded significant antioxidant load and lowed oxidative damage in female brains (Sobocanec, Balog, Sverko and Marotti 2003); (Uzun, Kayali and Cakatay 2010). Analysis of female rats after gonadectomy indicated that protection in the female brain against oxidative stress is probably mediated through ovarian sex hormones (Kume-Kick, Ferris, Russo-Menna and Rice 1996). Males are more susceptible than females to neurobehavioral changes induced by stress which in turn might be related to oxidative damage (Chakraborti, Gulati, Banerjee and Ray 2007). Gender difference has a significant influence on susceptibility to diseases (Ren 2007) including neurodegenerative disorders (Schuessel, Leutner, Cairns, Muller and Eckert 2004).

Most of the studies carried out to determine protein nitration (3-NT) have concentrated on the role of this process in cellular damage during physiological aging (Gokulrangan , et al. 2007)and neurodegenerative disorders (Pennathur , et al. 1999, Smith , et al. 1997) and during mitochondrial dysfunction (Bharath , et al. 2005, Clementi , et al. 1998, Murray , et al. 2003, Mythri, Jagatha, Pradhan, Andersen and Bharath 2007). For example, a-synuclein the major protein component of lewy bodies in PD undergoes protein nitration as determined by studies with the postmortem samples and experimental models of the disease (Good, Hsu, Werner, Perl and Olanow 1998, Przedborski , et al. 2001) and this is directly correlated with PD pathology. Similarly another protein, Parkin is suggested to be nitrosylated in animal models and human samples of PD (Chung , et al. 2004). However, based on our study, it needs to be further validated whether the nitration status of proteins obtained from studies using human postmortem samples indeed represents pathology or other non-disease related parameters such as vulnerable neuroanatomical areas affected, PMI, storage time and gender of the deceased. In our previous study (Chandana, Mythri, Mahadevan, Shankar and Srinivas Bharath 2009), we observed enhanced 3-NT levels with increasing PMI in substantia nigra (SN) unlike other areas and this could be a secondary effect due to the localization of dopaminergic system and neuromelanin that makes SN vulnerable to oxidative and nitrosative stress (Bharath, Hsu, Kaur, Rajagopalan and Andersen 2002). In the current study, we did not detect any change in the status of nitrated proteins with increasing PMI and agonal state in the three anatomical areas selected (Figure 18). However, MD region showed significant decrease in protein nitration signal with increasing storage time. Similar to the scenario regarding protein oxidation, it is possible that in MD, due to prolonged storage time, protein 3-NT epitopes are masked or degraded due to protein degradation or the nitrated proteins might be aggregated thereby forming insoluble deposits (Figure 18).

In an earlier study (Chandana, Mythri, Mahadevan, Shankar and Srinivas Bharath 2009), marginal but statistically insignificant increase in GFAP expression in SN with increasing PMI in contrast to other anatomical areas was observed, reflecting probable regional differences among the samples. Similarly, in the current study, we observed no difference in the GFAP signal with increasing PMI and agonal state. However, similar to the data regarding protein oxidation and nitration, GFAP levels were altered with increasing storage time in MD unlike FC and CB (Figure 19). We surmise that with storage, the GFAP, a fibrillar protein aggregates and this needs to be further investigated using different cellular fractions.

In conclusion, this section was aimed to understand whether pre and postmortem factors had a bearing on the postmortem preservation of oxidized and nitrated brain proteins and GFAP in three anatomical areas of postmortem human brain tissues stored for long periods. Tissue pH was not affected by the pre and postmortem factors indicating fairly well-preserved tissue quality for the study. Interestingly, storage time had a significant bearing on the stability of oxidized and nitrated proteins and GFAP reflecting alteration in the tissue quality and preservation dependent protein structure-function which needs to be kept in mind in biochemical studies using brain tissues. However, other factors such as PMI, agonal state and gender difference did not significantly influence the markers tested in our samples. Such studies aimed at analyzing individual markers are required to substantiate the reliability of biochemical studies using postmortem samples to understand the biochemistry of physiological and pathological events in the brain.

7.3 Effect of Premortem and Postmortem Factors on the Distribution and Preservation of Antioxidant Activities in the Cytosol and Synaptosomes of Human Brains

The differential distribution of antioxidant markers in subcellular compartments must to be studied to understand its influence on brain function and pathology. Among these markers, intracellular distribution of total GSH has been studied extensively in animal models. Schnellmann , et al. (1988) examined intracellular distribution of total GSH in rabbit renal tubules and found that irrespective of the in vivo GSH levels in the tubules, the mitochondrial GSH pool was unaltered. Although the mitochondrial GSH concentration accounted for ~ 10% of the total cellular GSH, depletion of total GSH in the tubules caused selective depletion of the cytosolic pool without affecting the mitochondrial GSH pool. Further, > 40% loss of mitochondrial GSH caused mitochondrial dysfunction, reflecting complicated GSH dynamics between cytosol and mitochondria and their role in mitochondrial function. In a related study Roychowdhury , et al. (2003) studied whether the mitochondrial and cytosolic GSH are differentially affected by endogenous nitric oxide (NO) in microglial cultures. Activated inducible nitric oxide synthase (iNOS) significantly depleted total GSH but the mitochondrial GSH was relatively unaffected. NO-mediated oxidative damage and mitochondrial dysfunction were more pronounced in cells where mitochondrial GSH was depleted compared to cytosolic GSH. Further, endogenous NO production did not compromise the mitochondrial GSH pool which, in turn, might explain the ability of these cells to resist the damaging effects of high-output NO production. Previously, we demonstrated that total GSH content in whole tissue extracts of FC from postmortem human brains was unaffected by premortem and postmortem factors. Similarly, total GSH in mitochondria and synaptosomes in FC from human brains was unaffected by premortem and postmortem factors.

In contrast, cytosolic GSH was significantly decreased with increasing age and PMI (Fig. 21). This is consistent with a previous study which investigated the effect of GSH depletion on the viability of synaptosomes during brain aging in mice. Although aging did not influence the GSH and ATP levels and the viability of mouse synaptosomes, depletion of cytosolic GSH decreased the ATP levels and viability of synaptosomes in aged mice but not in young animals. These results emphasize the importance of the cytosol GSH for the maintenance of the membrane integrity of synaptosomes during brain aging in mice. The antioxidant activities in cytosol and synaptosomes were mainly affected by age and to a lesser extent by other premortem and postmortem factors. Gender had the least effect on antioxidant activities tested (Table 9). On the other hand, other antioxidant activities such as SOD, GPx, and GST were altered in the subcellular fractions with increasing age. We observed opposite trends in the GPx activity with increasing age, with declining activity in cytosol probably compensated by an increase in synaptosomes (Fig. 22D).

The current study also observed several fold lower antioxidant activity in synaptosomes compared to cytosol. All activities were * 5 fold higher in the cytosol compared to synaptosomes, while GR activity was increased by ~20% in cytosol. This also indicates the selective vulnerability of synaptosomes to oxidative damage with implications for pathogenesis of degenerative pathology. This was explained by a recent study, which quantitated the distribution of oxidant and antioxidant markers in various subcellular (neuropil) fractions of FC from control, mild cognitive impairment (MCI), moderate AD, and advanced AD human brains (Ansari and Scheff 2010). All the neuropil fractions (cytosol, mitochondria, and synaptosomes) demonstrated pathology­dependent elevation in oxidant markers and depletion of antioxidant activities, but the highest oxidative damage was observed in synaptosomes. This might be considered as the mechanistic basis underlying the selective targeting of synaptosomes during neurodegeneration.

Table 9 shows the distribution of antioxidant activities in whole brain extracts and different subcellular fractions in FC of postmortem human brains. Although the antioxidant activities in the whole extracts of FC remained unaffected by premortem and postmortem factors, the subcellular fractions revealed significant alterations. Hence the data from whole cell or tissue extracts should be used cautiously, as it may not accurately reflect the changes in subcellular structures. For instance, while the SOD activity was unaltered with increasing age in total extracts of FC, the activity increased with age in synaptosomes (Fig. 20D). Similarly, the GSH level which was unaltered in the total extracts of FC with increasing age and PMI was significantly decreased in the cytosolic fraction from the same samples indicating that this decrease was masked due to the other fractions (Figs. 21B and 21D). GPx activity in cytosol and synaptosomes was altered with age, while this was not reflected in the whole extracts (Fig. 22D). Similarly, the GST activity which was significantly increased in the whole extracts with increasing age was due to elevation only in the synaptosomes compared to the cytosolic and mitochondrial fractions (Fig. 24D and Table 9). In some instances, the activity in total extract could be correlated with the opposing trends in subcellular fractions. For example, GPx activity revealed decrease in cytosolic and increase in synaptosomal fraction with age, while total extracts showed the activity to be unaltered (Fig. 22D). Interestingly, gender of the subjects did not influence any of the activities tested in the current study. In our previous study (Harish , et al. 2012), we observed that the activities of mitochondrial enzymes, such as citrate synthase and succinate dehydrogenase, were significantly higher in brains of female subjects compared to males.

In conclusion, the current study combined with the previous data reveal a complex distribution of antioxidant activities in FC of postmortem human brain samples which could also differ in various anatomical areas. Further, these activities could be influenced by some premortem and postmortem factors, and the effect could be different in different subcellular fractions. Compared to cytosol, the synaptosomes showed several fold decrease in antioxidant activity which might make it vulnerable to oxidative stress and contribute to evolution of neurodegenerative diseases.

7.4 Region-specific changes in redox status of human brains: Vulnerability to degeneration and trauma:

It has been suggested that that different anatomical regions in the mammalian brain show varied redox status and response to oxidative stress (Wang , et al. 2010). While certain anatomical regions are resistant to increased oxidative stress, certain others are more vulnerable. Analysis in the kainic acid model demonstrated selective vulnerability of different brain regions to oxidative damage (Candelario-Jalil , et al. 2001). However, there was no correlation between the lowered antioxidant status in regions with elevated markers of oxidative damage. Regions exhibiting differential redox status could display functional decline, cell death and age-associated degeneration (Wang and Michaelis 2010). For instance, in AD, there is a particular pattern of building up of oxidative stress in the hippocampus starting with the CA3, progressing to CA1 and then to other hippocampal and cortical areas (Cruz-Sanchez , et al. 2010). Mandal , et al. (2012) correlated the GSH content in the anatomical regions of healthy subjects and AD patients. Accordingly, parietal cortex had highest GSH while hippocampus had the lowest in healthy controls. AD patients displayed lower GSH in the right frontal cortex of AD patients compared to female controls while the left frontal cortex showed lower GSH compared to male controls. In our study, redox markers showed significant alteration across different brain regions (Figure 25-28). Total GSH was highest in CB followed by FC, while CD displayed ~ 25 % GSH compared to CB. On the other hand, HC and MO showed only ~5 % total GSH while MB had ~ 2% GSH.

Since aging is correlated with the lowered redox status of different regions, it is possible that different anatomical regions could show altered redox status when compared among different regions of the same age group. Such analysis in our study indicated that the pattern of the relative distribution of antioxidant markers in different brain regions was similar between young and old brains for GSH, GPx, SOD and GST (Figures 25, 26). On the other hand, GR and catalase showed variation in the relative distribution of antioxidant markers in different brain regions between young and old brains (Figure 25). Similarly, oxidant markers and GFAP signal (Figures 26-28) showed similar pattern in different brain regions between young and old brains. These data indicate that each biochemical pathway and marker marker in the brain could have specific pattern that could be maintained uniformly or unevenly across different ages. This emphasizes the existence of “biochemical/molecular signature” in the brain regions that could contribute to the anatomical heterogeneity of the cellular parameters.

This heterogeneity could also be contributed by physiological/ pathophysiological/ toxic stimuli. In human glioma, there are significant metabolic and mitochondrial differences and oxidative susceptibility of different cell populations of the tumor tissue (Santandreu , et al. 2008). The cancer cells showing higher antioxidant capacity within the tumor could resist chemotherapy and radiotherapy. Analysis of the susceptibility of neuronal from different brain regions to unconjugated bilirubin (UCB) indicated that hippocampus displayed highest ROS, lowered GSH and increased cell death compared to cortex and cerebellum (Vaz , et al. 2011). Hence oxidative injury and disruption of neurite network might increase the vulnerability of hippocampus to UCB. Interestingly, local differences in GSH content could account for different susceptibility between brain regions exposed to UCB. The factors contributing to selective susceptibility could be increased requirement for ROS signalling, low ATP content due to mitochondrial damage, elevated inflammatory response, impaired DNA repair, calcium dysregulation and excitotoxicity (Wang and Michaelis 2010). According to a study by Diedrich , et al. (2011), the mitophagy rate (mitochondrial damage and turnover) of a particular region could contribute to selective neuronal vulnerability in PD and this was most prominent in the midbrain. Understanding the basis of this disparity among the regions would help in developing interventions to protect such neuronal population (Wang and Michaelis 2010).

7.5 Role of oxidative stress in the injured regions in TBI

Although oxidative stress has been associated with TBI, most of the studies are carried out either in animal models or in the CSF/plasma samples of TBI patients. Comparative study of redox markers in the contused region vs. pericontusion TBI brains and controls are non-existent. Our study demonstrated extensive oxidative damage evidenced by altered redox markers in whole extracts and sub-cellular fractions from TBI brains thereby confirming the previous reports regarding the adverse role of ROS, antioxidant depletion, excitotoxicity and protein damage in TBI (Bayir , et al. 2005, Chong, Li and Maiese 2005). Elevated ROS has been detected in experimental TBI immediately after injury (Fabian, Dewitt and Kent 1998). Our study also showed consistent decrease of antioxidant enzymes (e.g., SOD and GSH metabolic enzymes in the contused sample (Figure 39) and these effects were profoundly affected by age (Figure 37). Temporal decrease in antioxidant enzymes such as SOD at 24 h after severe TBI has been reported (Cernak , et al. 2000).

Elevated lipid peroxidation was documented in severe TBI (Bayir , et al. 2004)based on the study on the CSF and plasma samples from neurotrauma patients (Clausen et al, 2012). Elevated MDA content could be prominently detected in plasma and CSF within 48-72h after injury (Bayir , et al. 2002). Quantitative lipidomics indicated elevated oxidation of lipids in TBI (Tyurin , et al. 2008). Our study showed elevated lipid peroxidation in contused tissue from TBI patients with increasing age. However, the status of lipid peroxidation was unchanged when correlated with either GCS, severity of the injury or midline shift.

Several studies have showed the implication of PN in post-TBI pathophysiology. Isoforms of NOS are known to be up-regulated during the first 24 h after TBI in rodents (Gahm, Holmin and Mathiesen 2000). The acute treatment of injured mice or rats with NOS inhibitors found to have a neuroprotective effect, establishing the neurotoxic role of PN after TBI (Wada, Chatzipanteli, Kraydieh, Busto and Dietrich 1998). PN-mediated damage has been documented in rodent TBI models including an increase in 3-NT levels and ADP ribosylation (Mesenge , et al. 1998). Further, elevated iNOS expression and elevated 3NT is associated with brain infarcts (Forster , et al. 1999). However, nitrosative stress did not have significant effect in our study since the status of total protein nitration was unchanged in the TBI tissue (Figure 41B, 42B, 43B). Bayir et al (2007) reported that MnSOD is a target of tyrosine nitration leading to lowered activity after TBI in mice and human subjects probably via PN. The data obtained from our study also suggested lowered SOD activity in the TBI samples (Figure 39C, 40A) which was probably due to decreased expression (Tables14, 15).

Peroxide metabolizing proteins such as Peroxiredoxin-5 expression are elevated in degenerative conditions such as multiple sclerosis (Holley , et al. 2007). Our study indicated that Peroxiredoxin, 1, 4, 5 and 6 were unchanged in the pericontusion. On the other hand, expression of Peroxiredoxin-2 and 4 was increased in contusion (Table 13) while the expression of peroxiredoxin 1, 5 and 6 were unchanged. However, western blot did not indicate elevated expression of peroxiredoxin-2 (Figure 50D).

The most common redox marker to be decreased in our study both in whole extracts from contusion was total GSH (Figure 39F). Total GSH was significantly decreased both in the synaptosomal and mitochondrial fractions both in contusion and pericontusion (Figure 40B) indicating that global depletion of GSH could contribute to TBI. This is supported by Lok , et al. (2011), who reported that gamma-glutamylcysteine ethyl ester (cell-permeable precursor of GSH) provided cytoprotection during injury and decreases blood-brain barrier permeability after experimental TBI.

Our study also indicated that elevated oxidative stress could lead to mitochondrial damage as indicated by lowered activity of metabolic enzymes (Figure 44). Mitochondrial dysfunction after experimental and human brain injury has been previously reported (Verweij , et al. 1997).

7.6 Proteomic analysis in TBI

Technical advances, especially in the field of proteomics have provided insights into novel processes and biomarkers in TBI (Papa , et al. 2008). This could assist in improved patient care, diagnostics and management of TBI (Zupanc 2007). More studies are required to analyze the relationship between biomarkers and severity of injury and clinical outcome in TBI patients (Papa , et al. 2008). There are reports regarding proteomic analysis in TBI, but these are limited to animal models and CSF/plasma samples intended to develop of diagnostic assays (Oli , et al. 2009).

Since the composition CSF changes following TBI, proteomic analysis of CSF could provide novel biomarkers for TBI. Accordingly, 2D analysis followed by MS analysis of TBI CSF vs. controls detected elevation in acute response proteins alpha 1 antitrypsin, haptoglobin 1 alpha 1, alpha2 and beta and other markers of fibrinolysis (Conti , et al. 2004). In another study on CSF proteomics in pediatric TBI patients (Gao , et al. 2007), proteins such as haptoglobin, prostaglandin synthase and cystatin were several fold increased in TBI. In an interesting proteomics study on TBI CSF (Sjodin , et al. 2010) using hexapeptide ligand libraries, shotgun proteomics and nano-LC-MS/MS analysis, 339 unique proteins were identified of which there were many proteins associated with degeneration/regeneration including neuron specific enolase, GFAP, creatine kinase B-type and s100 beta were detected in the study.

Using 2D gel and MS analysis Boutte , et al. (2012) identified 386 proteins with > 2 fold change of which 321 proteins were increased and 65 were decreased 24 h after injury. Most up regulated proteins were cytoskeletal, nuclein acid binding or kinases and involved in protein metabolism, signal transduction, development with role in neurite outgrowth and cell differentiation. Among these proteins, ubiquitin carboxyl- terminal hydrolase isozyme L1, tyrosine hydroxylase and syntaxin-6 were elevated in brain and CSF after PBBI. The only proteomic study on the human cortex after TBI (Yang , et al. 2009) indicated 138 overexpressed proteins associated with cytoskeleton, metabolism, electron transport, signal transduction, stress response, protein synthesis and turnover, transporter, cell cycle etc. However, high-throughput analysis using human brain tissue samples are non-existent.

According to our knowledge the current study is one of the first carried out on TBI brains with an intention to identify and quantitate differentially expressed proteins specific to contusion and pericontusion. Our study identified several differentially expressed proteins with 402 proteins over expressed in contusion, 73 proteins in pericontusion, while 119 and 84 proteins were under-expressed in contusion and pericontusion respectively (Figure 46, Table 13-16). It is interesting to note that there were proteins common to pericontusion and contusion, while there were proteins over expressed exclusively in the contusion or pericontusion (Figure 46C, D). The following section aims to discuss some of the specific proteins differentially expressed during TBI:

a. SOD: Our data indicated overall increase in oxidative stress as observed by down-regulation of SOD1 [Cu-Zn] and SOD2 [Mn] (Table 14, 16). We observed a sustained decrease in enzyme activity of SOD in the synaptosomal fraction and whole tissue extracts of injured brain tissue (Figure 39C, 40A). Other studies have also reported decrease in SOD activity without an alteration in the protein expression after TBI both in mice and humans (Bayir , et al. 2007). Peluffo , et al. (2005)have shown a strong neuronal down-regulation of SOD1 [Cu-Zn] following excitotoxic injury, which is relevant since many studies have demonstrated excitotoxic damage in TBI models (Moritani , et al. 2005, Palmer , et al. 1993). Decreased SOD2 expression has been reported after experimental focal cerebral ischemia in rats and gerbils (Bemeur , et al. 2004). Although the mechanism by which this down-regulation occurs is not clear, it appears that it could be mediated by oxidative stress, as PC12 cells treated with H2O2 rapidly down-regulate SOD [Cu/Zn] (Rojo , et al. 2004). SOD provides the first line of defense against (V- generated in mitochondria. SOD competes with NO for reaction with (V- and prevents generation of PN. Bayir , et al. (2007)has shown that SOD [Mn] as a target of nitration that is associated with a decrease in its enzymatic activity in mouse model TBI.

b. Synaptophysin (SYP): Since TBI pathophysiology involves brain damage and degeneration, expression and distribution of SYP is hypothesized to significantly change. Our data indicated 6.4 fold in contusion and 4.2 fold increase in SYP expression (Figure 51C, D). SYP, a major integral transmembrane protein of synaptic vesicles, provides a molecular marker for the synapse. Several studies indicated that SYP regulates endocytosis and exocytosis of neurotransmitters and participates in the formation and recycling of synaptic vesicles (Daly , et al. 2000). Densitometric quantitation of SYP western blot indicated significant increase in SYP levels in contusion and pericontusion regions compared to age matched controls (Fig 51). SYP immunoreactivity increased in the injured cortex and in the subcortical white matter with increasing severity of injury and time after trauma in rodent model of TBIShojo , et al. (2006). Increased SYP staining was accompanied with degeneration of neuronal cell bodies, their processes and terminals. Since TBI results in neural cell body atrophy and necrosis, synaptic terminals would also detach from degenerated neuronal cell bodies in the cortex (Murdoch , et al. 1998). Our study observed extensive ischemic changes in the neuronal cell body with vacuolation around cell body and dendrites indicating loss of synaptic connections (Fig 33, 34). Previous data Shojo and Kibayashi (2006) indicated increased SYP immunoreactivity in the cerebral cortex following TBI reflected inhibition of synaptic vesicle transportation and dysfunction of synapse, thus providing a histological substrate for brain dysfunctions. These results were consistent with our study where we observed down-regulated synaptic vesicle cycle proteins- SNAP25, Syntaxin and Complexin (Fig 49). Increased SYP in the injured region could also be interpreted as an injury induced neuronal repair response and synaptogenesis. Thompson , et al. (2006)found prolonged and sustained increases in SYP levels in the ipsilateral hippocampus following moderate CCI-TBI. Stroemer , et al. (1995) also demonstrated elevated SYP levels in the neocortex following permanent focal ischemia. Through electron microscopic studies they observed increased synaptogenesis and synaptic reorganization.

c. Chitinase: Chitinase and chitinase like proteins are members of mammalian family of glycoside hydrolases found to have a role in inflammation and tissue remodeling. They are expressed and secreted by chondrocytes, synovial cells, neutrophils, and macrophages during differentiation. Chitinase-like proteins have no chitin hydrolyzing activity and exert their biological effect through protein-protein or protein-carbohydrate interactions (Lee , et al. 2011). Our study reported significant up­regulation of Chitinase 3-like protein 1 (CHI3L1) and CHIT1, and the same was validated by western blot in contusion and pericontusion regions compared to controls (Figure 50A, B). Bonneh-Barkay , et al. (2010) demonstrated an increased CHI3L1 levels in the CSF of TBI patients which peaked at 4 days after injury. Further, there is a significant correlation between CSF CHI3L1 concentrations and the inflammatory cytokines IL-1P and TNF-a in the CSF. Increased plasma chitotriosidase was associated with increased macrophage activation and severity of stroke in patients with acute ischemic stroke (Sotgiu , et al. 2005). Increased CHI3L1 was also found in astrocytes in the pericontusion of the injury site and was associated with inflammation, as the inflammation resolved CHI3L1 expression also diminished (Bonneh-Barkay , et al. 2012). We could correlate inflammation with chitinase expression, as we found temporal changes in inflammation through oedematic changes in pericontusion region, (also, inflammation was evident with activation of microglia) (Figure 33, 36). Oedema in the pericontusion zone was decreased with increase in injury-surgery interval (4 hrs to 24 hrs; data not shown). Further experiments are required to establish the observed changes.

d. GFAP: In response to the injury, astrocytes up-regulate the expression of GFAP, which largely contributes to the reactive gliosis after brain injury. Several studies have demonstrated gliosis in TBI (Liu , et al. 2012). Buritica , et al. (2009) observed GFAP immunoreactivity in the white matter, in the gray-white matter transition, and around the perineural sectors with total neuronal loss in TBI. These findings may reflect dynamic activity as a consequence of the lesion that is associated with changes in the excitatory circuits of neighboring hyper-activated glutamatergic neurons, possibly due to the primary impact, or secondary events such as hypoxia-ischemia. We have also observed intense GFAP accumulation in a plaque like fashion in the contusion and pericontusion regions; these reflected an increase in synthesis of the fibrillary protein confirmed on Western blots (Fig 51A, B). Contribution of GFAP to TBI pathology is widely investigated. For instance, gliosis is observed as a cause for epileptogenesis as observed in TBI patients post injury (Buritica , et al. 2009). GFAP, is also a promising marker for TBI. Increased GFAP in serum was also found to predict poor outcome in TBI patients. After severe TBI, high levels of GFAP (>1.5microg/l) is strongly predictive of death or a poor outcome (Vos , et al. 2004). A correlation between serum GFAP levels and severity of both injury and outcome has been documented (Nylen , et al. 2006, Pelinka , et al. 2004).

e. Others: TBI initiates neuroinflammation which results in astrocyte and microglial activation and increased production of immune mediators. Following TBI there is infiltration of immune cells, in particular leukocytes; however, both astrocytes and microglia participate in mounting neuroinflammation, (observed by Iba 1 and HLA DR +ve cells; Figure 36). Our proteomic observations indicated that 5.8% of the up regulated proteins were immune response proteins. Immune response HLA class protein like HLA-C (7.4 fold in contusion), HLA-G (2.6 fold in contusion), HLA-A (2.1 fold in contusion). Complement protein Complement factor B’ was 2.5 fold over­expressed in contusion. We also observed through immunohistochemistry that CD68, a lysosomal protein expressed by microglia/macrophages was seen only in the injured cortex and not in the controls. Consistent with our results, Johnson , et al. (2013) observed extensive, densely packed, reactive microglia (CR3/43- and/or CD68- immunoreactive), in TBI brains and the pathology was not seen in control subjects. Many studies have consistently observed microglial activation (HLA DR +ve) in TBI (Hernandez-Ontiveros , et al. 2013).

In humans, vascular margination is seen incontusions by 24 h, and by 3-5 days there is monocytelymphocyteand lymphocyte infiltration, and both astrocytic and microglial activation (Holmin , et al. 1998). Microglial cells and macrophages can be seen associated with contusions (Gentleman , et al. 2004). Immunohistochemical studies of contusions frompost mortem brains have demonstrated increasing pericontusional CD68 immunoload, a marker of phagocytic activity, with survival time (Gentleman , et al. 2004).

7.7 Alteration in the glutathione metabolism in synaptic terminals but not in synaptic-free mitochondria in the frontal cortex of Parkinson's disease brains.

Even as previous studies have correlated the redox dynamics (Alam , et al. 1997, Alam , et al. 1997, Dexter , et al. 1989, Dexter , et al. 1989, Floor and Wetzel 1998, Good, Hsu, Werner, Perl and Olanow 1998, Jenner , et al. 1992, Sofic , et al. 1991, Yoritaka , et al. 1996) and mitochondrial status (Keeney, Xie, Capaldi and Bennett 2006, Schapira , et al. 1990) in PD brains compared to controls, the sub-cellular distribution of antioxidant markers in PD brains need to be investigated, as such studies in human samples are non-existent. The current study compared non-synaptic mitochondria, synaptosomes (which represent synaptic terminals) and cytosolic fractions from human PD brains. While the total extracts from the human brain represent neuronal and non-neuronal cells, analysis of synaptosomes represents only the neuronal physiology devoid of glial and vascular endothelial contribution. Since the bioenergetic capacities of neurochemically different synaptosomes do not vary significantly (Choi , et al. 2011), the synaptosomes used in the current study could be considered as a homogenous population. We observed that the synaptosomes and mitochondrial fractions in the control FC showed several fold lower antioxidant activity compared to cytosol (Figures 52 and 53). This is in agreement with our previous study (Harish , et al. 2012), which showed that the antioxidant activities such as SOD, GPx and GST in the neuronal cytosol and synaptosomes were altered by age. The neuropil fractions from subjects with mild cognitive impairment and AD displayed elevated oxidative stress and antioxidant depletion, with the highest oxidative damage observed in the synaptosomes (Ansari and Scheff 2010). While these data indicate the selective vulnerability of synaptosomes, elevated GSH and related activities in FC of PD brains (Figures 52 and 53) might be neuroprotective, consistent with a report (Pocernich , et al. 2001) that elevated brain GSH protected synaptosomes against acrolein-induced damage. While higher GSH content and related enzyme activities might provide the major antioxidant defense in the synaptosomes, elevated SOD activity might play an important role in the non- synaptic mitochondria. Interestingly, most of the other activities were relatively unaltered in the non-synaptic mitochondria (Figure 52 and 53). Two previous studies (Roychowdhury, Wolf, Keilhoff and Horn 2003, Schnellmann, Gilchrist and Mandel 1988) showed that under conditions that induce cellular GSH depletion, irrespective of the in vivo GSH levels, the mitochondrial GSH pool was unaltered thus signifying the mitochondrial mechanism underlying the ability to counter oxidative stress.

These results also indicate that the data from whole cell or tissue extracts might not reflect the status of different activities in individual compartments. Hence biochemical activity from total cell extracts should be interpreted cautiously. In a relevant study on aging human brains (Harish , et al. 2012), we showed that while the SOD activity and GSH content were unchanged with increasing age in total brain extracts, the parameters changed significantly in the neuropil fractions. In another study (Mythri , et al. 2011) carried out on the total extract of FC from PD brains, we showed that the SOD activity from PD brains was unaltered compared to controls, while the SOD activity was elevated in the non-synaptic mitochondria (Figure 52A), consistent with a previous study (Radunovic , et al. 1997) which demonstrated increased mitochondrial SOD activity in the motor cortex from PD cases but not in cases of amyotropic lateral sclerosis. Similarly, while the GR and GST activities in the total extract of FC were unaltered between PD and control brains (Mythri , et al. 2011) neuropil analysis revealed significantly elevated activities in the synaptosomes (Figure 53B and 53C). Total GSH was elevated by 3 fold in total extract of FC compared to control (Mythri , et al. 2011) while the highest relative elevation was observed in the synaptosomes (Figure 52B) compared to cytosol and non-synaptic mitochondria. On the other hand, the GPx activity elevated in the total extract of FC from PD brains was contributed only by the cytosolic and synaptosomal fractions while the activity was unchanged in the mitochondria (Figure 53A).

Pre /post postmortem factors might impinge on tissue integrity and quality (Harish , et al. 2012, Harish , et al. 2011) and on different biochemical parameters (Chandana, Mythri, Mahadevan, Shankar and Srinivas Bharath 2009).The average PMI of the control brain samples (13.8 h) used in the current study was nearly two-fold higher than in the PD samples (6.1 h), which might affect the antioxidant activities. However, our previous studies on human brains showed that increasing PMI (2.5 to 26 h) did not affect the antioxidant activities, GSH content and related enzymes in FC (Harish , et al. 2011). Previously, we have demonstrated that increasing PMI did not influence the postmortem stability of GFAP and oxidatively damaged proteins indifferent anatomical regions of human brains including FC (Harish , et al. 2011). These data indicate that the oxidant and antioxidant markers in FC are relatively unaffected by PMI and the differences observed between the control and PD samples in the study represent disease-related changes.

Most studies on redox dynamics in PD have concentrated on the nigrosriatum but studies on FC are limited (Gomez , et al. 2010).While the SN from PD brains displays GSH loss (Riederer 1989, Sofic, Lange, Jellinger and Riederer 1992), non-SN regions show elevated GSH content, which might protect mitochondria against oxidative damage (Mythri , et al. 2011). Elevated GSH could either be due to increased synthesis or leakage of intracellular GSH or by lowered breakdown. It was previously showed that the elevated GSH in FC of PD brains was due to lowered activity of gamma glutamyltranspeptidase (GGT), while the activity of the GSH synthesizing enzyme gamma glutamyl ligase (GCL) was unaltered (Mythri , et al. 2011). The current study showed that the GSH elevation in FC was not due to the oxidation of GSH to GSSG since both control and PD samples showed the same GSSG/total glutathione ratio (Figures 52C and 52D). Analysis of mitochondrial enzyme activities (Figure 57) indicated that the mitochondrial content in the synaptosomal fraction was at least 5 fold lower than the non-synaptic mitochondria. Since this phenomenon was seen both in the control and PD samples, it could be concluded that the selective vulnerability of the synaptosomes to oxidative damage in the controls and the relative changes in the antioxidant activities in PD samples is not directly associated with the mitochondrial content.

In conclusion, our data indicate that (i) the relative distribution of antioxidant markers including GSH in synaptic and non-synaptic regions significantly impinges on the degenerative and protective mechanisms in PD (ii) synaptosomal and mitochondrial fractions exhibit lower antioxidant activity compared to cytosol in FC from control brains indicating the susceptibility of synaptosomes against oxidative damage (iii) Elevated GSH content and related enzyme activities in FC of PD brains compared to controls might contribute to neuroprotection in PD.

7.8 Curcumin derivatives display improved protection against GSH depletion mediated oxidative stress in a dopaminergic neuronal cell line: Implications for PD and TBI.

The analysis of redox markers in TBI and PD has indicated the importance of GSH and emphasized that while GSH depletion is associated with degeneration and injury, elevated GSH could provide neuroprotection (Mythri et al, 2007, Jagatha et al, 2008). This could have therapeutic implications for degenerative diseases such as PD which involve GSH depletion in early degenerative stages (Bharath et al, 2002).

There have been significant advances in PD therapy including surgical and pharmacological interventions. Currently, there are several options for pharmacological treatment of PD with Levodopa (L-dopa) being the most popular drug. However, the occurrence of severe drug-induced side effects in chronic L-dopa therapy has allowed utilization of alternative molecules including dopamine receptor agonists, anti­cholinergic drugs, monoamine oxidase-B inhibitors, catechol-O-methyl transferase inhibitors etc. These therapeutic strategies are mainly symptomatic and strive to replenish striatal dopamine. But their ability to prevent or slowdown neurodegeneration of SN neurons has not been completely validated in humans. Therefore, there is scope for exploration of novel therapeutic molecules that prevent neurodegeneration and could be used as adjunctive therapies along with dopamine replacement.

Curcumin exhibits neuroprotective effect against MPTP (Rajeswari and Sabesan 2008) and 6-hydroxy dopamine (6-OHDA) in vivo (Zbarsky , et al. 2005). Curcumin protects mitochondria against PN and GSH depletion mediated toxicity in vitro and in vivo (Mythri, Jagatha, Pradhan, Andersen and Bharath 2007). Curcumin also binds to the redox-active metals, iron and copper thereby exerting neuroprotection against oxidative damage (Lavoie , et al. 2009). Interestingly, curcumin induces GSH synthesis in cells by activation of GCL activity in vivo thereby enhancing the antioxidant potential (Dickinson, Iles, Zhang, Blank and Forman 2003). Dickinson, Iles, Zhang, Blank and Forman (2003) demonstrated that, curcumin-induced elevation of GCL activity is probably by enhanced transcription of GCL genes via binding of specific transcription factor complexes to TRE and EpRE elements. Further, the authors showed that curcumin modulates the components of transcription factors that compose EpRE and AP-1 complexes which bind to these cis elements, thereby impacting the GCL expression. Lavoie , et al. (2009) demonstrated both in astrocytes and neuronal cells that curcumin caused a specific and significant increase in the expression of the gene coding for the modifier subunit of y-GCL (GCLM) which is required for the up­regulation of y -GCL activity. Based on these data and our previous reports (Jagatha, Mythri, Vali and Bharath 2008) we surmise that curcumin could be a potential candidate for adjunctive pharmacotherapy in PD.

However, there are several reports indicating poor bioavailability of curcumin due to the rapid systemic derivatization in the GI tract suggesting that small doses of curcumin are necessary for its neuroprotective effect (Ramassamy 2006). Limited bioavailability might be due to poor absorption, rapid metabolism and quick systemic elimination. In order to improve its bioavailability, the use of adjuvants like piperine to prevent glucuronidation, use of curcumin nanoparticles, curcumin structural analogues, liposomal curcumin or curcumin phospholipid complexes have been attempted (Anand , et al. 2007).

We have utilized diester derivatives of curcumin (di-piperoyl, di-valinoyl and di- glutamoyl) that improve its bioavailability (Dubey, Sharma, Narain, Misra and Pati 2008). These conjugates could be dissociated at the target site by cellular esterases releasing curcumin thereby functioning on as pro-drugs. Studies in animal models and human volunteers showed that piperine, an active natural compound from black pepper enhances the activity and bioavailability of curcumin several fold (Shoba , et al. 1998). Similarly, valinoyl ester prodrugs exhibit improved bioavailability (Sinko , et al. 1998). The diesters of curcumin used in our study have: (i) enhanced metabolic stability due to protection of phenolic hydroxyl groups thus delaying their glucuronidation (ii) improved cellular uptake due to transportation via amino acid carriers and (iii) better solubility due to increased hydrophilicity (Dubey, Sharma, Narain, Misra and Pati 2008).

We observed that all the three derivatives tested were either equal to or more potent compared to curcumin in countering the GSH depletion mediated oxidative stress. Moreover, the derivatives showed neuroprotection both during pre and post treatment suggesting that they could reverse the oxidative damage caused during GSH depletion. Among the three bioconjugates, D3 exhibited significant increase in GSH levels and showed maximum detoxification of ROS. The predictive study carried out to delineate the beneficial effects of D3 indicated the following: During GSH synthesis, the rate limiting step is the formation of the dipeptide gamma-glutamyl cysteine (Griffith 1999). This requires influx of glu (as glutamine) and cys (as cystine) from the extra­cellular matrix as shown in the schematic representation in figure 62(Vali , et al. 2007). Since it was not possible to introduce the glutamoyldiester of curcumin in our neuronal platform, we chose to increase the extracellular glu levels (thus increasing its influx into the neuronal cytosol) from 1 pM (control condition) to 3 uM along with increased levels of cytosolic curcumin. Consequently, the initial increase in GSH was basically due to increased neuronal glu without any significant changes in cystine uptake and intracellular cys levels. With increased glu in the pM range in the system, we observed initial elevation in cellular GSH. However, when the glutamate levels increased to 1-5 mM, cystine uptake in astrocytes was inhibited leading to reduction in neuronal cys thereby decreasing GSH levels (data not shown). Despite abundant glutamate levels, GSH levels dropped since cys here became rate limiting for synthesis.

Our study indicated that administration of D3 probably enhanced cellular GSH levels not only via increased accumulation of curcumin in the cell, but also by improved intracellular concentrations of glu in the non-toxic range. D3 not only exhibits improved bioavailability but also independently enhances GSH levels making it a specific pro-drug for diseases involving oxidative stress. In conclusion, compounds such as D3 with improved uptake, ROS-scavenging capacity and neuroprotective efficiency in vivo compared with curcumin with a capacity to cross blood brain barrier could serve as potential neuroprotective strategies in disorders such as PD.

8 Summary

1. Pre and postmortem factors including age influence antioxidant/oxidant markers in the human brain.

- SOD and catalase activities in the human brains show significant stability postmortem. Total GSH was altered in MO and CB which could be partly due to significant increase in MDA levels with increasing PMI. GST was the least altered enzyme and its activity was not affected by pre and postmortem factors.
- Histopathological analysis for alteration in neuronal morphology showed that CB was well preserved with no significant pathological features. The maximal pathological changes were observed in MO, while the changes were milder in FC.
- Total protein carbonyls were unaltered up to ~6 years at -80 [0]C and protein 3-NT levels were unaltered up to ~6.25 years at -80 [0]C. GFAP protein was intact up to ~7.4 years at -80 [0]C.
- Overall, antioxidant activities were several folds lower in synaptosomes compared to cytosol. The activities were affected mainly by the age and to a lesser extent by other pre/ postmortem factors.
- The brains displayed anatomical heterogeneity in the distribution of redox markers in different regions (CB, FC, CD, MB, MO and HC) between young (20±5 y) and old brains (70±10 y). Similar profile of differences was observed in GSH, GPx, GR, catalase, protein carbonyls, GFAP and MDA in young vs. old brains while SOD and catalase showed significant difference in the profile.

2. Biochemical and immunohistochemical characterization of contusion and pericontusion of human TBI

- Contused region of TBI showed compromised antioxidant activity and elevated oxidative stress. Among the neuropil fractions, non-synaptic and synaptic mitochondria from contusion and pericontusion were most affected in terms of redox capacity evidenced by decline in antioxidant enzyme activities, mitochondrial functional enzyme activities and elevated protein oxidation.
- Contused regions were mostly haemmorhagic with intervening parenchyma showing ischemic dark neurons with prominent gliosis. Pericontusion was oedematous with viable neurons. Dystrophic neurons, axonal alterations, reactive astrocytes and immune response were prominently observed in pericontusion.

3. Proteomic analysis in TBI

- In contused regions, 3254 proteins were identified and quantified. Among these, 402 proteins were over expressed and 119 proteins were under expressed in contusion compared to age-matched controls. In the pericontusion region, among the 3254 identified proteins, 73 proteins were over expressed, while 84 proteins were under expressed compared to age-matched controls.
- Gene Ontology analysis localized 61.1% of proteins to intracellular locations, 13.9% were protein complexes, 14.4% were of extracellular, and 6.3% were plasma membrane proteins. Among the total identified proteins, 462 (14%) were mitochondrial and 732 (22.4%) were synaptic proteins.
- Several proteins in KEAP1-NRF2, mitochondrial electron transport chain, spinal cord injury pathway, oxidative stress pathway, prostaglandin synthesis pathway, ubiquitin proteasome and synaptic vesicle pathway were significantly altered.
- Western blot analysis showed that CHI1 expression was up regulated. Observation of Iba 1 and HLA DR immunoreactive cells in injured tissue confirmed profound immune response which corroborated the proteomic results.
- The expression of peroxiredoxin-2 was unchanged, while Synaptophysin and GFAP were significantly over expressed both in contusion and pericontusion compared to controls.

4. Synaptic terminals in the FC of human PD brains display altered GSH metabolism.

- The antioxidant markers showed significant alteration in control brains across different sub-cellular fractions of FC with the synaptosomes showing least activity followed by mitochondria and cytosol.
- Synaptosomes from FC of PD brains showed relatively increased GSH content and higher related enzyme activities compared to controls
- Mitochondria from FC of PD brains showed higher SOD activity compared to controls.

5. Curcumin derivatives offer improved protection via elevated GSH in dopaminergic neurons with implications for PD therapy.

- Compared to the curcumin bioconjugates D1 and D2, D3 enhanced cellular GSH levels in the BSO model via elevated intracellular curcumin and glutamate thus making it a pro-drug for diseases involving oxidative stress.

9 Conclusions

- The redox status, mitochondrial physiology and proteomics in the human brain are highly dynamic and inter-related. These processes are regulated by pre/postmortem factors, aging, degeneration and injury. Nevertheless, oxidative and mitochondrial changes are common events that link aging, degeneration and injury mechanisms in the human brain.
- There is a persistence of signature pattern of redox markers in different anatomical regions of young and old brains thus making certain regions more susceptible to damage with functional and evolutionary significance.
- Biochemical and histopathological changes in the human brain need to be recorded to validate the data obtained from animal and cell models of disease. The effect of pre and postmortem factors need to be determined to obtain the absolute quantitative data of specific biomarkers.
- GSH dynamics could be the most sensitive to pre/postmortem changes and was significantly altered during aging, PD and injury. Elevation of cellular GSH by pharmacological means could be a viable option for adjunctive therapy in PD and TBI.
- Distinct biochemical, pathological and proteomic changes differentiate contusion from the pericontusion regions in the injured brain, thereby providing a handle to analyze primary and secondary events in TBI.

10 LIST OF PUBLICATIONS

1. Harish G, Mahadevan A, Srinivas Bharath MM, Shankar SK. Alteration in glutathione content and associated enzyme activities in the synaptic terminals but not in the non-synaptic mitochondria from the frontal cortex of Parkinson's disease brains. Neurochem Res 2013; 38: 186-200
2. Harish G, Venkateshappa C, Mahadevan A, Pruthi N, Bharath MM, Shankar SK. Mitochondrial function in human brains is affected by pre- and post mortem factors. Neuropathol Appl Neurobiol 2013; 39: 298-315
3. Harish G, Venkateshappa C, Mahadevan A, Pruthi N, Bharath MMS, Shankar SK. Effect of Storage Time, Postmortem Interval, Agonal State, and Gender on the Postmortem Preservation of Glial Fibrillary Acidic Protein and Oxidatively Damaged Proteins in Human Brains. Biopreservation and Biobanking 2011; 9: 379-87
4. Harish G, Venkateshappa C, Mahadevan A, Pruthi N, Srinivas Bharath MM, Shankar SK. Glutathione metabolism is modulated by postmortem interval, gender difference and agonal state in postmortem human brains. Neurochem Int 2011; 59: 1029-42
5. Harish G, Venkateshappa C, Mahadevan A, Pruthi N, Srinivas Bharath MM, Shankar SK. Effect of Premortem and Postmortem Factors on the Distribution and Preservation of Antioxidant Activities in the Cytosol and Synaptosomes of Human Brains. Biopreservation and Biobanking 2012; 10: 253-65
6. Harish G, Venkateshappa C, Mythri RB, Dubey SK, Mishra K, Singh N, Vali S, Bharath MM. Bioconjugates of curcumin display improved protection against glutathione depletion mediated oxidative stress in a dopaminergic neuronal cell line: Implications for Parkinson's disease. Bioorg Med Chem 2010; 18: 2631-8
7. Mythri R, Harish G, Dubey S, Misra K, Bharath M. Glutamoyl diester of the dietary polyphenol curcumin offers improved protection against peroxynitrite-mediated nitrosative stress and damage of brain mitochondria in vitro: implications for Parkinson's disease. Mol Cell Biochem 2011; 347: 135-43
8. Mythri RB, Harish G, Bharath MM. Therapeutic potential of natural products in Parkinson's disease. Recent Pat Endocr Metab Immune Drug Discov 2012; 6: 181-200
9. Mythri RB, Harish G, Dubey SK, Misra K, Bharath MM. Glutamoyl diester of the dietary polyphenol curcumin offers improved protection against peroxynitrite- mediated nitrosative stress and damage of brain mitochondria in vitro: implications for Parkinson's disease. Mol Cell Biochem 2011; 347: 135-43
10. Mythri RB, Veena J, Harish G, Shankaranarayana Rao BS, Srinivas Bharath MM. Chronic dietary supplementation with turmeric protects against 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine-mediated neurotoxicity in vivo: implications for Parkinson's disease. Br J Nutr 2011; 106: 63-72
11. Mythri RB, Venkateshappa C, Harish G, Mahadevan A, Muthane UB, Yasha TC, Srinivas Bharath MM, Shankar SK. Evaluation of markers of oxidative stress, antioxidant function and astrocytic proliferation in the striatum and frontal cortex of Parkinson's disease brains. Neurochem Res 2011; 36: 1452-63
12. Shankar SK, Mahadevan A, Harish G, Srinivas Bharath MM. Human Brain Tissue Repository: A National Facility Fostering Neuroscience Research. Proc Natl Acad Sci, India, Sect B Biol Sci 2013: 1-12
13. Venkateshappa C, Harish G, Mahadevan A, Srinivas Bharath MM, Shankar SK. Elevated Oxidative Stress and Decreased Antioxidant Function in the Human Hippocampus and Frontal Cortex with Increasing Age: Implications for Neurodegeneration in Alzheimer's Disease. Neurochem Res 2012:
14. Venkateshappa C, Harish G, Mythri RB, Mahadevan A, Bharath MM, Shankar SK. Increased oxidative damage and decreased antioxidant function in aging human substantia nigra compared to striatum: implications for Parkinson's disease. Neurochem Res 2012; 37: 358-69

11 References

1. Abbott LC, Nejad HH, Bottje WG, & Hassan AS (1990) Glutathione levels in specific brain regions of genetically epileptic (tg/tg) mice. Brain research bulletin 25(4):629-631.

2. Adams FS , et al. (1996) Characterization and transplantation of two neuronal cell lines with dopaminergic properties. Neurochem Res 21(5):619-627.

3. Adams JH, Graham DI, Murray LS, & Scott G (1982) Diffuse axonal injury due to nonmissile head injury in humans: an analysis of 45 cases. Ann Neurol 12(6):557-563.

4. Adelson PD, Whalen MJ, Kochanek PM, Robichaud P, & Carlos TM (1998) Blood brain barrier permeability and acute inflammation in two models of traumatic brain injury in the immature rat: a preliminary report. Acta neurochirurgica. Supplement 71:104-106.

5. Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121-126.

6. Agarwal S & Sohal RS (1994) Aging and proteolysis of oxidized proteins. Arch Biochem Biophys 309(1):24-28.

7. Aguilaniu H, Gustafsson L, Rigoulet M, & Nystrom T (2003) Asymmetric inheritance of oxidatively damaged proteins during cytokinesis. Science 299(5613):1751-1753.

8. Ahlgren S, Li GL, & Olsson Y (1996) Accumulation of beta-amyloid precursor protein and ubiquitin in axons after spinal cord trauma in humans: immunohistochemical observations on autopsy material. Acta Neuropathol 92(1):49-55.

9. Aitken RJ & Henkel RR (2011) Sperm cell biology: current perspectives and future prospects. Asian journal of andrology 13(1):3-5.

10. Aksenov MY, Aksenova MV, Butterfield DA, Geddes JW, & Markesbery WR (2001) Protein oxidation in the brain in Alzheimer's disease. Neuroscience 103(2):373-383.

11. Alam ZI , et al. (1997) A generalised increase in protein carbonyls in the brain in Parkinson's but not incidental Lewy body disease. J Neurochem 69(3):1326-1329.

12. Alam ZI , et al. (1997) Oxidative DNA damage in the parkinsonian brain: an apparent selective increase in 8-hydroxyguanine levels in substantia nigra. J Neurochem 69(3):1196-1203.

13. Albers DS & Beal MF (2000) Mitochondrial dysfunction and oxidative stress in aging and neurodegenerative disease. J Neural Transm Suppl 59:133-154.

14. Ali SS , et al. (2006) Gender differences in free radical homeostasis during aging: shorter-lived female C57BL6 mice have increased oxidative stress. Aging Cell 5(6):565-574.

15. Anand P, Kunnumakkara AB, Newman RA, & Aggarwal BB (2007) Bioavailability of curcumin: problems and promises. Mol Pharm 4(6):807-818.

16. Andersen JK , et al. (1996) Effect of buthionine sulfoximine, a synthesis inhibitor of the antioxidant glutathione, on the murine nigrostriatal neurons. J Neurochem 67(5):2164-2171.

17. Andreyev AY, Kushnareva YE, & Starkov AA (2005) Mitochondrial metabolism of reactive oxygen species. Biochemistry. Biokhimiia 70(2):200-214.

18. Ansari MA, Roberts KN, & Scheff SW (2008) Oxidative stress and modification of synaptic proteins in hippocampus after traumatic brain injury. Free Radic Biol Med 45(4):443-452.

19. Ansari MA & Scheff SW (2010) Oxidative stress in the progression of Alzheimer disease in the frontal cortex. J Neuropathol Exp Neurol 69(2):155-167.

20. Arriagada PV, Marzloff K, & Hyman BT (1992) Distribution of Alzheimer-type pathologic changes in nondemented elderly individuals matches the pattern in Alzheimer's disease. Neurology 42(9):1681-1688.

21. Ashok BT & Ali R (1999) The aging paradox: free radical theory of aging. Exp Gerontol 34(3):293- 303.

22. Astiz M, de Alaniz MJ, & Marra CA (2012) The oxidative damage and inflammation caused by pesticides are reverted by lipoic acid in rat brain. Neurochem Int 61(7):1231-1241.

23. Atlante A , et al. (2001) Glutamate neurotoxicity, oxidative stress and mitochondria. FEBS Lett 497(1):1-5.

24. Awasthi D , et al. (2003) Early gene expression in the rat cortex after experimental traumatic brain injury and hypotension. Neurosci Lett 345(1):29-32.

25. Bagnyukova TV, Storey, K.B., Lushchak, V.I. (2003) Induction of oxidative stress in Rana ridibunda during recovery from winter hibernation. J Therm Biol 28:21-28.

26. Bahn S , et al. (2001) Gene expression profiling in the post-mortem human brain--no cause for dismay. J Chem Neuroanat 22(1-2):79-94.

27. Balaban RS, Nemoto S, & Finkel T (2005) Mitochondria, oxidants, and aging. Cell 120(4):483-495.

28. Balazy M & Nigam S (2003) Aging, lipid modifications and phospholipases--new concepts. Ageing Res Rev 2(2):191-209.

29. Ballatori N , et al. (2009) Glutathione dysregulation and the etiology and progression of human diseases. Biol Chem 390(3):191-214.

30. Banerjee R, Starkov AA, Beal MF, & Thomas B (2009) Mitochondrial dysfunction in the limelight of Parkinson's disease pathogenesis. Biochim Biophys Acta 1792(7):651-663.

31. Bannister JV, Halliwell B, & O'Neill P (1985) Free Radicals in Biological Medicine (Harwood Academic).

32. Barger SW, Van Eldik LJ, & Mattson MP (1995) S100 beta protects hippocampal neurons from damage induced by glucose deprivation. Brain Res 677(1):167-170.

33. Barja G (1999) Mitochondrial oxygen radical generation and leak: sites of production in states 4 and 3, organ specificity, and relation to aging and longevity. J Bioenerg Biomembr 31(4):347-366.

34. Barja G & Herrero A (1998) Localization at complex I and mechanism of the higher free radical production of brain nonsynaptic mitochondria in the short-lived rat than in the longevous pigeon. J Bioenerg Biomembr 30(3):235-243.

35. Barker JE , et al. (1996) Depletion of brain glutathione results in a decrease of glutathione reductase activity; an enzyme susceptible to oxidative damage. Brain Res 716(1-2):118-122.

36. Bayir H , et al. (2005) Enhanced oxidative stress in iNOS-deficient mice after traumatic brain injury: support for a neuroprotective role of iNOS. J Cereb Blood Flow Metab 25(6):673-684.

37. Bayir H , et al. (2007) Neuronal NOS-mediated nitration and inactivation of manganese superoxide dismutase in brain after experimental and human brain injury. J Neurochem 101(1):168-181.

38. Bayir H , et al. (2002) Assessment of antioxidant reserves and oxidative stress in cerebrospinal fluid after severe traumatic brain injury in infants and children. Pediatric research 51(5):571-578.

39. Bayir H , et al. (2004) Marked gender effect on lipid peroxidation after severe traumatic brain injury in adult patients. J Neurotrauma 21(1):1-8.

40. Bazarian JJ, Zemlan FP, Mookerjee S, & Stigbrand T (2006) Serum S-100B and cleaved-tau are poor predictors of long-term outcome after mild traumatic brain injury. Brain injury : [BI] 20(7):759-765.

41. Beckman JS & Koppenol WH (1996) Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol 271(5 Pt 1):C1424-1437.

42. Bellander BM, Bendel O, Von Euler G, Ohlsson M, & Svensson M (2004) Activation of microglial cells and complement following traumatic injury in rat entorhinal-hippocampal slice cultures. J Neurotrauma 21(5):605-615.

43. Bellander BM , et al. (2011) Secondary insults following traumatic brain injury enhance complement activation in the human brain and release of the tissue damage marker S100B. Acta Neurochir (Wien) 153(1):90-100.

44. Bellander BM, Singhrao SK, Ohlsson M, Mattsson P, & Svensson M (2001) Complement activation in the human brain after traumatic head injury. J Neurotrauma 18(12):1295-1311.

45. Bellander BM, von Holst H, Fredman P, & Svensson M (1996) Activation of the complement cascade and increase of clusterin in the brain following a cortical contusion in the adult rat. J Neurosurg 85(3):468-475.

46. Bemeur C , et al. (2004) Expression of superoxide dismutase in hyperglycemic focal cerebral ischemia in the rat. Neurochem Int 45(8):1167-1174.

47. Bender A , et al. (2006) High levels of mitochondrial DNA deletions in substantia nigra neurons in aging and Parkinson disease. Nat Genet 38(5):515-517.

48. Benner EJ , et al. (2008) Nitrated alpha-synuclein immunity accelerates degeneration of nigral dopaminergic neurons. PLoS One 3(1):e1376.

49. Berger RP , et al. (2005) Serum neuron-specific enolase, S100B, and myelin basic protein concentrations after inflicted and noninflicted traumatic brain injury in children. J Neurosurg 103(1 Suppl):61-68.

50. Bernardi P , et al. (2006) The mitochondrial permeability transition from in vitro artifact to disease target. Febs j 273(10):2077-2099.

51. Bertoni-Freddari C , et al. (2004) Cytochrome oxidase activity in hippocampal synaptic mitochondria during aging: a quantitative cytochemical investigation. Ann N Y Acad Sci 1019:33­36.

52. Bertoni-Freddari C , et al. (1996) Synaptic structural dynamics and aging. Gerontology 42(3):170- 180.

53. Betarbet R , et al. (2000) Chronic systemic pesticide exposure reproduces features of Parkinson's disease. Nat Neurosci 3(12):1301-1306.

54. Bharath S & Andersen JK (2005) Glutathione depletion in a midbrain-derived immortalized dopaminergic cell line results in limited tyrosine nitration of mitochondrial complex I subunits: implications for Parkinson's disease. Antioxid Redox Signal 7(7-8):900-910.

55. Bharath S, Hsu M, Kaur D, Rajagopalan S, & Andersen JK (2002) Glutathione, iron and Parkinson's disease. Biochem Pharmacol 64(5-6):1037-1048.

56. Bharucha NE, Bharucha EP, Bharucha AE, Bhise AV, & Schoenberg BS (1988) Prevalence of Parkinson's disease in the Parsi community of Bombay, India. Arch Neurol 45(12):1321-1323.

57. Bindon JR (1989) Modern biological theories of aging. Edited by Huber R. Warner, Robert N. Butler, Richard L. Sprott, and Edward L. Schneider. New York: Raven Press. 1987. Aging, Vol. 31. xviii + 324 pp., figures, tables, index. $73.00 (cloth). American Journal of Physical Anthropology 78(3):449-450.

58. Birdsill AC, Walker DG, Lue L, Sue LI, & Beach TG (2010) Postmortem interval effect on RNA and gene expression in human brain tissue. Cell Tissue Bank.

59. Blennow K, Hampel H, Weiner M, & Zetterberg H (2010) Cerebrospinal fluid and plasma biomarkers in Alzheimer disease. Nature reviews. Neurology 6(3):131-144.

60. Bompart GJ, Prevot DS, & Bascands JL (1990) Rapid automated analysis of glutathione reductase, peroxidase, and S-transferase activity: application to cisplatin-induced toxicity. Clin Biochem 23(6):501-504.

61. Bonneh-Barkay D , et al. (2012) Astrocyte and macrophage regulation of YKL-40 expression and cellular response in neuroinflammation. Brain Pathol 22(4):530-546.

62. Bonneh-Barkay D , et al. (2010) YKL-40 expression in traumatic brain injury: an initial analysis. J Neurotrauma 27(7):1215-1223.

63. Boutte AM , et al. (2012) Proteomic analysis and brain-specific systems biology in a rodent model of penetrating ballistic-like brain injury. Electrophoresis 33(24):3693-3704.

64. Bower JH , et al. (2003) Head trauma preceding PD: a case-control study. Neurology 60(10):1610- 1615.

65. Boyland E & Chasseaud LF (1969) The role of glutathione and glutathione S-transferases in mercapturic acid biosynthesis. Adv Enzymol Relat Areas Mol Biol 32:173-219.

66. Braak H , et al. (2003) Staging of brain pathology related to sporadic Parkinson's disease. Neurobiol Aging 24(2):197-211.

67. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254.

68. Bramlett HM & Dietrich WD (2007) Progressive damage after brain and spinal cord injury: pathomechanisms and treatment strategies. Prog Brain Res 161:125-141.

69. Bredt DS (1999) Endogenous nitric oxide synthesis: biological functions and pathophysiology. Free Radic Res 31(6):577-596.

70. Brewer LD, Porter NM, Kerr DS, Landfield PW, & Thibault O (2006) Chronic 1alpha,25-(OH)2 vitamin D3 treatment reduces Ca2+ -mediated hippocampal biomarkers of aging. Cell Calcium 40(3):277-286.

71. Brouet I & Ohshima H (1995) Curcumin, an anti-tumour promoter and anti-inflammatory agent, inhibits induction of nitric oxide synthase in activated macrophages. Biochem Biophys Res Commun 206(2):533-540.

72. Bullock R , et al. (1998) Factors affecting excitatory amino acid release following severe human head injury. J Neurosurg 89(4):507-518.

73. Buritica E , et al. (2009) Changes in calcium-binding protein expression in human cortical contusion tissue. J Neurotrauma 26(12):2145-2155.

74. Butterfield D , et al. (2002) Nutritional approaches to combat oxidative stress in Alzheimer's disease. The Journal of nutritional biochemistry 13(8):444.

75. Butterfield DA, Howard BJ, Yatin S, Allen KL, & Carney JM (1997) Free radical oxidation of brain proteins in accelerated senescence and its modulation by N-tert-butyl-alpha-phenylnitrone. Proc Natl Acad Sci U S A 94(2):674-678.

76. Cadenas E & Davies KJ (2000) Mitochondrial free radical generation, oxidative stress, and aging. Free Radic Biol Med 29(3-4):222-230.

77. Cakatay U , et al. (2001) Relation of oxidative protein damage and nitrotyrosine levels in the aging rat brain. Exp Gerontol 36(2):221-229.

78. Calabrese V, Bates TE, & Stella AM (2000) NO synthase and NO-dependent signal pathways in brain aging and neurodegenerative disorders: the role of oxidant/antioxidant balance. Neurochem Res 25(9-10):1315-1341.

79. Calleja M , et al. (1993) Mitochondrial DNA remains intact during Drosophila aging, but the levels of mitochondrial transcripts are significantly reduced. J Biol Chem 268(25):18891-18897.

80. Candelario-Jalil E, Al-Dalain SM, Castillo R, Martinez G, & Fernandez OS (2001) Selective vulnerability to kainate-induced oxidative damage in different rat brain regions. J Appl Toxicol 21(5):403-407.

81. Carlberg I & Mannervik B (1985) Glutathione reductase. Methods Enzymol 113:484-490.

82. Carney JM , et al. (1991) Reversal of age-related increase in brain protein oxidation, decrease in enzyme activity, and loss in temporal and spatial memory by chronic administration of the spin­trapping compound N-tert-butyl-alpha-phenylnitrone. Proc Natl Acad Sci U S A 88(9):3633-3636.

83. Carrillo MC, Kanai S, Sato Y, & Kitani K (1992) Age-related changes in antioxidant enzyme activities are region and organ, as well as sex, selective in the rat. Mech Ageing Dev 65(2-3):187- 198.

84. Castellani RJ , et al. (2004) Contribution of redox-active iron and copper to oxidative damage in Alzheimer disease. Ageing Res Rev 3(3):319-326.

85. Cernak I , et al. (2000) Characterization of plasma magnesium concentration and oxidative stress following graded traumatic brain injury in humans. J Neurotrauma 17(1):53-68.

86. Chakraborti A, Gulati K, Banerjee BD, & Ray A (2007) Possible involvement of free radicals in the differential neurobehavioral responses to stress in male and female rats. Behav Brain Res 179(2):321-325.

87. Chan MM, Huang HI, Fenton MR, & Fong D (1998) In vivo inhibition of nitric oxide synthase gene expression by curcumin, a cancer preventive natural product with anti-inflammatory properties. Biochem Pharmacol 55(12):1955-1962.

88. Chanas SA , et al. (2002) Loss of the Nrf2 transcription factor causes a marked reduction in constitutive and inducible expression of the glutathione S-transferase Gsta1, Gsta2, Gstm1, Gstm2, Gstm3 and Gstm4 genes in the livers of male and female mice. Biochem J 365(Pt 2):405- 416.

89. Chance B, Sies H, & Boveris A (1979) Hydroperoxide metabolism in mammalian organs. Physiol Rev 59(3):527-605.

90. Chandana R, Mythri RB, Mahadevan A, Shankar SK, & Srinivas Bharath MM (2009) Biochemical analysis of protein stability in human brain collected at different post-mortem intervals. Indian J Med Res 129(2):189-199.

91. Chang CK, Chang CP, Liu SY, & Lin MT (2007) Oxidative stress and ischemic injuries in heat stroke. Prog Brain Res 162:525-546.

92. Chang L, Jiang CS, & Ernst T (2009) Effects of age and sex on brain glutamate and other metabolites. Magnetic resonance imaging 27(1):142-145.

93. Chen J , et al. (2006) Curcumin protects PC12 cells against 1-methyl-4-phenylpyridinium ion- induced apoptosis by bcl-2-mitochondria-ROS-iNOS pathway. Apoptosis 11(6):943-953.

94. Chen YR & Tan TH (1998) Inhibition of the c-Jun N-terminal kinase (JNK) signaling pathway by curcumin. Oncogene 17(2):173-178.

95. Cheng G, Kong RH, Zhang LM, & Zhang JN (2012) Mitochondria in traumatic brain injury and mitochondrial-targeted multipotential therapeutic strategies. British journal of pharmacology 167(4):699-719.

96. Chevyreva I, Faull RL, Green CR, & Nicholson LF (2008) Assessing RNA quality in postmortem human brain tissue. Exp Mol Pathol 84(1):71-77.

97. Chiaretti A , et al. (2009) NGF, DCX, and NSE upregulation correlates with severity and outcome of head trauma in children. Neurology 72(7):609-616.

98. Choi J , et al. (2006) Oxidative damage of DJ-1 is linked to sporadic Parkinson and Alzheimer diseases. J Biol Chem 281(16):10816-10824.

99. Choi SW , et al. (2011) Intrinsic bioenergetic properties and stress sensitivity of dopaminergic synaptosomes. J Neurosci 31(12):4524-4534.

100. Chong ZZ, Li F, & Maiese K (2005) Oxidative stress in the brain: novel cellular targets that govern survival during neurodegenerative disease. Prog Neurobiol 75(3):207-246.

101. Chung KK , et al. (2004) S-nitrosylation of parkin regulates ubiquitination and compromises parkin's protective function. Science 304(5675):1328-1331.

102. Chung YH, Shin CM, Joo KM, Kim MJ, & Cha CI (2002) Immunohistochemical study on the distribution of nitrotyrosine and neuronal nitric oxide synthase in aged rat cerebellum. Brain Res 951(2):316-321.

103. Church AJ & Andrew RD (2005) Spreading depression expands traumatic injury in neocortical brain slices. J Neurotrauma 22(2):277-290.

104. Cini M & Moretti A (1995) Studies on lipid peroxidation and protein oxidation in the aging brain. Neurobiol Aging 16(1):53-57.

105. Clausen A, Doctrow S, & Baudry M (2010) Prevention of cognitive deficits and brain oxidative stress with superoxide dismutase/catalase mimetics in aged mice. Neurobiol Aging 31(3):425- 433.

106. Clemens JA (2000) Cerebral ischemia: gene activation, neuronal injury, and the protective role of antioxidants. Free Radic Biol Med 28(10):1526-1531.

107. Clementi E, Brown GC, Feelisch M, & Moncada S (1998) Persistent inhibition of cell respiration by nitric oxide: crucial role of S-nitrosylation of mitochondrial complex I and protective action of glutathione. Proc Natl Acad Sci U S A 95(13):7631-7636.

108. Cohen G, Farooqui R, & Kesler N (1997) Parkinson disease: a new link between monoamine oxidase and mitochondrial electron flow. Proc Natl Acad Sci U S A 94(10):4890-4894.

109. Conti A , et al. (2004) Proteome study of human cerebrospinal fluid following traumatic brain injury indicates fibrin(ogen) degradation products as trauma-associated markers. J Neurotrauma 21(7):854-863.

110. Conway KA, Harper JD, & Lansbury PT (1998) Accelerated in vitro fibril formation by a mutant alpha-synuclein linked to early-onset Parkinson disease. Nat Med 4(11):1318-1320.

111. Cooke MS, Evans MD, Dizdaroglu M, & Lunec J (2003) Oxidative DNA damage: mechanisms, mutation, and disease. Faseb j 17(10):1195-1214.

112. Cornelius C , et al. (2013) Traumatic brain injury: oxidative stress and neuroprotection. Antioxid Redox Signal 19(8):836-853.

113. Corral-Debrinski M , et al. (1992) Mitochondrial DNA deletions in human brain: regional variability and increase with advanced age. Nat Genet 2(4):324-329.

114. Costello S, Cockburn M, Bronstein J, Zhang X, & Ritz B (2009) Parkinson's disease and residential exposure to maneb and paraquat from agricultural applications in the central valley of California. American journal of epidemiology 169(8):919-926.

115. Crecelius A , et al. (2008) Assessing quantitative post-mortem changes in the gray matter of the human frontal cortex proteome by 2-D DIGE. Proteomics 8(6):1276-1291.

116. Crompton M (1999) The mitochondrial permeability transition pore and its role in cell death. Biochem J 341 ( Pt 2):233-249.

117. Cruz-Sanchez FF, Girones X, Ortega A, Alameda F, & Lafuente JV (2010) Oxidative stress in Alzheimer's disease hippocampus: a topographical study. J Neurol Sci 299(1-2):163-167.

118. Csuka E , et al. (1999) IL-10 levels in cerebrospinal fluid and serum of patients with severe traumatic brain injury: relationship to IL-6, TNF-alpha, TGF-beta1 and blood-brain barrier function. Journal of neuroimmunology 101(2):211-221.

119. Cui J, Holmes EH, Greene TG, & Liu PK (2000) Oxidative DNA damage precedes DNA fragmentation after experimental stroke in rat brain. Faseb j 14(7):955-967.

120. Czigner A , et al. (2007) Kinetics of the cellular immune response following closed head injury. Acta Neurochir (Wien) 149(3):281-289.

121. Daly C, Sugimori M, Moreira JE, Ziff EB, & Llinas R (2000) Synaptophysin regulates clathrin- independent endocytosis of synaptic vesicles. Proceedings of the National Academy of Sciences 97(11):6120-6125.

122. Danielson SR & Andersen JK (2008) Oxidative and nitrative protein modifications in Parkinson's disease. Free Radic Biol Med 44(10):1787-1794.

123. Dauer W & Przedborski S (2003) Parkinson's Disease: Mechanisms and Models. Neuron 39(6):889-909.

124. Davis AE (2000) Mechanisms of traumatic brain injury: biomechanical, structural and cellular considerations. Crit Care Nurs Q 23(3):1-13.

125. Dayon L , et al. (2008) Relative quantification of proteins in human cerebrospinal fluids by MS/MS using 6-plex isobaric tags. Analytical chemistry 80(8):2921-2931.

126. Dei R , et al. (2002) Lipid peroxidation and advanced glycation end products in the brain in normal aging and in Alzheimer's disease. Acta Neuropathol 104(2):113-122.

127. Deiana L, Carru C, Pes G, & Tadolini B (1999) Spectrophotometric measurement of hydroperoxides at increased sensitivity by oxidation of Fe2+ in the presence of xylenol orange. Free Radic Res 31(3):237-244.

128. Demirkaya S , et al. (2001) Malondialdehyde, glutathione peroxidase and superoxide dismutase

in peripheral blood erythrocytes of patients with acute cerebral ischemia. Eur J Neurol 8(1):43- 51.

129. Dexter DT , et al. (1989) Basal lipid peroxidation in substantia nigra is increased in Parkinson's disease. J Neurochem 52(2):381-389.

130. Dexter DT & Jenner P (2013) Parkinson disease: from pathology to molecular disease mechanisms. Free Radic Biol Med 62:132-144.

131. Dexter DT , et al. (1989) Increased nigral iron content and alterations in other metal ions occurring in brain in Parkinson's disease. J Neurochem 52(6):1830-1836.

132. Dickinson DA, Iles KE, Zhang H, Blank V, & Forman HJ (2003) Curcumin alters EpRE and AP-1 binding complexes and elevates glutamate-cysteine ligase gene expression. Faseb j 17(3):473- 475.

133. Dickstein DL , et al. (2007) Changes in the structural complexity of the aged brain. Aging Cell 6(3):275-284.

134. Diedrich M , et al. (2011) Brain region specific mitophagy capacity could contribute to selective neuronal vulnerability in Parkinson's disease. Proteome Sci 9:59.

135. Dixon CE, Clifton GL, Lighthall JW, Yaghmai AA, & Hayes RL (1991) A controlled cortical impact model of traumatic brain injury in the rat. J Neurosci Methods 39(3):253-262.

136. Dongre AR, Opiteck G, Cosand WL, & Hefta SA (2001) Proteomics in the post-genome age. Biopolymers 60(3):206-211.

137. Dragunow M , et al. (1993) Is c-Jun involved in nerve cell death following status epilepticus and hypoxic-ischaemic brain injury? Brain Res Mol Brain Res 18(4):347-352.

138. Dringen R, Gutterer JM, & Hirrlinger J (2000) Glutathione metabolism in brain metabolic interaction between astrocytes and neurons in the defense against reactive oxygen species. Eur J Biochem 267(16):4912-4916.

139. Dubey A, Forster MJ, & Sohal RS (1995) Effect of the Spin-Trapping CompoundN-tert-Butyl-a- phenylnitrone on Protein Oxidation and Life Span. Archives of Biochemistry and Biophysics 324(2):249-254.

140. Dubey SK, Sharma AK, Narain U, Misra K, & Pati U (2008) Design, synthesis and characterization of some bioactive conjugates of curcumin with glycine, glutamic acid, valine and demethylenated piperic acid and study of their antimicrobial and antiproliferative properties. Eur J Med Chem 43(9):1837-1846.

141. Duncan GW (2011) The aging brain and neurodegenerative diseases. Clin Geriatr Med 27(4):629- 644.

142. Ekegren T, Hanrieder J, & Bergquist J (2008) Clinical perspectives of high-resolution mass spectrometry-based proteomics in neuroscience: exemplified in amyotrophic lateral sclerosis biomarker discovery research. Journal of mass spectrometry : JMS 43(5):559-571.

143. Eldadah BA & Faden AI (2000) Caspase pathways, neuronal apoptosis, and CNS injury. J Neurotrauma 17(10):811-829.

144. Elstner M , et al. (2011) Expression analysis of dopaminergic neurons in Parkinson's disease and aging links transcriptional dysregulation of energy metabolism to cell death. Acta Neuropathol 122(1):75-86.

145. Erraji-Benchekroun L , et al. (2005) Molecular aging in human prefrontal cortex is selective and continuous throughout adult life. Biol Psychiatry 57(5):549-558.

146. Esiri MM (2007) Ageing and the brain. The Journal of pathology 211(2):181-187.

147. Esterbauer H (1993) Cytotoxicity and genotoxicity of lipid-oxidation products. The American journal of clinical nutrition 57(5 Suppl):779S-785S; discussion 785S-786S.

148. Exner N, Lutz AK, Haass C, & Winklhofer KF (2012) Mitochondrial dysfunction in Parkinson/'s disease: molecular mechanisms and pathophysiological consequences. EMBO J 31(14):3038- 3062.

149. Fabian RH, Dewitt DS, & Kent TA (1998) The 21-aminosteroid U-74389G reduces cerebral superoxide anion concentration following fluid percussion injury of the brain. J Neurotrauma 15(6):433-440.

150. Faden AI (1993) Comparison of single and combination drug treatment strategies in experimental brain trauma. J Neurotrauma 10(2):91-100.

151. Faden AI (1996) Pharmacological treatment of central nervous system trauma. Pharmacology & toxicology 78(1):12-17.

152. Faden AI, Demediuk P, Panter SS, & Vink R (1989) The role of excitatory amino acids and NMDA receptors in traumatic brain injury. Science 244(4906):798-800.

153. Farkas O , et al. (2005) Spectrin breakdown products in the cerebrospinal fluid in severe head injury--preliminary observations. Acta Neurochir (Wien) 147(8):855-861.

154. Feeney DM, Boyeson MG, Linn RT, Murray HM, & Dail WG (1981) Responses to cortical injury: I. Methodology and local effects of contusions in the rat. Brain Res 211(1):67-77.

155. Ferris DC, Kume-Kick J, Russo-Menna I, & Rice ME (1995) Gender differences in cerebral ascorbate levels and ascorbate loss in ischemia. Neuroreport 6(11):1485-1489.

156. Flohe L & Gunzler WA (1984) Assays of glutathione peroxidase. Methods Enzymol 105:114-121.

157. Floor E & Wetzel MG (1998) Increased protein oxidation in human substantia nigra pars compacta in comparison with basal ganglia and prefrontal cortex measured with an improved dinitrophenylhydrazine assay. J Neurochem 70(1):268-275.

158. Floyd CL, Gorin FA, & Lyeth BG (2005) Mechanical strain injury increases intracellular sodium and reverses Na+/Ca2+ exchange in cortical astrocytes. Glia 51(1):35-46.

159. Floyd RA, West M, & Hensley K (2001) Oxidative biochemical markers; clues to understanding aging in long-lived species. Experimental Gerontology 36(4-6):619-640.

160. Foda MA & Marmarou A (1994) A new model of diffuse brain injury in rats. Part II: Morphological characterization. J Neurosurg 80(2):301-313.

161. Forster C, Clark HB, Ross ME, & Iadecola C (1999) Inducible nitric oxide synthase expression in human cerebral infarcts. Acta Neuropathol 97(3):215-220.

162. Franz G , et al. (2003) Amyloid beta 1-42 and tau in cerebrospinal fluid after severe traumatic brain injury. Neurology 60(9):1457-1461.

163. Frei B, England L, & Ames BN (1989) Ascorbate is an outstanding antioxidant in human blood plasma. Proc Natl Acad Sci U S A 86(16):6377-6381.

164. Freire MA (2012) Pathophysiology of neurodegeneration following traumatic brain injury. West Indian Med J 61(7):751-755.

165. Frugier T, Morganti-Kossmann MC, O'Reilly D, & McLean CA (2010) In situ detection of inflammatory mediators in post mortem human brain tissue after traumatic injury. J Neurotrauma 27(3):497-507.

166. Gabbita SP , et al. (2005) Cleaved-tau: a biomarker of neuronal damage after traumatic brain injury. J Neurotrauma 22(1):83-94.

167. Gahm C, Holmin S, & Mathiesen T (2000) Temporal profiles and cellular sources of three nitric oxide synthase isoforms in the brain after experimental contusion. Neurosurgery 46(1):169-177.

168. Galluzzi L , et al. (2008) Methods to dissect mitochondrial membrane permeabilization in the course of apoptosis. Methods Enzymol 442:355-374.

169. Ganea E & Harding JJ (2006) Glutathione-related enzymes and the eye. Current eye research 31(1):1-11.

170. Gant JC, Sama MM, Landfield PW, & Thibault O (2006) Early and simultaneous emergence of multiple hippocampal biomarkers of aging is mediated by Ca2+-induced Ca2+ release. J Neurosci 26(13):3482-3490.

171. Gao WM , et al. (2007) A gel-based proteomic comparison of human cerebrospinal fluid between

inflicted and non-inflicted pediatric traumatic brain injury. J Neurotrauma 24(1):43-53.

172. Gentleman SM , et al. (2004) Long-term intracerebral inflammatory response after traumatic brain injury. Forensic science international 146(2-3):97-104.

173. Gentleman SM , et al. (1995) Axonal injury: a universal consequence of fatal closed head injury? Acta Neuropathol 89(6):537-543.

174. Gerlach M, Reichmann H, & Riederer P (2005) Levodopa in the treatment of Parkinson's disease: current controversies. Mov Disord 20(5):643; author reply 643-644.

175. Giannakopoulos P, Hof PR, Mottier S, Michel JP, & Bouras C (1994) Neuropathological changes in the cerebral cortex of 1258 cases from a geriatric hospital: retrospective clinicopathological evaluation of a 10-year autopsy population. Acta Neuropathol 87(5):456-468.

176. Giasson BI , et al. (2000) Oxidative damage linked to neurodegeneration by selective alpha- synuclein nitration in synucleinopathy lesions. Science 290(5493):985-989.

177. Gilgun-Sherki Y, Melamed E, & Offen D (2001) Oxidative stress induced-neurodegenerative diseases: the need for antioxidants that penetrate the blood brain barrier. Neuropharmacology 40(8):959-975.

178. Giusto NM, Salvador GA, Castagnet PI, Pasquare SJ, & Ilincheta de Boschero MG (2002) Age- associated changes in central nervous system glycerolipid composition and metabolism. Neurochem Res 27(11):1513-1523.

179. Godbout JP , et al. (2005) Exaggerated neuroinflammation and sickness behavior in aged mice following activation of the peripheral innate immune system. Faseb j 19(10):1329-1331.

180. Godbout JP & Johnson RW (2006) Age and neuroinflammation: a lifetime of psychoneuroimmune consequences. Neurologic clinics 24(3):521-538.

181. Goedert M, Clavaguera F, & Tolnay M (2010) The propagation of prion-like protein inclusions in neurodegenerative diseases. Trends Neurosci 33(7):317-325.

182. Gokulrangan G, Zaidi A, Michaelis ML, & Schoneich C (2007) Proteomic analysis of protein nitration in rat cerebellum: effect of biological aging. J Neurochem 100(6):1494-1504.

183. Goldman SM , et al. (2006) Head injury and Parkinson's disease risk in twins. Ann Neurol 60(1):65-72.

184. Gomez-Villafuertes R, Mellstrom B, & Naranjo JR (2007) Searching for a role of NCX/NCKX exchangers in neurodegeneration. Mol Neurobiol 35(2):195-202.

185. Gomez A & Ferrer I (2010) Involvement of the cerebral cortex in Parkinson disease linked with G2019S LRRK2 mutation without cognitive impairment. Acta Neuropathol 120(2):155-167.

186. Good PF, Hsu A, Werner P, Perl DP, & Olanow CW (1998) Protein nitration in Parkinson's disease. J Neuropathol Exp Neurol 57(4):338-342.

187. Govindan A , et al. (2011) Histopathologic and immunohistochemical profile of spinal glioblastoma: a study of six cases. Brain Tumor Pathol 28(4):297-303.

188. Goyal A , et al. (2013) S100b as a prognostic biomarker in outcome prediction for patients with severe traumatic brain injury. J Neurotrauma 30(11):946-957.

189. Graham DI, McIntosh TK, Maxwell WL, & Nicoll JA (2000) Recent advances in neurotrauma. J Neuropathol Exp Neurol 59(8):641-651.

190. Granacher RP (2003) Traumatic Brain Injury: Methods for Clinical and Forensic Neuropsychiatric Assessment (Taylor & Francis).

191. Griffith OW (1999) Biologic and pharmacologic regulation of mammalian glutathione synthesis. Free Radic Biol Med 27(9-10):922-935.

192. Grote S, Bocker W, Mutschler W, Bouillon B, & Lefering R (2011) Diagnostic value of the Glasgow Coma Scale for traumatic brain injury in 18,002 patients with severe multiple injuries. J Neurotrauma 28(4):527-534.

193. Grunblatt E , et al. (2009) Tryptophan is a marker of human postmortem brain tissue quality. J Neurochem 110(5):1400-1408.

194. Grunblatt E , et al. (2010) Brain tryptophan rather than pH-value is altered as consequence of artificial postmortem interval and storage conditions. Neurochem Int 57(7):819-822.

195. Grune T , et al. (1998) Peroxynitrite increases the degradation of aconitase and other cellular proteins by proteasome. J Biol Chem 273(18):10857-10862.

196. Grune T, Shringarpure R, Sitte N, & Davies K (2001) Age-related changes in protein oxidation and proteolysis in mammalian cells. J Gerontol A Biol Sci Med Sci 56(11):B459-467.

197. Gu M, Cooper JM, Taanman JW, & Schapira AH (1998) Mitochondrial DNA transmission of the mitochondrial defect in Parkinson's disease. Ann Neurol 44(2):177-186.

198. Guerra A, et al. (2000) Acid phosphatase, genetic polymorphism and cardiovascular risk factors in a pediatric population. Revista portuguesa de cardiologia : orgao oficial da Sociedade Portuguesa de Cardiologia = Portuguese journal of cardiology : an official journal of the Portuguese Society of Cardiology 19(6):679-691.

199. Guillozet AL, Weintraub S, Mash DC, & Mesulam MM (2003) Neurofibrillary tangles, amyloid, and memory in aging and mild cognitive impairment. Arch Neurol 60(5):729-736.

200. Gururaj G (2002) Epidemiology of traumatic brain injuries: Indian scenario. Neurol Res 24(1):24- 28.

201. Guthenberg C, Alin P, & Mannervik B (1985) Glutathione transferase from rat testis. Methods Enzymol 113:507-510.

202. Halliwell B (2006) Reactive species and antioxidants. Redox biology is a fundamental theme of aerobic life. Plant physiology 141(2):312-322.

203. Halliwell B & Gutteridge JM (1986) Oxygen free radicals and iron in relation to biology and medicine: some problems and concepts. Arch Biochem Biophys 246(2):501-514.

204. Halliwell B, Gutteridge JM, & Cross CE (1992) Free radicals, antioxidants, and human disease: where are we now? J Lab Clin Med 119(6):598-620.

205. Hammond CL, Lee TK, & Ballatori N (2001) Novel roles for glutathione in gene expression, cell death, and membrane transport of organic solutes. Journal of hepatology 34(6):946-954.

206. Han D, Antunes F, Canali R, Rettori D, & Cadenas E (2003) Voltage-dependent anion channels control the release of the superoxide anion from mitochondria to cytosol. J Biol Chem 278(8):5557-5563.

207. Han X, Shen T, & Lou H (2007) Dietary Polyphenols and Their Biological Significance. International Journal of Molecular Sciences 8(9):950-988.

208. Harish G , et al. (2012) Effect of pre and postmortem factors on the distribution and preservation of antioxidant activities in the cytosol and synaptosomes of human brains Biopreservation and Biobanking.

209. Harish G , et al. (2012) Mitochondrial function in human brains is affected by pre and postmortem factors. Neuropathol Appl Neurobiol.

210. Harish G , et al. (2011) Effect of Storage Time, Postmortem Interval, Agonal State, and Gender on the Postmortem Preservation of Glial Fibrillary Acidic Protein and Oxidatively Damaged Proteins in Human Brains. Biopreservation and Biobanking 9(4):379-387.

211. Harish G , et al. (2011) Glutathione metabolism is modulated by postmortem interval, gender difference and agonal state in postmortem human brains. Neurochem Int 59(7):1029-1042.

212. Harish G , et al. (2010) Bioconjugates of curcumin display improved protection against glutathione depletion mediated oxidative stress in a dopaminergic neuronal cell line: Implications for Parkinson's disease. Bioorg Med Chem 18(7):2631-2638.

213. Harman D (1991) The aging process: major risk factor for disease and death. Proc Natl Acad Sci U S A 88(12):5360-5363.

214. Harman D (1956) Aging: a theory based on free radical and radiation chemistry. J Gerontol 11(3):298-300.

215. Harman D (2006) Alzheimer's disease pathogenesis: role of aging. Ann N Y Acad Sci 1067:454­460.

216. Harrison PJ , et al. (1995) The relative importance of premortem acidosis and postmortem interval for human brain gene expression studies: selective mRNA vulnerability and comparison with their encoded proteins. Neurosci Lett 200(3):151-154.

217. Head E , et al. (2002) Oxidative damage increases with age in a canine model of human brain aging. J Neurochem 82(2):375-381.

218. Hensley K, Robinson KA, Gabbita SP, Salsman S, & Floyd RA (2000) Reactive oxygen species, cell signaling, and cell injury. Free Radic Biol Med 28(10):1456-1462.

219. Hernandez-Ontiveros DG , et al. (2013) Microglia activation as a biomarker for traumatic brain injury. Frontiers in neurology 4:30.

220. Herrmann M , et al. (1999) Protein S-100B and neuron specific enolase as early neurobiochemical markers of the severity of traumatic brain injury. Restorative neurology and neuroscience 14(2):109-114.

221. Hill KE, McCollum GW, & Burk RF (1997) Determination of thioredoxin reductase activity in rat liver supernatant. Anal Biochem 253(1):123-125.

222. Hissin PJ & Hilf R (1976) A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem 74(1):214-226.

223. Hoehn MM & Yahr MD (1967) Parkinsonism: onset, progression and mortality. Neurology 17(5):427-442.

224. Hof PR, Cox K, & Morrison JH (1990) Quantitative analysis of a vulnerable subset of pyramidal neurons in Alzheimer's disease: I. Superior frontal and inferior temporal cortex. J Comp Neurol 301(1):44-54.

225. Hof PR , et al. (2002) Age-related changes in GluR2 and NMDAR1 glutamate receptor subunit protein immunoreactivity in corticocortically projecting neurons in macaque and patas monkeys. Brain Res 928(1-2):175-186.

226. Holley JE, Newcombe J, Winyard PG, & Gutowski NJ (2007) Peroxiredoxin V in multiple sclerosis lesions: predominant expression by astrocytes. Multiple sclerosis (Houndmills, Basingstoke, England) 13(8):955-961.

227. Holmin S & Mathiesen T (1999) Long-term intracerebral inflammatory response after experimental focal brain injury in rat. Neuroreport 10(9):1889-1891.

228. Holmin S , et al. (1997) Delayed cytokine expression in rat brain following experimental contusion. J Neurosurg 86(3):493-504.

229. Holmin S, Soderlund J, Biberfeld P, & Mathiesen T (1998) Intracerebral inflammation after human brain contusion. Neurosurgery 42(2):291-298; discussion 298-299.

230. Horowitz MP & Greenamyre JT (2010) Gene-environment interactions in Parkinson's disease: the importance of animal modeling. Clinical pharmacology and therapeutics 88(4):467-474.

231. Hortobagyi T , et al. (2007) Traumatic axonal damage in the brain can be detected using beta- APP immunohistochemistry within 35 min after head injury to human adults. Neuropathol Appl Neurobiol 33(2):226-237.

232. Houlden H & Singleton AB (2012) The genetics and neuropathology of Parkinson's disease. Acta Neuropathol 124(3):325-338.

233. Husi H, Ward MA, Choudhary JS, Blackstock WP, & Grant SG (2000) Proteomic analysis of NMDA receptor-adhesion protein signaling complexes. Nat Neurosci 3(7):661-669.

234. Hutson CB , et al. (2011) Traumatic brain injury in adult rats causes progressive nigrostriatal dopaminergic cell loss and enhanced vulnerability to the pesticide paraquat. J Neurotrauma 28(9):1783-1801.

235. Hyder AA, Amach OH, Garg N, & Labinjo MT (2006) Estimating the burden of road traffic injuries among children and adolescents in urban South Asia. Health Policy 77(2):129-139.

236. Hynd MR, Lewohl JM, Scott HL, & Dodd PR (2003) Biochemical and molecular studies using human autopsy brain tissue. J Neurochem 85(3):543-562.

237. Iglesias-Gonzalez J , et al. (2012) Differential toxicity of 6-hydroxydopamine in SH-SY5Y human neuroblastoma cells and rat brain mitochondria: protective role of catalase and superoxide dismutase. Neurochem Res 37(10):2150-2160.

238. Ignarro LJ (1990) Haem-dependent activation of guanylate cyclase and cyclic GMP formation by endogenous nitric oxide: a unique transduction mechanism for transcellular signaling. Pharmacology & toxicology 67(1):1-7.

239. Ishihara T , et al. (2001) Age-dependent induction of congophilic neurofibrillary tau inclusions in tau transgenic mice. The American journal of pathology 158(2):555-562.

240. Itoh K, Weis S, Mehraein P, & Muller-Hocker J (1996) Cytochrome c oxidase defects of the human substantia nigra in normal aging. Neurobiol Aging 17(6):843-848.

241. Iwasaki Y, Shiojima T, & Kinoshita M (1997) S100 beta prevents the death of motor neurons in newborn rats after sciatic nerve section. J Neurol Sci 151(1):7-12.

242. Iwunze MO & McEwan D (2004) Peroxynitrite interaction with curcumin solubilized in ethanolic solution. Cell Mol Biol (Noisy-le-grand) 50(6):749-752.

243. Jagatha B, Mythri RB, Vali S, & Bharath MM (2008) Curcumin treatment alleviates the effects of glutathione depletion in vitro and in vivo: therapeutic implications for Parkinson's disease explained via in silico studies. Free Radic Biol Med 44(5):907-917.

244. Jellinger KA (2012) Neuropathology of sporadic Parkinson's disease: evaluation and changes of concepts. Mov Disord 27(1):8-30.

245. Jenkins LW , et al. (1989) Increased vulnerability of the mildly traumatized rat brain to cerebral ischemia: the use of controlled secondary ischemia as a research tool to identify common or different mechanisms contributing to mechanical and ischemic brain injury. Brain Res 477(1- 2):211-224.

246. Jenkins LW , et al. (2002) Conventional and functional proteomics using large format two­dimensional gel electrophoresis 24 hours after controlled cortical impact in postnatal day 17 rats. J Neurotrauma 19(6):715-740.

247. Jenner P (2003) Oxidative stress in Parkinson's disease. Ann Neurol 53 Suppl 3:S26-36; discussion S36-28.

248. Jenner P (2004) Preclinical evidence for neuroprotection with monoamine oxidase-B inhibitors in Parkinson's disease. Neurology 63(7 Suppl 2):S13-22.

249. Jenner P, Dexter DT, Sian J, Schapira AH, & Marsden CD (1992) Oxidative stress as a cause of nigral cell death in Parkinson's disease and incidental Lewy body disease. The Royal Kings and Queens Parkinson's Disease Research Group. Ann Neurol 32 Suppl:S82-87.

250. Jennett B & Bond M (1975) Assessment of outcome after severe brain damage. Lancet 1(7905):480-484.

251. Jeyapaul J & Jaiswal AK (2000) Nrf2 and c-Jun regulation of antioxidant response element (ARE)- mediated expression and induction of gamma-glutamylcysteine synthetase heavy subunit gene. Biochem Pharmacol 59(11):1433-1439.

252. Jiang ZY, Woollard AC, & Wolff SP (1990) Hydrogen peroxide production during experimental protein glycation. FEBS Lett 268(1):69-71.

253. Jiao Y , et al. (2006) Iron chelation in the biological activity of curcumin. Free Radic Biol Med 40(7):1152-1160.

254. Jiao Y , et al. (2009) Curcumin, a cancer chemopreventive and chemotherapeutic agent, is a biologically active iron chelator. Blood 113(2):462-469.

255. Johnson JA, el Barbary A, Kornguth SE, Brugge JF, & Siegel FL (1993) Glutathione S-transferase isoenzymes in rat brain neurons and glia. J Neurosci 13(5):2013-2023.

256. Johnson VE , et al. (2013) Inflammation and white matter degeneration persist for years after a single traumatic brain injury. Brain 136(Pt 1):28-42.

257. Johnson VE, Stewart W, & Smith DH (2010) Traumatic brain injury and amyloid-[beta] pathology: a link to Alzheimer's disease? Nat Rev Neurosci 11(5):361-370.

258. Johnson VE, Stewart W, & Smith DH (2012) Widespread tau and amyloid-beta pathology many years after a single traumatic brain injury in humans. Brain Pathol 22(2):142-149.

259. Johnson WM, Wilson-Delfosse AL, & Mieyal JJ (2012) Dysregulation of glutathione homeostasis in neurodegenerative diseases. Nutrients 4(10):1399-1440.

260. Johnsson P (1996) Markers of cerebral ischemia after cardiac surgery. Journal of cardiothoracic and vascular anesthesia 10(1):120-126.

261. Johnston NL, Cervenak J, Shore AD, Torrey EF, & Yolken RH (1997) Multivariate analysis of RNA levels from postmortem human brains as measured by three different methods of RT-PCR. Stanley Neuropathology Consortium. J Neurosci Methods 77(1):83-92.

262. Kalaitzakis ME, Graeber MB, Gentleman SM, & Pearce RK (2008) The dorsal motor nucleus of the vagus is not an obligatory trigger site of Parkinson's disease: a critical analysis of alpha-synuclein staging. Neuropathol Appl Neurobiol 34(3):284-295.

263. Kampfl A , et al. (1996) mu-calpain activation and calpain-mediated cytoskeletal proteolysis following traumatic brain injury. J Neurochem 67(4):1575-1583.

264. Kang Y , et al. (1999) Brain gamma-glutamyl cysteine synthetase (GCS) mRNA expression patterns correlate with regional-specific enzyme activities and glutathione levels. J Neurosci Res 58(3):436-441.

265. Kannan K & Jain SK (2000) Oxidative stress and apoptosis. Pathophysiology : the official journal of the International Society for Pathophysiology / ISP 7(3):153-163.

266. Kavalci C , et al. (2007) The value of serum tau protein for the diagnosis of intracranial injury in minor head trauma. The American journal of emergency medicine 25(4):391-395.

267. Keeney PM, Xie J, Capaldi RA, & Bennett JP, Jr. (2006) Parkinson's disease brain mitochondrial complex I has oxidatively damaged subunits and is functionally impaired and misassembled. J Neurosci 26(19):5256-5264.

268. Keller JN, Hanni KB, & Markesbery WR (2000) Possible involvement of proteasome inhibition in aging: implications for oxidative stress. Mech Ageing Dev 113(1):61-70.

269. Ketonen LM (1998) Neuroimaging of the aging brain. Neurologic clinics 16(3):581-598.

270. Kim HG , et al. (2003) Age-related changes in the activity of antioxidant and redox enzymes in rats. Mol Cells 16(3):278-284.

271. Kim SJ , et al. (2008) Curcumin stimulates proliferation of embryonic neural progenitor cells and neurogenesis in the adult hippocampus. J Biol Chem 283(21):14497-14505.

272. Kingsbury AE , et al. (1995) Tissue pH as an indicator of mRNA preservation in human post­mortem brain. Brain Res Mol Brain Res 28(2):311-318.

273. Kishido T , et al. (2007) Decline in glutathione peroxidase activity is a reason for brain senescence: consumption of green tea catechin prevents the decline in its activity and protein oxidative damage in ageing mouse brain. Biogerontology 8(4):423-430.

274. Kitto GB & Lewis RG (1967) Purification and properties of tuna supernatant and mitochondrial malate dehydrogenases. Biochimica et Biophysica Acta (BBA) - Enzymology 139(1):1-15.

275. Kohen R, Misgav R, & Ginsburg I (1991) The SOD like activity of copper:carnosine, copper:anserine and copper:homocarnosine complexes. Free radical research communications 12-13 Pt 1:179-185.

276. Kordower JH, Chu Y, Hauser RA, Freeman TB, & Olanow CW (2008) Lewy body-like pathology in long-term embryonic nigral transplants in Parkinson's disease. Nat Med 14(5):504-506.

277. Kroppenstedt SN , et al. (1999) Effect of cerebral perfusion pressure on contusion volume following impact injury. J Neurosurg 90(3):520-526.

278. Kumar A, Bodhinathan K, & Foster TC (2009) Susceptibility to Calcium Dysregulation during Brain Aging. Front Aging Neurosci 1:2.

279. Kumar A & Loane DJ (2012) Neuroinflammation after traumatic brain injury: opportunities for therapeutic intervention. Brain, behavior, and immunity 26(8):1191-1201.

280. Kumar A, Naidu PS, Seghal N, & Padi SS (2007) Effect of curcumin on intracerebroventricular colchicine-induced cognitive impairment and oxidative stress in rats. J Med Food 10(3):486-494.

281. Kumar V (2006) Potential medicinal plants for CNS disorders: an overview. Phytother Res 20(12):1023-1035.

282. Kume-Kick J, Ferris DC, Russo-Menna I, & Rice ME (1996) Enhanced oxidative stress in female rat brain after gonadectomy. Brain Res 738(1):8-14.

283. Kushnareva Y, Murphy AN, & Andreyev A (2002) Complex I-mediated reactive oxygen species generation: modulation by cytochrome c and NAD(P)+ oxidation-reduction state. Biochem J 368(Pt 2):545-553.

284. LaFrance R, Brustovetsky N, Sherburne C, Delong D, & Dubinsky JM (2005) Age-related changes in regional brain mitochondria from Fischer 344 rats. Aging Cell 4(3):139-145.

285. Lam PY, Yin F, Hamilton RT, Boveris A, & Cadenas E (2009) Elevated neuronal nitric oxide synthase expression during ageing and mitochondrial energy production. Free Radic Res 43(5):431-439.

286. Lang-Rollin IC, Rideout HJ, Noticewala M, & Stefanis L (2003) Mechanisms of caspase­independent neuronal death: energy depletion and free radical generation. J Neurosci 23(35):11015-11025.

287. Langston JW (2006) The Parkinson's complex: parkinsonism is just the tip of the iceberg. Ann Neurol 59(4):591-596.

288. Langston JW, Ballard P, Tetrud JW, & Irwin I (1983) Chronic Parkinsonism in humans due to a product of meperidine-analog synthesis. Science 219(4587):979-980.

289. Langston JW & Ballard PA, Jr. (1983) Parkinson's disease in a chemist working with 1-methyl-4- phenyl-1,2,5,6-tetrahydropyridine. N Engl J Med 309(5):310.

290. Lao CD , et al. (2006) Dose escalation of a curcuminoid formulation. BMC Complement Altern Med 6:10.

291. LaPlaca MC , et al. (2001) Pharmacologic inhibition of poly(ADP-ribose) polymerase is neuroprotective following traumatic brain injury in rats. J Neurotrauma 18(4):369-376.

292. LaVoie MJ & Hastings TG (1999) Dopamine quinone formation and protein modification associated with the striatal neurotoxicity of methamphetamine: evidence against a role for extracellular dopamine. J Neurosci 19(4):1484-1491.

293. LaVoie MJ, Ostaszewski BL, Weihofen A, Schlossmacher MG, & Selkoe DJ (2005) Dopamine covalently modifies and functionally inactivates parkin. Nat Med 11(11):1214-1221.

294. Lavoie S , et al. (2009) Curcumin, quercetin, and tBHQ modulate glutathione levels in astrocytes and neurons: importance of the glutamate cysteine ligase modifier subunit. J Neurochem 108(6):1410-1422.

295. Lee CG , et al. (2011) Role of chitin and chitinase/chitinase-like proteins in inflammation, tissue remodeling, and injury. Annu Rev Physiol 73:479-501.

296. Lee CK, Weindruch R, & Prolla TA (2000) Gene-expression profile of the ageing brain in mice. Nat Genet 25(3):294-297.

297. Levine RL , et al. (1990) Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol 186:464-478.

298. Lewen A , et al. (2001) Oxidative stress-dependent release of mitochondrial cytochrome c after traumatic brain injury. J Cereb Blood Flow Metab 21(8):914-920.

299. Lewis DA (2002) The human brain revisited: opportunities and challenges in postmortem studies of psychiatric disorders. Neuropsychopharmacology 26(2):143-154.

300. Li JZ , et al. (2004) Systematic changes in gene expression in postmortem human brains associated with tissue pH and terminal medical conditions. Hum Mol Genet 13(6):609-616.

301. Lifshitz J, Kelley BJ, & Povlishock JT (2007) Perisomatic thalamic axotomy after diffuse traumatic brain injury is associated with atrophy rather than cell death. J Neuropathol Exp Neurol 66(3):218-229.

302. Lifshitz J, Sullivan PG, Hovda DA, Wieloch T, & McIntosh TK (2004) Mitochondrial damage and dysfunction in traumatic brain injury. Mitochondrion 4(5-6):705-713.

303. Lim GP , et al. (2001) The curry spice curcumin reduces oxidative damage and amyloid pathology in an Alzheimer transgenic mouse. J Neurosci 21(21):8370-8377.

304. Lin MT & Beal MF (2006) Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443(7113):787-795.

305. Lin MT, Simon DK, Ahn CH, Kim LM, & Beal MF (2002) High aggregate burden of somatic mtDNA point mutations in aging and Alzheimer's disease brain. Hum Mol Genet 11(2):133-145.

306. Lin Y & Wen L (2013) Inflammatory response following diffuse axonal injury. International journal of medical sciences 10(5):515-521.

307. Ling ZD , et al. (2000) Striatal trophic activity is reduced in the aged rat brain. Brain Res 856(1- 2):301-309.

308. Lisacek F , et al. (2004) Shaping biological knowledge: applications in proteomics. Comparative and functional genomics 5(2):190-195.

309. Liu L, Chen XL, Yang JK, Ren ZG, & Wang S (2012) Gliosis after traumatic brain injury in conditional ephrinB2-knockout mice. Chinese medical journal 125(21):3831-3835.

310. Liu RM (2002) Down-regulation of gamma-glutamylcysteine synthetase regulatory subunit gene expression in rat brain tissue during aging. J Neurosci Res 68(3):344-351.

311. Loane DJ & Faden AI (2010) Neuroprotection for traumatic brain injury: translational challenges and emerging therapeutic strategies. Trends in pharmacological sciences 31(12):596-604.

312. Lok J , et al. (2011) gamma-glutamylcysteine ethyl ester protects cerebral endothelial cells during injury and decreases blood-brain barrier permeability after experimental brain trauma. J Neurochem 118(2):248-255.

313. Lu T , et al. (2004) Gene regulation and DNA damage in the ageing human brain. Nature 429(6994):883-891.

314. Luk KC , et al. (2009) Exogenous alpha-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells. Proc Natl Acad Sci U S A 106(47):20051-20056.

315. Luo P, Fei F, Zhang L, Qu Y, & Fei Z (2011) The role of glutamate receptors in traumatic brain injury: implications for postsynaptic density in pathophysiology. Brain research bulletin 85(6):313-320.

316. Luo Y, Hattori A, Munoz J, Qin ZH, & Roth GS (1999) Intrastriatal dopamine injection induces apoptosis through oxidation-involved activation of transcription factors AP-1 and NF-kappaB in rats. Mol Pharmacol 56(2):254-264.

317. Lupien SJ, McEwen BS, Gunnar MR, & Heim C (2009) Effects of stress throughout the lifespan on the brain, behaviour and cognition. Nat Rev Neurosci 10(6):434-445.

318. Malinska D , et al. (2010) Complex III-dependent superoxide production of brain mitochondria contributes to seizure-related ROS formation. Biochim Biophys Acta 1797(6-7):1163-1170.

319. Mandal PK, Tripathi M, & Sugunan S (2012) Brain oxidative stress: detection and mapping of anti-oxidant marker 'Glutathione' in different brain regions of healthy male/female, MCI and Alzheimer patients using non-invasive magnetic resonance spectroscopy. Biochem Biophys Res Commun 417(1):43-48.

320. Marimuthu A , et al. (2013) Identification of head and neck squamous cell carcinoma biomarker candidates through proteomic analysis of cancer cell secretome. Biochim Biophys Acta 1834(11):2308-2316.

321. Marklund N, Bakshi A, Castelbuono DJ, Conte V, & McIntosh TK (2006) Evaluation of pharmacological treatment strategies in traumatic brain injury. Curr Pharm Des 12(13):1645- 1680.

322. Marmarou A , et al. (1994) A new model of diffuse brain injury in rats. Part I: Pathophysiology and biomechanics. J Neurosurg 80(2):291-300.

323. Martinez A, Portero-Otin M, Pamplona R, & Ferrer I (2010) Protein targets of oxidative damage in human neurodegenerative diseases with abnormal protein aggregates. Brain Pathol 20(2):281-297.

324. Masel BE & DeWitt DS (2010) Traumatic brain injury: a disease process, not an event. J Neurotrauma 27(8):1529-1540.

325. Masson F , et al. (2001) Epidemiology of severe brain injuries: a prospective population-based study. J Trauma 51(3):481-489.

326. Mather M & Rottenberg H (2000) Aging enhances the activation of the permeability transition pore in mitochondria. Biochem Biophys Res Commun 273(2):603-608.

327. Mattson MP & Magnus T (2006) Ageing and neuronal vulnerability. Nat Rev Neurosci 7(4):278- 294.

328. Mattson MP, Son TG, & Camandola S (2007) Viewpoint: mechanisms of action and therapeutic potential of neurohormetic phytochemicals. Dose Response 5(3):174-186.

329. Maxwell WL & Graham DI (1997) Loss of axonal microtubules and neurofilaments after stretch­injury to guinea pig optic nerve fibers. J Neurotrauma 14(9):603-614.

330. McCord JM & Fridovich I (1969) Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J Biol Chem 244(22):6049-6055.

331. McCormack AL, Mak SK, & Di Monte DA (2012) Increased alpha-synuclein phosphorylation and nitration in the aging primate substantia nigra. Cell Death Dis 3:e315.

332. McIlwain CC, Townsend DM, & Tew KD (2006) Glutathione S-transferase polymorphisms: cancer incidence and therapy. Oncogene 25(11):1639-1648.

333. McIntosh TK, Noble L, Andrews B, & Faden AI (1987) Traumatic brain injury in the rat: characterization of a midline fluid-percussion model. Central nervous system trauma : journal of the American Paralysis Association 4(2):119-134.

334. McKeating EG, Andrews PJ, & Mascia L (1998) Relationship of neuron specific enolase and protein S-100 concentrations in systemic and jugular venous serum to injury severity and outcome after traumatic brain injury. Acta neurochirurgica. Supplement 71:117-119.

335. Medvedev ZA (1990) An attempt at a rational classification of theories of ageing. Biol Rev Camb Philos Soc 65(3):375-398.

336. Mesenge C , et al. (1998) Protective effect of melatonin in a model of traumatic brain injury in mice. J Pineal Res 25(1):41-46.

337. Metting Z, Wilczak N, Rodiger LA, Schaaf JM, & van der Naalt J (2012) GFAP and S100B in the acute phase of mild traumatic brain injury. Neurology 78(18):1428-1433.

338. Miller CL, Diglisic S, Leister F, Webster M, & Yolken RH (2004) Evaluating RNA status for RT-PCR in extracts of postmortem human brain tissue. Biotechniques 36(4):628-633.

339. Minambres E , et al. (2008) Cerebral apoptosis in severe traumatic brain injury patients: an in vitro, in vivo, and postmortem study. J Neurotrauma 25(6):581-591.

340. Mishra S, Narain U, Mishra R, & Misra K (2005) Design, development and synthesis of mixed bioconjugates of piperic acid-glycine, curcumin-glycine/alanine and curcumin-glycine-piperic acid and their antibacterial and antifungal properties. Bioorg Med Chem 13(5):1477-1486.

341. Miyoshi N, Oubrahim H, Chock PB, & Stadtman ER (2006) Age-dependent cell death and the role of ATP in hydrogen peroxide-induced apoptosis and necrosis. Proc Natl Acad Sci U S A 103(6):1727-1731.

342. Mondello S , et al. (2010) alphaII-spectrin breakdown products (SBDPs): diagnosis and outcome in severe traumatic brain injury patients. J Neurotrauma 27(7):1203-1213.

343. Monoranu CM , et al. (2009) pH measurement as quality control on human post mortem brain tissue: a study of the BrainNet Europe consortium. Neuropathol Appl Neurobiol 35(3):329-337.

344. Monoranu CM , et al. (2010) Methyl- and acetyltransferases are stable epigenetic markers postmortem. Cell Tissue Bank.

345. Moore DJ, West AB, Dawson VL, & Dawson TM (2005) Molecular pathophysiology of Parkinson's disease. Annual review of neuroscience 28:57-87.

346. Morganti-Kossmann MC, Rancan M, Stahel PF, & Kossmann T (2002) Inflammatory response in acute traumatic brain injury: a double-edged sword. Current opinion in critical care 8(2):101- 105.

347. Moritani T, Smoker WR, Sato Y, Numaguchi Y, & Westesson P-LA (2005) Diffusion-weighted imaging of acute excitotoxic brain injury. American journal of neuroradiology 26(2):216-228.

348. Morrison B, 3rd, Cater HL, Benham CD, & Sundstrom LE (2006) An in vitro model of traumatic brain injury utilising two-dimensional stretch of organotypic hippocampal slice cultures. J Neurosci Methods 150(2):192-201.

349. Morrison B, 3rd, Elkin BS, Dolle JP, & Yarmush ML (2011) In vitro models of traumatic brain injury. Annual review of biomedical engineering 13:91-126.

350. Morrison RS , et al. (2002) Proteomic analysis in the neurosciences. Molecular & cellular proteomics : MCP 1(8):553-560.

351. Mougenot AL , et al. (2012) Prion-like acceleration of a synucleinopathy in a transgenic mouse model. Neurobiol Aging 33(9):2225-2228.

352. Mukhin AG, Ivanova SA, Allen JW, & Faden AI (1998) Mechanical injury to neuronal/glial cultures in microplates: role of NMDA receptors and pH in secondary neuronal cell death. J Neurosci Res 51(6):748-758.

353. Mukhin AG, Ivanova SA, Knoblach SM, & Faden AI (1997) New in vitro model of traumatic neuronal injury: evaluation of secondary injury and glutamate receptor-mediated neurotoxicity. J Neurotrauma 14(9):651-663.

354. Murdoch I, Perry EK, Court JA, Graham DI, & Dewar D (1998) Cortical cholinergic dysfunction after human head injury. J Neurotrauma 15(5):295-305.

355. Murphy EJ & Horrocks LA (1993) A model for compression trauma: pressure-induced injury in cell cultures. J Neurotrauma 10(4):431-444.

356. Murray J, Taylor SW, Zhang B, Ghosh SS, & Capaldi RA (2003) Oxidative damage to mitochondrial complex I due to peroxynitrite: identification of reactive tyrosines by mass spectrometry. J Biol Chem 278(39):37223-37230.

357. Mustacich D & Powis G (2000) Thioredoxin reductase. Biochem J 346 Pt 1:1-8.

358. Mythri RB, Harish G, Dubey SK, Misra K, & Bharath MM (2011) Glutamoyl diester of the dietary polyphenol curcumin offers improved protection against peroxynitrite-mediated nitrosative stress and damage of brain mitochondria in vitro: implications for Parkinson's disease. Mol Cell Biochem 347(1-2):135-143.

359. Mythri RB, Jagatha B, Pradhan N, Andersen J, & Bharath MM (2007) Mitochondrial complex I inhibition in Parkinson's disease: how can curcumin protect mitochondria? Antioxid Redox Signal 9(3):399-408.

360. Mythri RB, Veena J, Harish G, Shankaranarayana Rao BS, & Srinivas Bharath MM (2011) Chronic dietary supplementation with turmeric protects against 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine-mediated neurotoxicity in vivo: implications for Parkinson's disease. Br J Nutr 106(1):63-72.

361. Mythri RB , et al. (2011) Evaluation of markers of oxidative stress, antioxidant function and astrocytic proliferation in the striatum and frontal cortex of Parkinson's disease brains. Neurochem Res 36(8):1452-1463.

362. Napankangas JP , et al. (2012) Superoxide production during ischemia-reperfusion in the perfused rat heart: a comparison of two methods of measurement. Journal of molecular and cellular cardiology 53(6):906-915.

363. Narotam PK , et al. (1998) Traumatic brain contusions: a clinical role for the kinin antagonist CP- 0127. Acta Neurochir (Wien) 140(8):793-802; discussion 802-793.

364. Navarro A & Boveris A (2010) Brain mitochondrial dysfunction in aging, neurodegeneration, and Parkinson's disease. Front Aging Neurosci 2.

365. Navarro A & Boveris A (2007) The mitochondrial energy transduction system and the aging process. American journal of physiology. Cell physiology 292(2):C670-686.

366. Navarro A & Boveris A (2004) Rat brain and liver mitochondria develop oxidative stress and lose enzymatic activities on aging. American journal of physiology. Regulatory, integrative and comparative physiology 287(5):R1244-1249.

367. Navarro A , et al. (2005) Vitamin E at high doses improves survival, neurological performance, and brain mitochondrial function in aging male mice. American journal of physiology. Regulatory, integrative and comparative physiology 289(5):R1392-1399.

368. Navarro A , et al. (2008) Hippocampal mitochondrial dysfunction in rat aging. American journal of physiology. Regulatory, integrative and comparative physiology 294(2):R501-509.

369. Negre-Salvayre A, Coatrieux C, Ingueneau C, & Salvayre R (2008) Advanced lipid peroxidation end products in oxidative damage to proteins. Potential role in diseases and therapeutic prospects for the inhibitors. British journal of pharmacology 153(1):6-20.

370. Neselius S , et al. (2012) CSF-biomarkers in Olympic boxing: diagnosis and effects of repetitive head trauma. PLoS One 7(4):e33606.

371. Nordberg J & Arner ES (2001) Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic Biol Med 31(11):1287-1312.

372. Nunomura A , et al. (2006) Oxidative damage to RNA in neurodegenerative diseases. J Biomed Biotechnol 2006(3):82323.

373. Nygaard O, Langbakk B, & Romner B (1998) Neuron-specific enolase concentrations in serum and cerebrospinal fluid in patients with no previous history of neurological disorder. Scandinavian journal of clinical and laboratory investigation 58(3):183-186.

374. Nylen K , et al. (2006) Increased serum-GFAP in patients with severe traumatic brain injury is related to outcome. J Neurol Sci 240(1-2):85-91.

375. O'Donnell VB , et al. (1999) Nitration of unsaturated fatty acids by nitric oxide-derived reactive nitrogen species peroxynitrite, nitrous acid, nitrogen dioxide, and nitronium ion. Chemical research in toxicology 12(1):83-92.

376. Ohashi T , et al. (2002) Rapid oxidation of dichlorodihydrofluorescin with heme and hemoproteins: formation of the fluorescein is independent of the generation of reactive oxygen species. FEBS Lett 511(1-3):21-27.

377. Ohkawa H, Ohishi N, & Yagi K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95(2):351-358.

378. Okonkwo DO , et al. (2013) GFAP-BDP as an Acute Diagnostic Marker in Traumatic Brain Injury: Results from the Prospective Transforming Research and Clinical Knowledge in Traumatic Brain Injury Study. J Neurotrauma 30(17):1490-1497.

379. Oli MW, Hayes RL, Robinson G, & Wang KK (2009) Traumatic brain injury biomarkers: from pipeline to diagnostic assay development. Methods Mol Biol 566:293-302.

380. Ondruschka B , et al. (2013) S100B and NSE as Useful Postmortem Biochemical Markers of Traumatic Brain Injury in Autopsy Cases. J Neurotrauma.

381. Ono K & Yamada M (2006) Antioxidant compounds have potent anti-fibrillogenic and fibril­destabilizing effects for alpha-synuclein fibrils in vitro. J Neurochem 97(1):105-115.

382. Opii WO , et al. (2007) Proteomic identification of oxidized mitochondrial proteins following experimental traumatic brain injury. J Neurotrauma 24(5):772-789.

383. Ota M , et al. (2006) Age-related decline of dopamine synthesis in the living human brain measured by positron emission tomography with L-[beta-11C]DOPA. Life Sci 79(8):730-736.

384. Owen L & Sunram-Lea SI (2011) Metabolic agents that enhance ATP can improve cognitive functioning: a review of the evidence for glucose, oxygen, pyruvate, creatine, and L-carnitine. Nutrients 3(8):735-755.

385. Packer L, Witt EH, & Tritschler HJ (1995) alpha-Lipoic acid as a biological antioxidant. Free Radic Biol Med 19(2):227-250.

386. Pal R, Miranda M, & Narayan M (2011) Nitrosative stress-induced Parkinsonian Lewy-like aggregates prevented through polyphenolic phytochemical analog intervention. Biochem Biophys Res Commun 404(1):324-329.

387. Palmer AM , et al. (1993) Traumatic brain injury-induced excitotoxicity assessed in a controlled cortical impact model. J Neurochem 61(6):2015-2024.

388. Pan MH, Lin-Shiau SY, & Lin JK (2000) Comparative studies on the suppression of nitric oxide synthase by curcumin and its hydrogenated metabolites through down-regulation of IkappaB kinase and NFkappaB activation in macrophages. Biochem Pharmacol 60(11):1665-1676.

389. Pandey N, Strider J, Nolan WC, Yan SX, & Galvin JE (2008) Curcumin inhibits aggregation of alpha-synuclein. Acta Neuropathol 115(4):479-489.

390. Papa L , et al. (2008) Use of biomarkers for diagnosis and management of traumatic brain injury patients. Expert opinion on medical diagnostics 2(8):937-945.

391. Papaioannou N , et al. (2001) Immunohistochemical investigation of the brain of aged dogs. I. Detection of neurofibrillary tangles and of 4-hydroxynonenal protein, an oxidative damage product, in senile plaques. Amyloid 8(1):11-21.

392. Pardue S, Zimmerman AL, & Morrison-Bogorad M (1994) Selective postmortem degradation of inducible heat shock protein 70 (hsp70) mRNAs in rat brain. Cell Mol Neurobiol 14(4):341-357.

393. Parker WD, Jr., Parks JK, & Swerdlow RH (2008) Complex I deficiency in Parkinson's disease frontal cortex. Brain Res 1189:215-218.

394. Pearce RK, Owen A, Daniel S, Jenner P, & Marsden CD (1997) Alterations in the distribution of glutathione in the substantia nigra in Parkinson's disease. J Neural Transm 104(6-7):661-677.

395. Pelinka LE , et al. (2004) Glial fibrillary acidic protein in serum after traumatic brain injury and multiple trauma. J Trauma 57(5):1006-1012.

396. Peluffo H, Acarin L, Faiz M, Castellano B, & Gonzalez B (2005) Cu/Zn superoxide dismutase expression in the postnatal rat brain following an excitotoxic injury. J Neuroinflammation 2(1):12.

397. Pennathur S, Jackson-Lewis V, Przedborski S, & Heinecke JW (1999) Mass spectrometric quantification of 3-nitrotyrosine, ortho-tyrosine, and o,o'-dityrosine in brain tissue of 1-methyl- 4-phenyl-1,2,3, 6-tetrahydropyridine-treated mice, a model of oxidative stress in Parkinson's disease. J Biol Chem 274(49):34621-34628.

398. Pennington RJ (1961) Biochemistry of dystrophic muscle. Mitochondrial succinate-tetrazolium reductase and adenosine triphosphatase. Biochem J 80:649-654.

399. Perry EK, Perry RH, & Tomlinson BE (1982) The influence of agonal status on some neurochemical activities of postmortem human brain tissue. Neurosci Lett 29(3):303-307.

400. Peterson C & Cotman CW (1989) Strain-dependent decrease in glutamate binding to the N- methyl-D-aspartic acid receptor during aging. Neurosci Lett 104(3):309-313.

401. Petrosillo G, Matera M, Casanova G, Ruggiero FM, & Paradies G (2008) Mitochondrial dysfunction in rat brain with aging Involvement of complex I, reactive oxygen species and cardiolipin. Neurochem Int 53(5):126-131.

402. Phizicky E, Bastiaens PI, Zhu H, Snyder M, & Fields S (2003) Protein analysis on a proteomic scale. Nature 422(6928):208-215.

403. Pike BR , et al. (2001) Accumulation of non-erythroid alpha II-spectrin and calpain-cleaved alpha II-spectrin breakdown products in cerebrospinal fluid after traumatic brain injury in rats. J Neurochem 78(6):1297-1306.

404. Pocernich CB, Cardin AL, Racine CL, Lauderback CM, & Butterfield DA (2001) Glutathione elevation and its protective role in acrolein-induced protein damage in synaptosomal membranes: relevance to brain lipid peroxidation in neurodegenerative disease. Neurochem Int 39(2):141-149.

405. Pocernich CB, La Fontaine M, & Butterfield DA (2000) In-vivo glutathione elevation protects against hydroxyl free radical-induced protein oxidation in rat brain. Neurochem Int 36(3):185- 191.

406. Posmantur RM, Newcomb JK, Kampfl A, & Hayes RL (2000) Light and confocal microscopic studies of evolutionary changes in neurofilament proteins following cortical impact injury in the rat. Exp Neurol 161(1):15-26.

407. Post B, Merkus MP, de Bie RM, de Haan RJ, & Speelman JD (2005) Unified Parkinson's disease rating scale motor examination: are ratings of nurses, residents in neurology, and movement disorders specialists interchangeable? Mov Disord 20(12):1577-1584.

408. Povlishock JT, Hayes RL, Michel ME, & McIntosh TK (1994) Workshop on animal models of traumatic brain injury. J Neurotrauma 11(6):723-732.

409. Power JH & Blumbergs PC (2009) Cellular glutathione peroxidase in human brain: cellular distribution, and its potential role in the degradation of Lewy bodies in Parkinson's disease and dementia with Lewy bodies. Acta Neuropathol 117(1):63-73.

410. Prasad KN , et al. (1994) Establishment and characterization of immortalized clonal cell lines from fetal rat mesencephalic tissue. In Vitro Cell Dev Biol Anim 30A(9):596-603.

411. Preece P & Cairns NJ (2003) Quantifying mRNA in postmortem human brain: influence of gender, age at death, postmortem interval, brain pH, agonal state and inter-lobe mRNA variance. Brain Res Mol Brain Res 118(1-2):60-71.

412. Priyadarsini KI , et al. (2003) Role of phenolic O-H and methylene hydrogen on the free radical reactions and antioxidant activity of curcumin. Free Radic Biol Med 35(5):475-484.

413. Przedborski S , et al. (2001) Oxidative post-translational modifications of alpha-synuclein in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. J Neurochem 76(2):637-640.

414. Przedborski S & Vila M (2003) The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model: a tool to explore the pathogenesis of Parkinson's disease. Ann N Y Acad Sci 991:189-198.

415. Qureshi S, Shah AH, & Ageel AM (1992) Toxicity studies on Alpinia galanga and Curcuma longa. Planta Med 58(2):124-127.

416. Raabe A , et al. (1998) Correlation of computed tomography findings and serum brain damage markers following severe head injury. Acta Neurochir (Wien) 140(8):787-791; discussion 791­782.

417. Raabe A, Grolms C, & Seifert V (1999) Serum markers of brain damage and outcome prediction in patients after severe head injury. Br J Neurosurg 13(1):56-59.

418. Radi R (2013) Protein tyrosine nitration: biochemical mechanisms and structural basis of functional effects. Acc Chem Res 46(2):550-559.

419. Radi R (2012) Protein Tyrosine Nitration: Biochemical Mechanisms and Structural Basis of Functional Effects. Accounts of Chemical Research 46(2):550-559.

420. Radunovic A, Porto WG, Zeman S, & Leigh PN (1997) Increased mitochondrial superoxide dismutase activity in Parkinson's disease but not amyotrophic lateral sclerosis motor cortex. Neurosci Lett 239(2-3):105-108.

421. Ragaisis V (2002) [Brain contusion: morphology, pathogenesis and treatment]. Medicina (Kaunas, Lithuania) 38(3):243-249; quiz 354.

422. Raghupathi R (2004) Cell Death Mechanisms Following Traumatic Brain Injury. Brain Pathology 14(2):215-222.

423. Raghupathi R , et al. (2002) Mild traumatic brain injury induces apoptotic cell death in the cortex that is preceded by decreases in cellular Bcl-2 immunoreactivity. Neuroscience 110(4):605-616.

424. Raghupathi R, Graham DI, & McIntosh TK (2000) Apoptosis after traumatic brain injury. J Neurotrauma 17(10):927-938.

425. Rajeswari A (2006) Curcumin protects mouse brain from oxidative stress caused by 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine. Eur Rev Med Pharmacol Sci 10(4):157-161.

426. Rajeswari A & Sabesan M (2008) Inhibition of monoamine oxidase-B by the polyphenolic compound, curcumin and its metabolite tetrahydrocurcumin, in a model of Parkinson's disease induced by MPTP neurodegeneration in mice. Inflammopharmacology 16(2):96-99.

427. Ramassamy C (2006) Emerging role of polyphenolic compounds in the treatment of neurodegenerative diseases: a review of their intracellular targets. Eur J Pharmacol 545(1):51- 64.

428. Razdan S, Kaul RL, Motta A, Kaul S, & Bhatt RK (1994) Prevalence and pattern of major neurological disorders in rural Kashmir (India) in 1986. Neuroepidemiology 13(3):113-119.

429. Reed TT , et al. (2009) Proteomic identification of nitrated brain proteins in traumatic brain- injured rats treated postinjury with gamma-glutamylcysteine ethyl ester: insights into the role of elevation of glutathione as a potential therapeutic strategy for traumatic brain injury. J Neurosci Res 87(2):408-417.

430. Reiter RJ (1995) Oxidative processes and antioxidative defense mechanisms in the aging brain. Faseb j 9(7):526-533.

431. Ren J (2007) Influence of gender on oxidative stress, lipid peroxidation, protein damage and apoptosis in hearts and brains from spontaneously hypertensive rats. Clinical and Experimental Pharmacology and Physiology 34(5-6):432-438.

432. Riederer BM (1989) Antigen preservation tests for immunocytochemical detection of cytoskeletal proteins: influence of aldehyde fixatives. J Histochem Cytochem 37(5):675-681.

433. Riederer BM, Zagon IS, & Goodman SR (1986) Brain spectrin(240/235) and brain spectrin(240/235E): two distinct spectrin subtypes with different locations within mammalian neural cells. J Cell Biol 102(6):2088-2097.

434. Rigobello MP, Folda A, Scutari G, & Bindoli A (2005) The modulation of thiol redox state affects the production and metabolism of hydrogen peroxide by heart mitochondria. Arch Biochem Biophys 441(2):112-122.

435. Robertson CL (2004) Mitochondrial dysfunction contributes to cell death following traumatic brain injury in adult and immature animals. J Bioenerg Biomembr 36(4):363-368.

436. Robertson CL , et al. (2001) Increased adenosine in cerebrospinal fluid after severe traumatic brain injury in infants and children: association with severity of injury and excitotoxicity. Critical care medicine 29(12):2287-2293.

437. Rojo AI, Salinas M, Martin D, Perona R, & Cuadrado A (2004) Regulation of Cu/Zn-superoxide dismutase expression via the phosphatidylinositol 3 kinase/Akt pathway and nuclear factor- kappaB. J Neurosci 24(33):7324-7334.

438. Ross SA, Cunningham RT, Johnston CF, & Rowlands BJ (1996) Neuron-specific enolase as an aid to outcome prediction in head injury. Br J Neurosurg 10(5):471-476.

439. Rossi F, Bellavite P, Berton G, Grzeskowiak M, & Papini E (1985) Mechanism of production of toxic oxygen radicals by granulocytes and macrophages and their function in the inflammatory process. Pathol Res Pract 180(2):136-142.

440. Roychowdhury S, Wolf G, Keilhoff G, & Horn TF (2003) Cytosolic and mitochondrial glutathione in microglial cells are differentially affected by oxidative/nitrosative stress. Nitric Oxide 8(1):39- 47.

441. Rubbo H , et al. (2000) Nitric oxide reaction with lipid peroxyl radicals spares alpha-tocopherol during lipid peroxidation. Greater oxidant protection from the pair nitric oxide/alpha-tocopherol than alpha-tocopherol/ascorbate. J Biol Chem 275(15):10812-10818.

442. Rustandi RR, Drohat AC, Baldisseri DM, Wilder PT, & Weber DJ (1998) The Ca(2+)-dependent interaction of S100B(beta beta) with a peptide derived from p53. Biochemistry 37(7):1951-1960.

443. Saito A, Hayashi T, Okuno S, Ferrand-Drake M, & Chan PH (2003) Overexpression of copper/zinc superoxide dismutase in transgenic mice protects against neuronal cell death after transient focal ischemia by blocking activation of the Bad cell death signaling pathway. J Neurosci 23(5):1710-1718.

444. Sambrook J & Russell D, W. (2001) Molecular Cloning - A Laboratory Manual (Cold Spring Harbour Laboratory Press, New York) Third Ed.

445. Santandreu FM , et al. (2008) Differences in mitochondrial function and antioxidant systems between regions of human glioma. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology 22(5-6):757-768.

446. Sastre J , et al. (1998) A Ginkgo biloba extract (EGb 761) prevents mitochondrial aging by protecting against oxidative stress. Free Radic Biol Med 24(2):298-304.

447. Sastre J, Pallardo FV, & Vina J (2003) The role of mitochondrial oxidative stress in aging. Free Radic Biol Med 35(1):1-8.

448. Sawada H , et al. (2002) Estradiol protects dopaminergic neurons in a MPP+Parkinson's disease model. Neuropharmacology 42(8):1056-1064.

449. Schalen W, Messeter K, & Nordstrom CH (1991) Cerebral vasoreactivity and the prediction of outcome in severe traumatic brain lesions. Acta anaesthesiologica Scandinavica 35(2):113-122.

450. Schapira AH (2008) Mitochondria in the aetiology and pathogenesis of Parkinson's disease. Lancet Neurol 7(1):97-109.

451. Schapira AH , et al. (1990) Mitochondrial complex I deficiency in Parkinson's disease. J Neurochem 54(3):823-827.

452. Schinder AF, Olson EC, Spitzer NC, & Montal M (1996) Mitochondrial dysfunction is a primary event in glutamate neurotoxicity. J Neurosci 16(19):6125-6133.

453. Schmidt OI, Heyde CE, Ertel W, & Stahel PF (2005) Closed head injury--an inflammatory disease? Brain Res Brain Res Rev 48(2):388-399.

454. Schmidt RH, Scholten KJ, & Maughan PH (2000) Cognitive impairment and synaptosomal choline uptake in rats following impact acceleration injury. J Neurotrauma 17(12):1129-1139.

455. Schnellmann RG, Gilchrist SM, & Mandel LJ (1988) Intracellular distribution and depletion of glutathione in rabbit renal proximal tubules. Kidney Int 34(2):229-233.

456. Schrag A, Ben-Shlomo Y, & Quinn NP (2000) Cross sectional prevalence survey of idiopathic Parkinson's disease and Parkinsonism in London. Bmj 321(7252):21-22.

457. Schuessel K, Leutner S, Cairns NJ, Muller WE, & Eckert A (2004) Impact of gender on upregulation of antioxidant defence mechanisms in Alzheimer's disease brain. J Neural Transm 111(9):1167-1182.

458. Schumacher M , et al. (2003) Steroid hormones and neurosteroids in normal and pathological aging of the nervous system. Prog Neurobiol 71(1):3-29.

459. Shacter E (2000) Quantification and significance of protein oxidation in biological samples. Drug Metab Rev 32(3-4):307-326.

460. Shan X, Jones DP, Hashmi M, & Anders MW (1993) Selective depletion of mitochondrial glutathione concentrations by (R,S)-3-hydroxy-4-pentenoate potentiates oxidative cell death. Chemical research in toxicology 6(1):75-81.

461. Shankar TN, Shantha NV, Ramesh HP, Murthy IA, & Murthy VS (1980) Toxicity studies on turmeric (Curcuma longa): acute toxicity studies in rats, guineapigs & monkeys. Indian J Exp Biol 18(1):73-75.

462. Shepard SR, Ghajar JB, Giannuzzi R, Kupferman S, & Hariri RJ (1991) Fluid percussion barotrauma chamber: a new in vitro model for traumatic brain injury. The Journal of surgical research 51(5):417-424.

463. Shetty PK, Galeffi F, & Turner DA (2011) Age-Induced Alterations in Hippocampal Function and Metabolism. Aging Dis 2(3):196-218.

464. Shibutani S, Takeshita M, & Grollman AP (1991) Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG. Nature 349(6308):431-434.

465. Shin CM , et al. (2002) Age-related changes in the distribution of nitrotyrosine in the cerebral cortex and hippocampus of rats. Brain Res 931(2):194-199.

466. Shoba G , et al. (1998) Influence of piperine on the pharmacokinetics of curcumin in animals and human volunteers. Planta Med 64(4):353-356.

467. Shohami E, Bass R, Wallach D, Yamin A, & Gallily R (1996) Inhibition of tumor necrosis factor alpha (TNFalpha) activity in rat brain is associated with cerebroprotection after closed head injury. J Cereb Blood Flow Metab 16(3):378-384.

468. Shohami E, Ginis I, & Hallenbeck JM (1999) Dual role of tumor necrosis factor alpha in brain injury. Cytokine & growth factor reviews 10(2):119-130.

469. Shojo H & Kibayashi K (2006) Changes in localization of synaptophysin following fluid percussion injury in the rat brain. Brain Res 1078(1):198-211.

470. Shreiber DI , et al. (1999) Experimental investigation of cerebral contusion: histopathological and immunohistochemical evaluation of dynamic cortical deformation. J Neuropathol Exp Neurol 58(2):153-164.

471. Shults CW (2006) Lewy bodies. Proc Natl Acad Sci U S A 103(6):1661-1668.

472. Sieg F, Wahle P, & Pape HC (1999) Cellular reactivity to mechanical axonal injury in an organotypic in vitro model of neurotrauma. J Neurotrauma 16(12):1197-1213.

473. Silver JM, McAllister TW, & Yudofsky SC (2011) Textbook of Traumatic Brain Injury (American Psychiatric Pub.).

474. Singhal B, Lalkaka J, & Sankhla C (2003) Epidemiology and treatment of Parkinson's disease in India. Parkinsonism & Related Disorders 9, Supplement 2(0):105-109.

475. Singleton AB , et al. (2003) alpha-Synuclein locus triplication causes Parkinson's disease. Science 302(5646):841.

476. Sinko PJ & Balimane PV (1998) Carrier-mediated intestinal absorption of valacyclovir, the L-valyl ester prodrug of acyclovir: 1. Interactions with peptides, organic anions and organic cations in rats. Biopharm Drug Dispos 19(4):209-217.

477. Sitte N, Merker K, Von Zglinicki T, Davies KJ, & Grune T (2000) Protein oxidation and degradation during cellular senescence of human BJ fibroblasts: part II--aging of nondividing cells. Faseb j 14(15):2503-2510.

478. Sivanandam TM & Thakur MK (2012) Traumatic brain injury: a risk factor for Alzheimer's disease. Neuroscience and biobehavioral reviews 36(5):1376-1381.

479. Sjodin MO, Bergquist J, & Wetterhall M (2010) Mining ventricular cerebrospinal fluid from patients with traumatic brain injury using hexapeptide ligand libraries to search for trauma biomarkers. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 878(22):2003-2012.

480. Sloane JA, Hollander W, Moss MB, Rosene DL, & Abraham CR (1999) Increased microglial activation and protein nitration in white matter of the aging monkey. Neurobiol Aging 20(4):395-405.

481. Smeyne M & Smeyne RJ (2013) Glutathione metabolism and Parkinson's disease. Free Radic Biol Med 62:13-25.

482. Smith C , et al. (2013) The neuroinflammatory response in humans after traumatic brain injury. Neuropathol Appl Neurobiol 39(6):654-666.

483. Smith CD , et al. (1991) Excess brain protein oxidation and enzyme dysfunction in normal aging and in Alzheimer disease. Proc Natl Acad Sci U S A 88(23):10540-10543.

484. Smith CD , et al. (1991) Excess brain protein oxidation and enzyme dysfunction in normal aging and in Alzheimer disease. Proceedings of the National Academy of Sciences 88(23):10540-10543.

485. Smith DH , et al. (1997) Progressive atrophy and neuron death for one year following brain trauma in the rat. J Neurotrauma 14(10):715-727.

486. Smith DH, Uryu K, Saatman KE, Trojanowski JQ, & McIntosh TK (2003) Protein accumulation in traumatic brain injury. Neuromolecular medicine 4(1-2):59-72.

487. Smith MA, Richey Harris PL, Sayre LM, Beckman JS, & Perry G (1997) Widespread peroxynitrite- mediated damage in Alzheimer's disease. J Neurosci 17(8):2653-2657.

488. Smith RG, Betancourt L, & Sun Y (2005) Molecular endocrinology and physiology of the aging central nervous system. Endocr Rev 26(2):203-250.

489. Sobocanec S, Balog T, Sverko V, & Marotti T (2003) Sex-dependent antioxidant enzyme activities and lipid peroxidation in ageing mouse brain. Free Radic Res 37(7):743-748.

490. Sofic E, Lange KW, Jellinger K, & Riederer P (1992) Reduced and oxidized glutathione in the substantia nigra of patients with Parkinson's disease. Neurosci Lett 142(2):128-130.

491. Sofic E, Paulus W, Jellinger K, Riederer P, & Youdim MB (1991) Selective increase of iron in substantia nigra zona compacta of parkinsonian brains. J Neurochem 56(3):978-982.

492. Sohal RS (2002) Oxidative stress hypothesis of aging. Free Radic Biol Med 33(5):573-574.

493. Sohal RS, Agarwal S, Dubey A, & Orr WC (1993) Protein oxidative damage is associated with life expectancy of houseflies. Proc Natl Acad Sci U S A 90(15):7255-7259.

494. Sotgiu S , et al. (2005) Chitotriosidase in patients with acute ischemic stroke. Eur Neurol 54(3):149-153.

495. Sparks DL, Liu H, Scheff SW, Coyne CM, & Hunsaker JC, 3rd (1993) Temporal sequence of plaque formation in the cerebral cortex of non-demented individuals. J Neuropathol Exp Neurol 52(2):135-142.

496. Spiteller G (2001) Lipid peroxidation in aging and age-dependent diseases. Experimental Gerontology 36(9):1425-1457.

497. Squier TC (2001) Oxidative stress and protein aggregation during biological aging. Exp Gerontol 36(9):1539-1550.

498. Srere PA (1969) [1] Citrate synthase: [EC 4.1.3.7. Citrate oxaloacetate-lyase (CoA-acetylating)]. Methods in Enzymology, ed John ML (Academic Press), Vol Volume 13, pp 3-11.

499. St-Pierre J, Buckingham JA, Roebuck SJ, & Brand MD (2002) Topology of superoxide production from different sites in the mitochondrial electron transport chain. J Biol Chem 277(47):44784- 44790.

500. Stadtman ER (2006) Protein oxidation and aging. Free Radic Res 40(12):1250-1258.

501. Stadtman ER (2001) Protein oxidation in aging and age-related diseases. Ann N Y Acad Sci 928:22-38.

502. Stan AD , et al. (2006) Human postmortem tissue: what quality markers matter? Brain Res 1123(1):1-11.

503. Staniek K & Nohl H (2000) Are mitochondria a permanent source of reactive oxygen species? Biochim Biophys Acta 1460(2-3):268-275.

504. Stanimirovic D, Djuricic BM, & Mrsulja BB (1988) Glutathione reductase during and after brain ischemia in gerbils. Metabolic brain disease 3(4):293-296.

505. Starkov AA, Chinopoulos C, & Fiskum G (2004) Mitochondrial calcium and oxidative stress as mediators of ischemic brain injury. Cell Calcium 36(3-4):257-264.

506. Starkov AA , et al. (2004) Mitochondrial alpha-ketoglutarate dehydrogenase complex generates reactive oxygen species. J Neurosci 24(36):7779-7788.

507. Sternbach GL (2000) The Glasgow coma scale. J Emerg Med 19(1):67-71.

508. Stoica BA & Faden AI (2010) Cell death mechanisms and modulation in traumatic brain injury. Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics 7(1):3-12.

509. Stridh MH , et al. (2010) Enhanced glutathione efflux from astrocytes in culture by low extracellular ca(2+) and curcumin. Neurochem Res 35(8):1231-1238.

510. Stroemer RP, Kent TA, & Hulsebosch CE (1995) Neocortical neural sprouting, synaptogenesis, and behavioral recovery after neocortical infarction in rats. Stroke; a journal of cerebral circulation 26(11):2135-2144.

511. Subramaniam R , et al. (1997) The lipid peroxidation product, 4-hydroxy-2-trans-nonenal, alters the conformation of cortical synaptosomal membrane proteins. J Neurochem 69(3):1161-1169.

512. Sullivan PG , et al. (2004) Proteasome inhibition alters neural mitochondrial homeostasis and mitochondria turnover. J Biol Chem 279(20):20699-20707.

513. Sullivan PG, Rabchevsky AG, Waldmeier PC, & Springer JE (2005) Mitochondrial permeability transition in CNS trauma: cause or effect of neuronal cell death? J Neurosci Res 79(1-2):231-239.

514. Sullivan PG, Thompson MB, & Scheff SW (1999) Cyclosporin A attenuates acute mitochondrial dysfunction following traumatic brain injury. Exp Neurol 160(1):226-234.

515. Sultana R, Perluigi M, & Butterfield DA (2006) Protein oxidation and lipid peroxidation in brain of subjects with Alzheimer's disease: insights into mechanism of neurodegeneration from redox proteomics. Antioxid Redox Signal 8(11-12):2021-2037.

516. Sulzer D (2007) Multiple hit hypotheses for dopamine neuron loss in Parkinson's disease. Trends Neurosci 30(5):244-250.

517. Sutton RL, Lescaudron L, & Stein DG (1993) Unilateral cortical contusion injury in the rat: vascular disruption and temporal development of cortical necrosis. J Neurotrauma 10(2):135- 149.

518. Takahashi T, Yamashita H, Nakamura T, Nagano Y, & Nakamura S (2002) Tyrosine 125 of alpha- synuclein plays a critical role for dimerization following nitrative stress. Brain Res 938(1-2):73- 80.

519. Tanner CM & Goldman SM (1996) Epidemiology of Parkinson's disease. Neurologic clinics 14(2):317-335.

520. Teasdale GM, Pettigrew LE, Wilson JT, Murray G, & Jennett B (1998) Analyzing outcome of treatment of severe head injury: a review and update on advancing the use of the Glasgow Outcome Scale. J Neurotrauma 15(8):587-597.

521. Tecoma ES, Monyer H, Goldberg MP, & Choi DW (1989) Traumatic neuronal injury in vitro is attenuated by NMDA antagonists. Neuron 2(6):1541-1545.

522. Thaik-Oo M , et al. (2002) Estimation of postmortem interval from hypoxic inducible levels of vascular endothelial growth factor. J Forensic Sci 47(1):186-189.

523. Thakur MK & Sharma PK (2006) Aging of brain: role of estrogen. Neurochem Res 31(11):1389- 1398.

524. Thibault O, Gant JC, & Landfield PW (2007) Expansion of the calcium hypothesis of brain aging and Alzheimer's disease: minding the store. Aging Cell 6(3):307-317.

525. Thiyagarajan M & Sharma SS (2004) Neuroprotective effect of curcumin in middle cerebral artery occlusion induced focal cerebral ischemia in rats. Life Sci 74(8):969-985.

526. Thompson HJ , et al. (2005) Lateral fluid percussion brain injury: a 15-year review and evaluation. J Neurotrauma 22(1):42-75.

527. Thompson SN, Gibson TR, Thompson BM, Deng Y, & Hall ED (2006) Relationship of calpain- mediated proteolysis to the expression of axonal and synaptic plasticity markers following traumatic brain injury in mice. Experimental neurology 201(1):253-265.

528. Tibbling G, Link H, & Ohman S (1977) Principles of albumin and IgG analyses in neurological disorders. I. Establishment of reference values. Scandinavian journal of clinical and laboratory investigation 37(5):385-390.

529. Tietze F (1969) Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem 27(3):502-522.

530. Tieu K (2011) A guide to neurotoxic animal models of Parkinson's disease. Cold Spring Harbor perspectives in medicine 1(1):a009316.

531. Toescu EC (2005) Normal brain ageing: models and mechanisms. Philos Trans R Soc Lond B Biol Sci 360(1464):2347-2354.

532. Tohgi H , et al. (1999) Alterations of 3-nitrotyrosine concentration in the cerebrospinal fluid during aging and in patients with Alzheimer's disease. Neurosci Lett 269(1):52-54.

533. Tomita H , et al. (2004) Effect of agonal and postmortem factors on gene expression profile: quality control in microarray analyses of postmortem human brain. Biol Psychiatry 55(4):346- 352.

534. Trojanowski JQ (2003) Rotenone neurotoxicity: a new window on environmental causes of Parkinson's disease and related brain amyloidoses. Exp Neurol 179(1):6-8.

535. Trotter SA, Brill LB, 2nd, & Bennett JP, Jr. (2002) Stability of gene expression in postmortem brain revealed by cDNA gene array analysis. Brain Res 942(1-2):120-123.

536. Trounce IA, Kim YL, Jun AS, & Wallace DC (1996) Assessment of mitochondrial oxidative phosphorylation in patient muscle biopsies, lymphoblasts, and transmitochondrial cell lines. Methods Enzymol 264:484-509.

537. Trumpower BL (1990) The protonmotive Q cycle. Energy transduction by coupling of proton translocation to electron transfer by the cytochrome bc1 complex. J Biol Chem 265(20):11409- 11412.

538. Tsao R (2010) Chemistry and Biochemistry of Dietary Polyphenols. Nutrients 2(12):1231-1246.

539. Tsujimoto Y & Shimizu S (2007) Role of the mitochondrial membrane permeability transition in cell death. Apoptosis : an international journal on programmed cell death 12(5):835-840.

540. Turrens JF (2003) Mitochondrial formation of reactive oxygen species. J Physiol 552(Pt 2):335- 344.

541. Turrens JF, Alexandre A, & Lehninger AL (1985) Ubisemiquinone is the electron donor for superoxide formation by complex III of heart mitochondria. Arch Biochem Biophys 237(2):408- 414.

542. Tyurin VA , et al. (2008) Oxidative lipidomics of programmed cell death. Methods Enzymol 442:375-393.

543. Uchida K , et al. (1998) Protein-bound acrolein: Potential markers for oxidative stress. Proceedings of the National Academy of Sciences 95(9):4882-4887.

544. Ulmann L, Mimouni V, Roux S, Porsolt R, & Poisson JP (2001) Brain and hippocampus fatty acid composition in phospholipid classes of aged-relative cognitive deficit rats. Prostaglandins Leukot Essent Fatty Acids 64(3):189-195.

545. Uzun H, Kayali R, & Qakatay U (2010) The chance of gender dependency of oxidation of brain proteins in aged rats. Archives of Gerontology and Geriatrics 50(1):16-19.

546. Vajragupta O , et al. (2003) Manganese complexes of curcumin and its derivatives: evaluation for the radical scavenging ability and neuroprotective activity. Free Radic Biol Med 35(12):1632- 1644.

547. Vali S , et al. (2007) Integrating glutathione metabolism and mitochondrial dysfunction with implications for Parkinson's disease: a dynamic model. Neuroscience 149(4):917-930.

548. Valko M , et al. (2007) Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 39(1):44-84.

549. van Meerloo J, Kaspers GJ, & Cloos J (2011) Cell sensitivity assays: the MTT assay. Methods Mol Biol 731:237-245.

550. Vanguilder HD & Freeman WM (2011) The hippocampal neuroproteome with aging and cognitive decline: past progress and future directions. Front Aging Neurosci 3:8.

551. Vaz AR , et al. (2011) Selective vulnerability of rat brain regions to unconjugated bilirubin. Mol Cell Neurosci 48(1):82-93.

552. Vaziri ND, Lee YS, Lin CY, Lin VW, & Sindhu RK (2004) NAD(P)H oxidase, superoxide dismutase, catalase, glutathione peroxidase and nitric oxide synthase expression in subacute spinal cord injury. Brain Res 995(1):76-83.

553. Veiga S, Melcangi RC, Doncarlos LL, Garcia-Segura LM, & Azcoitia I (2004) Sex hormones and brain aging. Exp Gerontol 39(11-12):1623-1631.

554. Venditti P, Di Stefano L, & Di Meo S (2013) Mitochondrial metabolism of reactive oxygen species. Mitochondrion 13(2):71-82.

555. Venkatesan C, Chrzaszcz M, Choi N, & Wainwright MS (2010) Chronic upregulation of activated microglia immunoreactive for galectin-3/Mac-2 and nerve growth factor following diffuse axonal injury. J Neuroinflammation 7:32.

556. Venkateshappa C, Harish G, Mahadevan A, Srinivas Bharath MM, & Shankar SK (2012) Elevated Oxidative Stress and Decreased Antioxidant Function in the Human Hippocampus and Frontal Cortex with Increasing Age: Implications for Neurodegeneration in Alzheimer's Disease. Neurochemical research.

557. Venkateshappa C , et al. (2012) Increased oxidative damage and decreased antioxidant function in aging human substantia nigra compared to striatum: implications for Parkinson's disease. Neurochemical research 37(2):358-369.

558. Vergun O, Votyakova TV, & Reynolds IJ (2003) Spontaneous changes in mitochondrial membrane potential in single isolated brain mitochondria. Biophysical journal 85(5):3358-3366.

559. Verweij BH , et al. (1997) Mitochondrial dysfunction after experimental and human brain injury

and its possible reversal with a selective N-type calcium channel antagonist (SNX-111). Neurol Res 19(3):334-339.

560. Vickers JC , et al. (1994) Alterations in neurofilament protein immunoreactivity in human hippocampal neurons related to normal aging and Alzheimer's disease. Neuroscience 62(1):1-13.

561. Vina J, Borras C, & Miquel J (2007) Theories of ageing. IUBMB Life 59(4-5):249-254.

562. Vina J, Sastre J, Pallardo F, & Borras C (2003) Mitochondrial theory of aging: importance to explain why females live longer than males. Antioxid Redox Signal 5(5):549-556.

563. Vos PE , et al. (2010) GFAP and S100B are biomarkers of traumatic brain injury: an observational cohort study. Neurology 75(20):1786-1793.

564. Vos PE , et al. (2004) Glial and neuronal proteins in serum predict outcome after severe traumatic brain injury. Neurology 62(8):1303-1310.

565. Wada K, Chatzipanteli K, Kraydieh S, Busto R, & Dietrich WD (1998) Inducible nitric oxide synthase expression after traumatic brain injury and neuroprotection with aminoguanidine treatment in rats. Neurosurgery 43(6):1427-1436.

566. Wall SB, Oh J-Y, Diers AR, & Landar A (2012) Oxidative modification of proteins: An emerging mechanism of cell signaling. Frontiers in Physiology 3.

567. Wang CX & Shuaib A (2002) Involvement of inflammatory cytokines in central nervous system injury. Prog Neurobiol 67(2):161-172.

568. Wang J, Du XX, Jiang H, & Xie JX (2009) Curcumin attenuates 6-hydroxydopamine-induced cytotoxicity by anti-oxidation and nuclear factor-kappa B modulation in MES23.5 cells. Biochem Pharmacol 78(2):178-183.

569. Wang MS, Boddapati S, Emadi S, & Sierks MR (2010) Curcumin reduces alpha-synuclein induced cytotoxicity in Parkinson's disease cell model. BMC Neurosci 11:57.

570. Wang SL , et al. (2009) Curcumin, a potential inhibitor of up-regulation of TNF-alpha and IL-6 induced by palmitate in 3T3-L1 adipocytes through NF-kappaB and JNK pathway. Biomed Environ Sci 22(1):32-39.

571. Wang X & Michaelis EK (2010) Selective neuronal vulnerability to oxidative stress in the brain. Front Aging Neurosci 2:12.

572. Wei YH & Lee HC (2002) Oxidative stress, mitochondrial DNA mutation, and impairment of antioxidant enzymes in aging. Exp Biol Med (Maywood) 227(9):671-682.

573. Weis S , et al. (2007) Quality control for microarray analysis of human brain samples: The impact of postmortem factors, RNA characteristics, and histopathology. J Neurosci Methods 165(2):198- 209.

574. Whitfield PC, Thomas EO, & Summers F (2009) Head Injury: A Multidisciplinary Approach (Cambridge University Press).

575. Wild AC, Moinova HR, & Mulcahy RT (1999) Regulation of gamma-glutamylcysteine synthetase subunit gene expression by the transcription factor Nrf2. J Biol Chem 274(47):33627-33636.

576. Wilder PT, Rustandi RR, Drohat AC, & Weber DJ (1998) S100B(betabeta) inhibits the protein kinase C-dependent phosphorylation of a peptide derived from p53 in a Ca2+-dependent manner. Protein Sci 7(3):794-798.

577. Williams RA, Kelly SM, Mottram JC, & Coombs GH (2003) 3-Mercaptopyruvate sulfurtransferase of Leishmania contains an unusual C-terminal extension and is involved in thioredoxin and antioxidant metabolism. J Biol Chem 278(3):1480-1486.

578. Williams S , et al. (2001) In situ DNA fragmentation occurs in white matter up to 12 months after head injury in man. Acta Neuropathol 102(6):581-590.

579. Willoughby KA , et al. (2004) S100B protein is released by in vitro trauma and reduces delayed neuronal injury. J Neurochem 91(6):1284-1291.

580. Winterbourn CC, Vissers MC, & Kettle AJ (2000) Myeloperoxidase. Current opinion in hematology 7(1):53-58.

581. Woertgen C, Rothoerl RD, Metz C, & Brawanski A (1999) Comparison of clinical, radiologic, and serum marker as prognostic factors after severe head injury. J Trauma 47(6):1126-1130.

582. Xiong Y, Gu Q, Peterson PL, Muizelaar JP, & Lee CP (1997) Mitochondrial dysfunction and calcium perturbation induced by traumatic brain injury. J Neurotrauma 14(1):23-34.

583. Xiong Y, Mahmood A, & Chopp M (2013) Animal models of traumatic brain injury. Nat Rev Neurosci 14(2):128-142.

584. Xu Y , et al. (2007) Curcumin reverses impaired hippocampal neurogenesis and increases serotonin receptor 1A mRNA and brain-derived neurotrophic factor expression in chronically stressed rats. Brain Res 1162:9-18.

585. Yakovlev AG , et al. (1997) Activation of CPP32-like caspases contributes to neuronal apoptosis and neurological dysfunction after traumatic brain injury. J Neurosci 17(19):7415-7424.

586. Yamamoto M (2001) Depression in Parkinson's disease: its prevalence, diagnosis, and neurochemical background. Journal of neurology 248 Suppl 3:III5-11.

587. Yamazaki Y, Yada K, Morii S, Kitahara T, & Ohwada T (1995) Diagnostic significance of serum neuron-specific enolase and myelin basic protein assay in patients with acute head injury. Surg Neurol 43(3):267-270; discussion 270-261.

588. Yan LJ, Levine RL, & Sohal RS (1997) Oxidative damage during aging targets mitochondrial aconitase. Proc Natl Acad Sci U S A 94(21):11168-11172.

589. Yang F , et al. (2005) Curcumin inhibits formation of amyloid beta oligomers and fibrils, binds plaques, and reduces amyloid in vivo. J Biol Chem 280(7):5892-5901.

590. Yang W, Ang LC, & Strong MJ (2005) Tau protein aggregation in the frontal and entorhinal cortices as a function of aging. Brain research. Developmental brain research 156(2):127-138.

591. Yang X , et al. (2009) Expressive proteomics profile changes of injured human brain cortex due to acute brain trauma. Brain injury : [BI] 23(10):830-840.

592. Yasha TC, Shankar L, Santosh V, Das S, & Shankar SK (1997) Histopathological & immunohistochemical evaluation of ageing changes in normal human brain. Indian J Med Res 105:141-150.

593. Yasojima K, McGeer EG, & McGeer PL (2001) High stability of mRNAs postmortem and protocols for their assessment by RT-PCR. Brain Res Brain Res Protoc 8(3):212-218.

594. Ye SF, Hou ZQ, Zhong LM, & Zhang QQ (2007) Effect of curcumin on the induction of glutathione S-transferases and NADP(H):quinone oxidoreductase and its possible mechanism of action. Yao Xue Xue Bao 42(4):376-380.

595. Yi JH & Hazell AS (2006) Excitotoxic mechanisms and the role of astrocytic glutamate transporters in traumatic brain injury. Neurochem Int 48(5):394-403.

596. Yoritaka A , et al. (1996) Immunohistochemical detection of 4-hydroxynonenal protein adducts in Parkinson disease. Proc Natl Acad Sci U S A 93(7):2696-2701.

597. Yu S , et al. (2010) Curcumin prevents dopaminergic neuronal death through inhibition of the c- Jun N-terminal kinase pathway. Rejuvenation Res 13(1):55-64.

598. Zasler N, Katz D, & Zafonte RD (2007) Brain Injury Medicine: Principles and Practice (Demos Medical Publishing).

599. Zasler ND, Katz DI, & Zafonte RD (2013) Brain injury medicine : principles and practice (Demos Medical Pub, New York) 2nd Ed.

600. Zbarsky V , et al. (2005) Neuroprotective properties of the natural phenolic antioxidants curcumin and naringenin but not quercetin and fisetin in a 6-OHDA model of Parkinson's disease. Free Radic Res 39(10):1119-1125.

601. Zecca L, Zucca FA, Wilms H, & Sulzer D (2003) Neuromelanin of the substantia nigra: a neuronal black hole with protective and toxic characteristics. Trends Neurosci 26(11):578-580.

602. Zemlan FP , et al. (2002) C-tau biomarker of neuronal damage in severe brain injured patients: association with elevated intracranial pressure and clinical outcome. Brain Res 947(1):131-139.

603. Zetterberg H , et al. (2008) Clinical proteomics in neurodegenerative disorders. Acta Neurol Scand 118(1):1-11.

604. Zhang J, Dawson VL, Dawson TM, & Snyder SH (1994) Nitric oxide activation of poly(ADP-ribose) synthetase in neurotoxicity. Science 263(5147):687-689.

605. Zhu Y, Carvey PM, & Ling Z (2006) Age-related changes in glutathione and glutathione-related enzymes in rat brain. Brain Res 1090(1):35-44.

606. Zoratti M, Szabo I, & De Marchi U (2005) Mitochondrial permeability transitions: how many doors to the house? Biochim Biophys Acta 1706(1-2):40-52.

607. Zupanc GK (2007) Proteomics of traumatic brain injury and regeneration. Proteomics. Clinical applications 1(11):1362-1372.

608. Zurek J & Fedora M (2012) The usefulness of S100B, NSE, GFAP, NF-H, secretagogin and Hsp70 as a predictive biomarker of outcome in children with traumatic brain injury. Acta Neurochir (Wien) 154(1):93-103; discussion 103.

[...]