Studies on the mechanisms of the beneficial effects of herba leonuri and leonurine on traumatic brain injury in rat

xvi List of Tables Table 1-1: Description of common experimental rodent models of closed head injury Table 3-1: EC50 of VC, pHL and Leo for the eliminationof DPPH radicals Table 3-2: Th

STUDIES ON THE MECHANISMS OF THE

BENEFICIAL EFFECTS OF HERBA LEONURI AND LEONURINE ON TRAUMATIC BRAIN INJURY IN RAT

CHEW SHIN YI B.Sc (Merit), NUS

In particular, I would like to express my deep and sincere gratitude to my supervisor,

Associate Professor Tan Kwong Huat, Benny, for his detailed review and

constructive comments which have been of great value for me I appreciate his understanding and encouragement throughout the course of my research

I am also deeply grateful to my supervisor, Professor Zhu Yi-Zhun, who introduced

me to the field of natural products and traumatic brain injury His extensive discussions and untiring help on my research project have been very helpful I thank him for my research stipend which made it possible for me to complete this project, especially without a research scholarship

I am grateful to Professor Wong Tsun Hon, Peter (Head of the Department of Pharmacology), as well as Associate Professor Tan Kwong Huat, Benny (Acting

head of the Department of Pharmacology) for facilitating requests and approvals My

ii

thanks also go to all staff of the Department for their kindness and timely help at any

point of my study Special mention goes to Mdm Xu XiaoGuang for lending me the rat housing in her lab and Mrs Ting Wee Lee for demonstrating the dissection of rat

brain

From A/P Tan’s lab, my deepest gratitude goes especially to Ms Annie Hsu, for her

encouragement, friendship and assistance throughout the whole project She has also taught me many techniques in animal work and biochemical assays Next, I warmly

thank Dr Ong Khang Wei, for introducing me to tissue sectioning, H&E staining,

immunohistochemistry (IHC) and western blot He is also a great friend who will help

me with troubleshooting, and share his opinions or ideas on my project

From Prof Zhu’s lab, I express my warm and sincere thanks to Dr Wang Hong, Dr

Wong Wan Hui, Dr Sonja Koh for their support, concern and friendship The many

discussions we had during lab meetings were often occasions for new discoveries In this way, they have contributed valuable advice and insights which have been of great help in this study

I would like to thank DSO National Laboratories, Kent Ridge, for the usage of their

fluid percussion device, and the staff of A/P Lu Jia’s lab for their kind help Firstly, I

sincerely thank Associate Professor Lu Jia for approving my access to work in DSO and assigning her staff to train me Next, I thank Ms Tan Li Li for arranging my DSO

orientation, helping me to book research facilities and prepare animal anesthetic

promptly Lastly, I thank Mr Ng Kian Chye and Ms Mary Kan for showing me how

to use the fluid percussion device and answering all my queries

iii

I wish to extend my appreciation to DSO Animal Holding Unit (AHU), Kent Ridge,

which provides excellent research facilities for animal study I am thankful to AHU

laboratory staff Parvathi and Foong Yen, for their assistance, friendship and

extremely positive attitude towards me I also wish to extend my appreciation to the

staff of Animal Holding Unit (AHU), NUS for preparing painkiller and antibiotics

for my rat experiments

I would like to thank Mrs Ng Geok Lan and Miss Pan Feng from Department of

Anatomy, NUS for their excellent technical assistance in histology Their experience

in histology work assisted me in solving problems and getting nice results In

particular, I sincerely thank Assistant Professor Srinivasan Dinesh Kumar, previously a senior lecturer in Department of Anatomy, NUS for guiding me

personally with histology, organizing my data and suggesting new directions for my research project

The financial support from research grant MD-NUS/JPP/09/10, NUS MINDEF

Joint Applied R&D Cooperation Programme (JPP) is gratefully acknowledged

Without friends, life as a graduate student would not be the same My friends have given me a powerful source of inspiration and energy However, it is not possible to list all of them here Their support in this research, whether directly or indirectly, is greatly appreciated

iv

Last but not least, I owe my loving thanks to my family members for their understanding and encouragement Without their moral support, it would have been impossible for me to stop working full time to complete my masters I would also like

to thank all staff and students from A/P Tan’s and Prof Zhu’s lab, for making the working environment one that is very pleasant to work in

v

Table of Contents

Acknowledgements i

Table of Contents v

List of Abbreviations xi

List of Tables xvi

List of Figures xvii

List of Publications xx

Summary xxi

Objectives and Structure of Thesis 1

CHAPTER 1 GENERAL INTRODUCTION 5

1.1: Traumatic Brain Injury (TBI) and Changes Following TBI 6

1.1.1 TBI 6

1.1.2 Pathophysiology of TBI 8

1.1.2.1 Primary and Secondary Injury 8

1.1.2.2 Excitotoxicity 9

1.1.2.3 Oxidative Stress 10

1.1.2.4 Inflammation 13

1.1.2.5 Apoptosis 14

vi

1.1.3 Animal Models of TBI 18

1.1.3.1 Weight-drop models 19

1.1.3.2 Fluid percussion injury (FPI) models 21

1.1.3.3 Controlled cortical impact (CCI) injury model 22

1.2: Pharmacological Management of TBI 25

1.2.1 Control of intracranial pressure and cerebral edema 25

1.2.2 N-methyl-D-aspartate (NMDA) receptor antagonists 26

1.2.3 Calcium channel blocking agents 26

1.2.4 Free radical scavengers 27

1.2.5 Anti-inflammatory agents 29

1.2.6 Apoptosis and caspase inhibitors 31

1.2.7 Neurotrophic factors 32

1.2.8 Poly(ADP-ribose) polymerase (PARP) inhibitors 34

1.2.9 Multipotential drugs 34

1.2.10 Herbal Medicines for TBI 35

1.3: Traditional Chinese Medicine (TCM) 37

1.3.1 Herba leonuri and pHL 37

1.3.2 Leo 41

vii

CHAPTER 2 MATERIALS AND METHODS 43

2.1: Materials 44

2.1.1 Test compounds (pHL and Leo) 44

2.1.1.1 pHL 44

2.1.1.2 Leo 44

2.1.2 Animals 45

2.1.3 Chemicals 45

2.2: Methods 45

2.2.1 Experimental protocol I 45

2.2.1.1 Objectives 45

2.2.1.2 Experimental design 46

2.2.2 Experimental protocol II 47

2.2.2.1 Objectives 47

2.2.2.2 Experimental design 47

2.2.3 Experimental protocol III 48

2.2.3.1 Objectives 48

2.2.3.2 Experimental design 49

2.2.4 Experimental techniques 49

viii

2.2.4.1 Lateral fluid-percussive brain injury (FPI) 49

2.2.4.2 Hematoxylin and Eosin staining 50

2.2.4.3 TUNEL (TdT-mediated dUTP Nick-End Labeling) assay 50

2.2.4.4 Immunohistochemical staining 51

2.2.4.5 Biochemical analysis 52

2.2.4.6 Western blot analysis 53

2.2.4.7 DPPH (2,2-diphenyl-1-picrylhydrazyl) antioxidant assay 54

2.2.5 Statistical Analysis 55

CHAPTER 3 RESULTS 56

3.1: Results of experiment I: Cerebral protection of pHL extract on rats with TBI 57

3.1.1 Pharmacological and functional outcome studies 57

3.1.1.1 Effects of pHL on changes in general brain morphology following TBI 57

3.1.1.2 Effects of pHL on morphologic alterations in the hippocampus following TBI 59

3.1.1.3 Effects of pHL on neuronal loss, astrocyte and microglia gliosis following TBI 61

3.1.2 Biochemical and molecular approaches 67

ix

3.1.2.1 Effects of pHL on the activities of SOD, CAT, GPx and GST in the cortex following TBI 673.1.2.2 Effects of pHL treatment on neuronal apoptosis following TBI 69

3.2: Results of experiment II: Leo protects rats with TBI through antioxidant and anti-apoptotic mechanisms 71

3.2.1 Biochemical and molecular approaches 71

3.2.1.1 Effects of Leo on the activities of SOD, CAT, GPx and GST in the cortex following TBI 71

3.2.1.2 Effects of Leo treatment on neuronal apoptosis following TBI 73

3.3: Investigating antioxidant capacity of pHL and Leo in-vitro and also

antioxidant and anti-apoptotic properties in TBI rats 75

3.3.1 Comparing the antioxidant effects of pHL and Leo 75

3.3.1.1 DPPH radical-scavenging activities of pHL, Leo and VC (positive

control) 75

3.3.1.2 EC50 values of VC, pHL and Leo for DPPH assay 77

3.3.1.3 Comparison of the effects of pHL and Leo on the activities of SOD, CAT, GPx and GST in the cortex following TBI 783.3.2 Comparing the anti-apoptotic effects of pHL and Leo 79

3.3.2.1 Comparison of the effects of pHL and Leo on the expression of

apoptosis-related proteins in the hippocampus following TBI 79

x

CHAPTER FOUR DISCUSSION 81

CHAPTER FIVE CONCLUSIONS, FUTURE STUDIES AND PERSPECTIVES 91

5.1 Conclusion 92

5.2 Limitations of study 94

5.3 Future studies 95

5.3.1 Varying treatment strategies 95

5.3.1.1 Isolating other active ingredients in pHL for study in TBI 95

5.3.1.2 Combination of pHL or Leo with other secondary injury therapies or western drugs 96

5.3.2 Investigating the pathways involved in TBI 96

5.3.2.1 pHL or Leo on the expression of apoptotic pathway proteins at earlier time points of TBI 96

5.3.2.2 The role of iInflammation in TBI 96

5.4 Future perspectives 97

AMPA α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid

Bax Bcl2-associated X protein

BBB Blood-brain barrier

Bcl-2 B-cell lymphoma 2

Bcl-xL B-cell lymphoma-extra large

BDNF Brain-derived neurotrophic factor

BSA Bovine serum albumin

CAT Catalase

CBF Cerebral blood flow

CCI Controlled cortical impact

CFP Central fluid percussion

CHI Closed head injury

CNS Central nervous system

COX Cyclooxygenase

CsA Cyclosporine A

DAPI 4',6-diamidino-2-phenylindole

DISC Death-inducing signaling complex

DNA Deoxyribonucleic acid

DPPH 2,2-diphenyl-1-picrylhydrazyl

EAA Excitatory amino acids

ECL Enhanced luminol-based chemiluminescence

xii

ECG Electrocardiography

ELAM Endothelial leukocyte adhesion molecule

ESR Electron spin resonance

ETC Electron transport chain

FasL Fas ligand

FPI Fluid percussion injury

FRAP Ferric Reducing Ability of Plasma

GAPDH Glyceraldehyde 3-phosphate dehydrogenase

GDNF Glial cell-derived neurotrophic factor

GFAP Glial fibrillary acidic protein

GPx Glutathione peroxidase

GSH Glutathione

GST Glutathione-S-transferase

H&E Hematoxylin and Eosin

HIF-1α Hypoxia inducible factor-1 alpha

HL Herba leonuri

HPLC High performance liquid chromatography

HRP Horseradish peroxidise

IACUC Institutional animal care & use committee

ICAD Inhibitor of caspase-activated deoxyribonuclease

ICAM Intracellular adhesion molecule

ICP Intracranial pressure

ICV Intracerebroventricular

IGF-1 Insulin-like growth factor

IHC Immunohistochemical staining

IL Interleukin

LFP Lateral fluid percussion

LFPI Lateral fluid percussion injury

LOC Loss of consciousness

MCAO Middle cerebral artery occlusion

MDA Malondialdehyde

mGluR Metabotropic glutamate receptor

mRNA Messenger ribonucleic acid

MnSOD Manganese superoxide dismutase

MPTP Mitochondrial permeability transition pore

NAD Nicotinamide adenine dinucleotide

NeuN Neuronal nuclei

NGF Nerve growth factor

ORAC Oxygen Radical Absorbance Capacity

PARP Poly ADP-ribose polymerase

PBS Phosphate buffered saline

PEG-SOD Polyethylene glycol-conjugated superoxide dismutase

PTA Post-traumatic amnesia

RCTs Randomized controlled trials

(rh)IL-1ra Recombinant human interleukin-1 receptor antagonist

ROS Reactive oxygen species

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SMAC Second mitochondria-derived activator of caspases

SOD Superoxide dismutase

TAA Total antioxidant activity

TBI Traumatic Brain Injury

TBS Tris-(hydroxymethyl)-aminomethane buffered saline

TCM Traditional Chinese medicine

TE Trolox Equivalents

TEAC Trolox Equivalent Antioxidant Capacity

TGF-a Transforming growth factor-a

TNFα Tumour necrosis factor-alpha

TNFBP Tumor necrosis factor-alpha binding protein

TNFR Tumor necrosis factor receptor

TUNEL Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling

VCAM Vascular adhesion molecule

VEGF Vascular endothelial growth factor

xv z-DEVDfmk N-benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone

z-VADfmk N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone

xvi

List of Tables

Table 1-1: Description of common experimental rodent models of closed head injury

Table 3-1: EC50 of VC, pHL and Leo for the eliminationof DPPH radicals

Table 3-2: The percentage increase in SOD, CAT, GPx and GST activities in TBI/pHL and TBI/Leo compared with TBI group

Table 3-3: A summary of the percentage expression of Bax, Bcl-xL, cleaved PARP and procaspase-3 in TBI/pHL and TBI/Leo compared with TBI group

xvii

List of Figures

Figure 1-1: A diagram to illustrate the sequence of events following TBI

Figure 1-2: A diagram to summarise post traumatic excitotoxicity leading to cell death

Figure 1-3: A schematic representation of major intracellular pathways in the generation of free radicals after CNS injury

Figure 1-4: A schematic diagram of apoptosis

Figure 1-5: HL (Chinese Motherwort)

Figure 1-6: The 5 known compounds from pHL

Figure 2-1: Mass spectrum of pHL

Figure 2-2: A flow chart to represent the experimental outline in the pilot study of pHL

Figure 2-3: A flow chart to represent the experimental outline in the pilot study of Leo

Figure 3-1: H&E staining of the cerebral cortex

Figure 3-2: TUNEL staining of the cerebral cortex

xviii

Figure 3-3: (a) Representative light micrographs of H&E stained sections in rats of each experimental group (b) Quantitative assessment of the percentage of dark-stained nuclei and distorted nerve cells in each experimental group

Figure 3-4: (a) Representative photomicrographs of NeuN-stained sections in rats of each experimental group (b) Quantitative assessment of the number of NeuN-stained cells in each experimental group

Figure 3-5: (a) Representative photomicrographs of GFAP-stained sections in rats of each experimental group (b) Quantitative assessment of the number of GFAP-stained cells in each experimental group

Figure 3-6: (a) Representative photomicrographs of Cd11b-stained sections in rats of each experimental group (b) Quantitative assessment of the number of Cd11b-stained cells in each experimental group

Figure 3-7: Effects of pHL on the antioxidant enzyme activities in the cortex Bar charts showing the activities of SOD (a), CAT (b), GPx (c) and GST (d) in each experimental group

Figure 3-8: (a) Representative western-blot bands of Bax, Bcl-xL, cleaved PARP, procaspase-3 and GAPDH in each experimental group Bar charts showing the expression levels of Bax (b), Bcl-xL (c), cleaved PARP (d) and procaspase-3 (e) in each experimental group after normalising with GAPDH

Figure 3-9: Effects of Leo on the antioxidant enzyme activities in the cortex Bar charts showing the activities of SOD (a), CAT (b), GPx (c) and GST (d) in each experimental group

Figure 3-10: (a) Representative western-blot bands of Bax, Bcl-xL, cleaved PARP, procaspase-3 and GAPDH in each experimental group Bar charts showing the expression levels of Bax (b), Bcl-xL (c), cleaved PARP (d) and procaspase-3 (e) in each experimental group after normalising with GAPDH

xix

Figure 3-11: Graphs showing elimination rate of DPPH radicals against concentration

of test compound used at different time points The graph for positive control VC in (a), pHL in (b) and Leo in (c)

xx

List of Publications

1 Shin Yi Chew, Annie Hsu, Srinivasan Dinesh Kumar, Yi Zhun Zhu, Benny

Kwong Huat Tan Neuroprotective Effects of Purified Herba leonuri extract against

Traumatic Brain Injury (Manuscript under review)

xxi

Summary

Purified Herba leonuri (pHL) is a compound isolated from the Chinese Motherwort

plant It has been shown to have a broad spectrum of pharmacological properties but

has not been tested for any beneficial effects in traumatic brain injury (TBI) The first

part of this study aims to investigate the effects of pHL on different parameters of damaged brain tissue following TBI in the rat The rats were given orally, pHL (400mg/kg) or vehicle, daily for one week starting from the day after TBI induction Sham-operated and vehicle-treated animals were used as control groups At the end of the treatment period, the animals were sacrificed and brain samples were collected for analysis The lesion area was measured and the number of apoptotic cells in the cortex were estimated The number of apoptotic-like cells, neurons, astrocytes and microglia

in the hippocampus were also counted The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) in the brain were measured In addition, the expressions of Bax, Bcl-xL, PARP and caspase-3 in the brain tissue were quantified The results showed that there was reduced lesion area and number of apoptotic cells in the injured cortex A significant reduction in the number of apoptotic hippocampal cells, neuronal loss, astrocytes and microglia was observed in the pHL-treated group compared with the vehicle group pHL significantly increased the activities of SOD, CAT and GPx in brain tissue but did not affect the activity of GST Furthermore, the expressions of Bax and PARP were significantly reduced while the expressions of Bcl-xL and caspase-3 were significantly increased with pHL treatment compared to vehicle

xxii

The second part of this study aims to investigate the effects of Leonurine (Leo) on TBI Leo was synthesized from syringic acid by carbonylation, reaction with thionyl

chloride (SOCl2), and the Gabriel reaction The rats were given orally, Leo (60mg/kg)

or vehicle, daily for one week starting from the day after TBI induction operated and vehicle-treated animals were used as control groups At the end of the treatment period, the animals were sacrificed and brain samples were collected for analysis In this study, only antioxidant activities and anti-apoptotic effects were chosen for observation Leo increased the activities of SOD, CAT, GPx and GST in brain tissue but only the increase in SOD was significant Furthermore, the expressions of Bax and PARP were significantly reduced while the expressions of Bcl-xL and caspase-3 were significantly increased with Leo treatment compared to vehicle

Sham-The third part of this study aims to compare the effects of pHL and Leo on TBI Using DPPH free radical scavenging assay, the antioxidant capacity of both compounds were determined The antioxidant activities and anti-apoptotic effects were also compared pHL has a higher antioxidant capacity as compared to Leo Similarly, pHL has better antioxidant and anti-apoptotic effects than Leo, as it shows a higher percentage increase/decrease of the treatment outcome compared to the TBI group The difference in activities of SOD, CAT and GPx was significant between both treatment groups Furthermore, there is also significant difference in the expressions

of PARP, Bcl-xL and caspase-3 between both treatment groups

xxiii

In summary, our data show that both pHL and Leo confer protection to brain tissue following TBI This protection may be mediated through antioxidative and antiapoptotic mechanisms However, the protective effects of pHL are better and this may be due to its higher antioxidant capacity, which is able to reduce oxidative stress and hence apoptosis more effectively Further studies are required to give an in-depth

understanding of the mechanism underlying the protective effects of pHL and Leo in

TBI

1

Objectives and Structure of Thesis

1) Objectives

The main objectives of this work are three-fold:

1.1 Studies to verify the possible therapeutic potential of pHL in rats subjected

to TBI:

In experiment I, a pilot study was conducted to observe the effect of pHL on rats subjected to TBI via a few parameters: lesion area and number of terminal TUNEL-positive apoptotic cells on the cortex, the number of apoptotic-like cells, neurons, astrocytes and microglia in the hippocampus Antioxidant measurements and anti-apoptotic assessment were carried out to identify the possible protective mechanisms

of pHL

The following parameters were measured:

• The lesion area and the number of apoptotic cells in the cortex

• The number of apoptotic-like cells, neurons, astrocytes and microglia in the hippocampus

• SOD, CAT, GPx and GST activities in the brain tissue to identify the effects

of pHL on antioxidant mechanisms

• Expressions of BAX, Bcl-xL, procaspase-3 and cleaved PARP in the brain

tissue to identify the effects of pHL on anti-apoptotic mechanisms

2

1.2 Studies to address the effects of Leo on antioxidant activity and the

expression of apoptotic pathway proteins in rats with TBI:

In experiment II, a key compound of pHL (Leo) was targeted to identify if it is one of the active ingredients of pHL for neuroprotection

The following parameters were measured:

• SOD, CAT, GPx and GST activities in brain tissue to identify the effects of Leo on antioxidant mechanisms

• Expressions of BAX, Bcl-xL, procaspase-3 and cleaved PARP in brain tissue

to identify the effects of Leo on anti-apoptotic mechanisms

1.3 Studies to compare pHL and Leo on anti-oxidant and anti-apoptotic effects

in rats with TBI:

In experiment III, the antioxidant capacity of both compounds were evaluated by the DPPH free radical scavenging assay This will determine which compound has a higher antioxidant capacity In particular, the antioxidant and anti-apoptotic effects demonstrated in experiment I and experiment II will be compared

2) Structure of thesis

This study is reported as follows:

Chapter 1: General Introduction

This chapter starts with an introduction of TBI and changes associated with it in section 1 A brief TBI epidemiology is presented, followed by types of TBI and its classifications and symptoms Next, a review of the scientific literature relevant to

3

TBI pathophysiology, involvement of excitotoxicity, oxidative stress, inflammation and apoptosis is introduced Lastly, the different types of animal model used in TBI will be described in detail

In section 2, pharmacological management of TBI will be reviewed along with the description of primary successes of clinical trial on TBI therapy, and their limitation

of usage on patients Current therapies include drugs to control intracranial pressure and edema, NMDA receptor antagonists, calcium channel blocking and anti-inflammatory agents, free radical scavengers, inhibitors of apoptosis, neurotrophic factors, multipotential drugs and herbal medicines

In section 3, the importance of study on the potential neuroprotective effects of natural products, particularly TCM are highlighted We also reviewed the rationale of focusing on Chinese Herbs as potential therapeutic agent with a few examples The

later part of this section introduces Herba leonuri, pHL and Leo in more details

Chapter 2: Materials and Methods

This chapter explains the three experimental protocols:

Experimental protocol I: Cerebral Protection of pHL on rats with TBI

Experimental protocol II: Leo protects rats with TBI through antioxidant and

4

Chapter 3: Results

Results obtained from the three experimental protocols are presented in this chapter

Chapter 4: Discussion

Discussion based on three parts of the experiment is brought out in this chapter

Chapter 5: Conclusion and Future Studies

This chapter concludes the whole thesis with an explanation of the outcomes of this project in relation to the initial objectives Limitations of the study will also be discussed The possible areas of research which could be further investigated and therapeutic expectations in the future are addressed

5

CHAPTER 1

GENERAL INTRODUCTION

Human TBI can be caused by an enormous heterogeneity of forces which impact the head (Kunz et al., 2010) Two major forms of TBI have been classified in humans: closed and penetrating; the nature of forces which act on the head as well as the amount of mechanical energy transmitted determine the type of TBI (Morales et al., 2005b) Closed TBI is further sub-classified into static and dynamic loading, depending on the velocity of the force transmission process (Morales et al., 2005b) The more common mechanism causing TBI is dynamic loading The purpose of categorizing TBI helps to isolate a distinct pattern of pathophysiological events which cause brain injury after trauma

7

TBI can be classified into mild, moderate and severe categories (Saatman et al., 2008) The Glasgow Coma Scale (GCS) is the most commonly used system to classify TBI severity It grades a person’s level of consciousness on a scale of 3-15 based on verbal, motor and eye-opening reactions to stimuli It is generally agreed that a TBI with a GCS of 13 or above is mild, 9-12 is moderate and 8 or below is severe (Parikh et al., 2007) This grading system has its limitations in predicting outcomes so other parameters have been used to judge the severity of TBI These parameters include duration of post-traumatic amnesia (PTA), duration of loss of consciousness (LOC) and checking for swelling and/or focal lesions by neuroimaging The seriousness of TBI is often underestimated because physical impairments are frequently mild or absent while the more disabling problems of cognitive and behavioral impairments are often overlooked or misdiagnosed by medical professionals Therefore, the after effects of TBI may be a long term burden to people where impairments or disabilities are present

The symptoms of TBI depend on whether it is a diffuse or focal injury and also the part of the brain which is affected It also depends on the severity of the injury With mild TBI, the patient remains conscious or lose consciousness for a few seconds or minutes Other symptoms of mild TBI include headache, vomiting, nausea, lack of motor coordination, dizziness and difficulty balancing (Kushner, 1998) Cognitive and emotional symptoms include behavioral or mood changes, confusion and having trouble with memory and concentration These symptoms may be present in both mild and moderate TBI In moderate or severe TBI, a person may show more serious symptoms like persistent headaches, repeated vomiting or nausea, convulsions,

8

slurred speech, weakness or numbness in the limbs, loss of coordination or agitation

In addition, there are long-term symptoms like changes in social behavior, deficits in social judgment and cognitive changes especially problems with sustained attention, processing speed and executive functioning (Busch et al., 2005; Kim, 2002; McDonald et al., 2003; Ponsford et al., 2008)

1.1.2 Pathophysiology of TBI

1.1.2.1 Primary and Secondary Injury

After TBI, the damage of brain tissue can be caused by primary and secondary injury mechanisms The primary injury refers to the direct effects of mechanical injury on the brain tissue A primary injury can incur focal and/or diffuse damage to the brain Examples of focal injuries are epidural, subdural or intracerebral hematomas and brain contusions, while diffuse damage refers to diffuse axonal injuries (DAI) (Kunz

et al., 2010) Primary injury usually causes skull fracture and abruptly disrupts the brain parenchyma, with shearing and tearing of blood vessels and brain tissue (Gentleman et al., 1995a; Povlishock and Christman, 1995b) This will then trigger a cascade of events characterized by activation of molecular and cellular responses, which lead to secondary injury (Leker and Shohami, 2002) Secondary injury takes hours or days to surface and is known to be a complex process The initial events include damage to the blood–brain barrier, release of inflammatory factors, overload

of free radicals, excessive release of the neurotransmitter glutamate (excitotoxicity), influx of calcium and sodium ions into neurons and mitochondria dysfunction (Park E Fau - Bell et al.) As a result, neurons can potentially be killed when the injured axons

in the brain’s white matter separate from their cell bodies Other factors which

9

subsequently contribute to secondary injury are reduced blood flow to the brain, ischemia, cerebral hypoxia, cerebral edema and raised intracranial pressure (Jain, 2008) Together, the primary and secondary events eventually lead to brain damage

Figure 1-1: A diagram to illustrate the sequence of events following TBI [Adapted from (Jain, 2008)]

1.1.2.2 Excitotoxicity

After TBI, excitatory amino acid neurotransmitters such as glutamate and aspartate are released in an uncontrolled manner to the injured areas Within minutes after neurons are exposed to glutamate, ionophoric NMDA and AMPA receptors are activated, the membrane is depolarized and this leads to the influx of calcium, sodium and water into cells of the lesioned region (Obrenovitch and Urenjak, 1997; Palmer et

10

al., 1993) This will cause cytotoxic edema and massive disruption of ionic homeostasis due to the lack of energy stores in the traumatized region Intracellular calcium level which is also elevated, causes an increase in cellular oxidative stress which leads to cell damage It activates various enzymes such as lipases, proteases and endonucleases that may damage DNA, cell proteins and lipids and cause cell death

Figure 1-2: A diagram to summarise post traumatic excitotoxicity leading to cell death [Adapted from (Ringel and Schmid-Elsaesser, 2001)]

1.1.2.3 Oxidative Stress

Oxidative stress is the state of imbalance between two opposing antagonistic forces, reactive oxygen species (ROS) and antioxidant, in which the effects of former predominate over the compensating action of latter (Fernandez-Checa et al., 1997)

11

Nitric oxide (NO˙) and superoxide (O2˙¯) are two major free radicals responsible for oxidative stress These two free radicals could react with each other to produce powerful oxidant peroxynitrite (ONOO¯) Other ROS includes hydrogen peroxide (H2O2) and hydroxyl radical (OH˙) As reported by Zhu et al., there are many possible mechanisms of free radical production Besides the basal level generation of O2˙¯ by the mitochondria, disruption of the mitochondria electron transport chain can result in autoxidation of flavoprotein and ubisemiquinone to form O2˙¯ (Zhu et al., 2004) Endothelial cells also produce free radicals such as NO˙ which is a major component

of endothelial-derived relaxing factor (Zhu et al., 2004)

The brain is very vulnerable to oxidative damage due to its high membrane surface to cytoplasm ratio; non-replicating neurons; relatively low antioxidant capacity and repair mechanism activity; high rate of oxidative metabolite activity and intensive production of reactive oxygen metabolites (Evans, 1993; Reiter, 1995) Prolonged elevations of intracellular calcium results in the formation of superoxide anion radicals by the respiratory chain, as well as by cytosolic enzymes, such as xanthine oxidase (Juurlink and Paterson, 1998) In the extracellular compartment, autoxidation

of catecholamines is an alternate pathway for free radical production This leads to an increase in oxidative stress

The increased production of ROS is due to excitotoxicity and exhaustion of the endogenous antioxidant system (e.g SOD, GPx, CAT) This leads to peroxidation of cellular and vascular structures, protein oxidation, cleavage of deoxyribonucleic acid (DNA) and inhibition of the mitochondrial electron transport chain (ETC) (Chong et

12

al., 2005; Shao et al., 2006) The brain contains high levels of redox-active metals such as iron, copper and manganese During trauma, the mobilization of these metals may occur and get exposed to reducing agents As a result, highly toxic radicals are produced and cause oxidative damage (Shohami et al., 1997) Free radicals also block mitochondrial respiration and facilitate the formation of mitochondrial permeability transition pore (MPTP), leading to mitochondrial swelling and cell death As a result

of oxidative stress, inflammatory processes and early or late apoptotic programmes are induced (Chong et al., 2005)

Figure 1-3: A schematic representation of major intracellular pathways in the generation of free radicals after CNS injury XDH, xanthine dehydrogenase; XO, xanthine oxidase; NOS, nitric oxide synthase (neuronal, inducible and endothelial); COX-2, cyclooxygenase-2; CuZnSOD, copper-zinc superoxide dismutase; GSPx, glutathione peroxidise [Adapted from (Lewen et al., 2000)]

13

1.1.2.4 Inflammation

TBI induces a complex array of inflammatory tissue responses After a traumatic insult, cellular mediators including proinflammatory cytokines, prostaglandins and free radicals are released As early as 1 hour after traumatic insults, proinflammatory cytokines such as tumour necrosis factor-alpha (TNFα), interleukin-1 (IL-1) and interleukin-6 (IL-6) are activated and secreted (Shohami et al., 1994a; Taupin et al., 1993) These processes induce chemokines and adhesion molecules and subsequently recruit immune and glial cells in a parallel and synergistic manner (Lucas et al., 2006; Potts et al., 2006) For example, activated polymorphonuclear leukocytes can adhere

to endothelial cell layers and infiltrate injured tissue along with macrophages and cell lymphocytes (Zhang et al., 2006) Cellular adhesion molecules such as intracellular adhesion molecule (ICAM), endothelial leukocyte adhesion molecule (ELAM), vascular adhesion molecules (VCAM-1) and tissue metalloproteinases are also upregulated and facilitate the penetration of leukocytes through the blood brain barrier (BBB) (Pantoni et al., 1998) In response to these inflammatory processes, injured and adjacent tissue will be eliminated, and within hours, days or weeks, astrocytes will produce microfilaments and neurotropines to synthesize scar tissue (Fabricius et al., 2006) The direct release of neurotoxic mediators or indirect release

T-of nitric oxide and cytokines in the affected region affects the extent T-of tissue damage In addition, the release of vasoconstrictors (prostaglandins and leukotrienes), the destruction of microvasculature through adhesion of leucocytes and platelets, the BBB lesion and edema formation further reduce tissue perfusion and aggravate secondary brain injury (Werner and Engelhard, 2007)

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Inflammatory response after injury is found to have detrimental effects in the early phase (within hours), but there have been studies to show that this response is

beneficial in the late (days-weeks) phase In vitro studies have demonstrated

detrimental effects of TNFα, causing neuronal, endothelial, glial cell damage and induction of apoptosis (Hisahara et al., 1997; Westmoreland et al., 1996)

In a study of TNFα-deficient (TNF -/-) mice, attenuation of cognitive and neurological motor deficits was observed in the first week following TBI However,

up to 4 weeks post-injury, TNF -/- mice were significantly worse in cognitive and neurological motor function when compared to wild-type controls This suggests that early, but not late inhibition of TNFα might improve the outcome and recovery following TBI (Scherbel et al., 1999) It was also reported that IL-6 is neuroprotective, promoting survival and differentiation of neurons and inducing neurotrophin expression in response to central nervous system (CNS) injury (Kossmann et al., 1996; Munoz-Fernandez and Fresno, 1998) The role of IL-6 and TNFα remains elusive as both cytokines may be attributed with neuroprotective and neurotoxic properties Another cytokine interleukin-10 (IL-10) which is involved in immunoregulation and anti-inflammation helps to protect cells against damage (Bethea et al., 1999; Knoblach and Faden, 1998)

1.1.2.5 Apoptosis

Cells dying after brain trauma can either die of necrosis or apoptosis Necrosis occurs

in response to severe mechanical or ischemic/hypoxic tissue damage, along with excessive release of excitatory amino acid neurotransmitters and metabolic failure, which disrupt cell viability Necrosis is irreversible massive cell death characterized

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by shrunken cells with darkened nuclei, swelling of cytoplasm and organelles and loss

of membrane integrity which results in cell lysis and release of cellular content that causes local inflammation to surrounding tissue (Taoufik and Probert, 2008) In contrast, apoptosis is an orderly process of energy dependent programmed cell death characterized by morphological features such as cell shrinkage, membrane blebbing, chromatin condensation and DNA fragmentation (Nakka et al., 2008) Cells undergoing apoptosis are morphologically intact immediately after the primary insult but only show changes hours or days later Apoptotic cells will be recognized and removed by phagocytosis to avoid inflammation and minimize the damage and disruption of neighbouring cells (Taylor et al., 2008) A more unique morphological characteristic of neuron undergoing apoptotsis is the neurite fragment (dendrites and axons) that occurs during the early cell death process (Taoufik and Probert, 2008) Mixed morphologies of apoptosis and necrosis observed could be explained by the initiation of apoptosis which is later overtaken by the molecular event associated with necrosis (Roy and Sapolsky, 1999)

The balance between numerous pro- and anti-apoptotic factors may contribute to the induction of apoptosis, this includes the formation of free radicals, increase in

excitatory amino acids and intracellular Ca2+, Bcl proteins, p53 and other transcription factors (Raghupathi et al., 2000) Apoptosis in the brain is regulated by both caspase-dependent and caspase-independent mechanisms Caspases are aspartate-specific cysteine proteases constitutively expressed in the brain and are activated by intrinsic and extrinsic signals (Galluzzi et al., 2009; Raghupathi et al., 2000; Yuan and Yankner, 2000) The two pathways, extrinsic pathway and intrinsic pathway are shown in (Figure 1-4) Extrinsic pathway initiates apoptosis through the engagement