Mechanisms of neurodegeneration and stem cell migration a study of molecular signals in the model of axotomy

Expression of chemokine receptors CXCR4, CCR2, CCR5 and CX3CR1 in the neural stem cells isolated from the subventricular zone of the adult rat brain.. Interactions of chemokines and ch

MIGRATION: A STUDY OF MOLECULAR SIGNALS

AFTER PERIPHERAL NERVE INJURIES

JI JUN FENG MBBS

A THESIS SUBMITTED FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

DEPARTMENT OF ANATOMY FACULTY OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE

2004

ACKNOWLEDGMENTS

I would like to express my deepest appreciation to my supervisor, Associate

Professor Samuel Sam Wah Tay, Department of Anatomy, National University of

Singapore, for his innovative ideas, invaluable guidance, constant encouragement,

infinite patience, and friendly critics throughout this study

I am greatly indebted to Assistant Professors S Thameem Dheen and He Bei

Ping, Department of Anatomy, National University of Singapore, for their valuable

suggestions and friendly help during this study

I am very grateful to Professor Ling Eng Ang, Head of Anatomy Department,

National University of Singapore, for his constant support and encouragement to me, and also for his full support in using the excellent research facilities

I must also acknowledge my gratitude to Mrs Ng Geok Lan, Mrs Yong Eng

Siang and the late Miss Margaret Sim for their excellent technical assistance; Mr Yick Tuck Yong for his constant assistance in computer work; Mr P Gobalakrishnan for his

constant guidance in photomicrography; Mr Tajuddin B Marican Ali for his help in

animal operation; Mr Lim Beng Hock for looking after the experimental animals; Miss

Teu Cheng Hong Kate for her assistance in cell culture work; and Mdm Ang Lye Gek Carolyne, Mdm Diljit Kaur, Mdm Teo Li Ching Violet for their secretarial assistance

I would like to thank all other staff members and my fellow postgraduate students

at Department of Anatomy, National University of Singapore for their help and support

I would like to express my special thanks to my friends, Drs.Ouyang Hongwei and Wang Zhuo at Department of Orthopaedic Surgery, National University Hospital for

their friendly help and advice

Certainly, without the financial support of the National University of Singapore, which offered a research grant (R181-000-059-213), this work would not have been

brought to a reality

I would like to take this opportunity to express my heartfelt thanks to my parents and sister for their full and endless support for my study

Finally, I am greatly indebted to my wife, Mdm Yu Xiao Liang for her

understanding and encouragement during this study

This thesis is dedicated to

my beloved family

PUBLICATIONS

Various portions of the present study have been published or accepted for publication

International Jounals:

1 Ji J, Dheen ST, Tay SSW (2002) Molecular analysis of vagal motoneuronal

degeneration after right vagotomy J Neurosci Res 69 (3): 406-17

2 Ji JF, He BP, Dheen ST, Tay SSW (2004) Expression of chemokine receptors

CXCR4, CCR2, CCR5 and CX3CR1 in the neural stem cells isolated from the

subventricular zone of the adult rat brain Neurosci Lett 355 (3): 236-40

3 Ji JF, He BP, Dheen ST, Tay SSW (2004) Interactions of chemokines and

chemokine receptors mediate the migration of bone marrow stromal cells to the

impaired sites in the brain after hypoglossal nerve avulsion Stem Cells 22 (3):

415-27

4 Ji JF, Dheen ST, Tay SSW Expressions of cytokines and chemokines in the dorsal

motor nucleus of the vagus of the rat after right vagotomy (Submitted)

Conference papers:

1 Ji JF, Dheen ST, Tay SSW (2002) Upregulation of inducible nitric oxide synthase

and its mRNA expression in vagal motor nuclei following right vagotomy in rat

Australia Neuroscience Society Meeting, Sydney, Australia

2 Ji JF, Dheen ST, Tay SSW (2002) Activation of apoptotic and

N-methyl-D-aspartate (NMDA) receptor-calcium-neuronal nitric oxide synthase (nNOS)

pathways in the vagal motor nuclei of rats after right vagotomy Annual Meetings of

Experimental Biology, New Orleans, LA, USA

3 Ji JF, Dheen ST, Tay SSW (2002) Site-specific migration of transplanted

mesenchymal stem cells into the hypoglossal nucleus after unilateral avulsion of

hypoglossal nerve Society for Neuroscience 32 nd Annual Meeting, Orlando, Florida, USA

4 Ji JF, He BP, Dheen ST, Tay SSW (2003) Expressions of chemokine receptors on

neural stem cells from adult rat brains Society for Neuroscience 33 rd Annual Meeting, New Orleans, LA, USA

5 Ji JF, He BP, Dheen ST, Tay SSW (2004) Expression of cytokines in the dorsal

motor nucleus of the vagus nerve after vagotomy 4 th ASEAN Microscopy Conference, Hanoi, Vietnam

TABLE OF CONTENTS

ACKNOWLEDGEMENTS……… i

DEDICATIONS………iii

PUBLICATIONS……… iv

TABLE OF CONTENTS……….vi

ABBREVIATIONS……….xvi

SUMMARY……… xx

CHAPTER 1: INTRODUCTION……….1

1 General introduction: Animal model of axotomy to study neurodegeneration 2

2 Neuronal and glial responses to axotomy……… 2

2.1 Axonal reaction……… 2

2.1.1 Morphological changes in nerve fibre………2

2.1.2 Changes in axonal transport………3

2.2 Perikaryal alteration……… 4

2.2.1 Morphological changes……… 4

2.2.2 Metabolic changes……… 5

2.3 Glial cell reaction……… 6

2.4 Fate of axotomized neurons 7

3 Neuronal death after axotomy……… 8

3.1 Apoptosis 8

3.1.1 Discovery of apoptosis……… 8

3.1.2 Morphological characteristics of apoptotic cell death………8

3.1.3 Apoptosis in the model of axotomy……… 9

3.2 Necrosis………9

4 Mechanisms of neurodegeneration………10

4.1 Involvement of Cytokines 10

4.1.1 Pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) ……… 11

4.1.2 Interleukin-6 (IL-6)……… 13

4.1.3 Transforming growth factor-beta 1 (TGF-β1)……… 14

4.2 Role of Nitric oxide (NO) in neurodegeneration.……… 14

4.2.1 Historical perspective of NO.……… 14

4.2.2 Isoforms of nitric oxide synthase (NOS).……….15

4.2.3 Biological functions of NO.……… 16

4.2.4 Roles of NO in the nervous system.……… 16

4.2.5 Roles of NO in the model of axotomy.……….19

4.3 Involvement of apoptosis associated molecules.………19

4.3.1 Bcl-2 and Bax.……… 19

4.3.1.1 Discovery of Bcl-2 and Bax 19

4.3.1.2 Functions of Bcl-2 and Bax in apoptosis.………20

4.3.1.3 Bcl-2 and Bax in the model of axotomy.……….21

4.3.2 Caspase-3.……….22

4.3.2.1 Discovery of caspases.……….22

4.3.2.2 Functions of caspase-3 in apoptosis.………23

4.3.2.3 Caspase-3 in the model of axotomy.………23

4.4 N-methyl-D-aspartate receptor (NMDAR)-calcium-neuronal nitric

oxide synthase (nNOS) pathway in neurodegeneration………24

4.4.1 Functions of NMDAR ……… 24

4.4.2 Calbindin D28 K (CB) 25

4.4.3 nNOS ………26

4.5 Chemokines/chemokine receptors……….27

4.5.1 Historical discovery of chemokines/chemokine receptors……… 27

4.5.2 Chemokines/chemokine receptors in the normal central nervous system (CNS) 28

4.5.2.1 Chemokines/chemokine receptors and brain development……….28

4.5.2.2 Chemokines/chemokine receptors in the normal adult brain…… 29

4.5.3 Roles of chemokines/chemokine receptors in neurodegeneration……… 30

4.5.3.1 Stromal Cell-Derived Factor 1 (SDF-1)……….30

4.5.3.2 Fractalkine……… 31

4.5.3.3 Monocyte Chemoattractant Protein 1 (MCP-1)……… 32

5 Animal model of the present study for molecular analysis of neurodegeneration: vagotomy………34

5.1 Structure and function of the vagus nerve system.……….34

5.2 Degeneration of the vagal motoneurons in the model of vagotomy……… 37

6 Cell replacement in the CNS: potential neuroregeneration by stem cells……….38

6.1 Mesenchymal Stem Cells (MSCs)……… 38

6.1.1 Historical discovery of MSCs……… 38

6.1.2 Biological characteristics of MSCs……… 39

6.1.2.1 Heterogeneity of MSCs………39

6.1.2.2 Expression of cytokines by MSCs……… 40

6.1.2.3 Proliferation and multipotent differentiation of MSCs………40

6.1.3 Transdifferentiation of MSCs into neurons and glia……….41

6.1.4 Therapeutic potential of MSCs to treat CNS diseases and injuries……… 42

6.1.5 Directed migration of transplanted MSCs………43

6.1.6 Mechanisms of cell migration: role of chemokines/chemokine receptors 43

6.1.6.1 SDF-1/CXCR4……….44

6.1.6.2 Fractalkine/CX3CR1………45

6.1.6.3 MCP-1/CCR2……… 45

6.1.7 Animal model of the present study of MSCs migration: unilateral avulsion of the hypoglossal nerve….……… 46

6.1.7.1 Structure of the hypoglossal nerve system……… 46

6.1.7.2 Unilateral avulsion of the hypoglossal nerve……… 47

6.2 Adult neural stem or progenitor cells………48

6.2.1 Historical discovery of neural stem or progenitor cells in the adult brain 48

6.2.2 Neural stem or progenitor cells in the subventricular zone (SVZ)……… 49

6.2.2.1 Origin of the postnatal SVZ………49

6.2.2.2 Architecture of the SVZ……… 49

6.2.2.3 Identity of neural stem or progenitor cells in the SVZ………… 50

6.2.3 Migration of endogenous neural stem or progenitor cells……… 51

6.2.3.1 Migration of endogenous neural stem or progenitor cells in the SVZ……… ……….51

6.2.3.2 Migration of transplanted neural stem or progenitor cells……… 52

6.2.4 Mechanisms of the migration of neural stem or progenitor cells in the SVZ ……… 53

6.2.4.1 Cell contact-mediating molecules……… 53

6.2.4.2 Soluble factors………54

6.2.5 Roles of chemokines in the migration of neural stem or progenitor cells 54

7 Aims of the present study……… 55

7.1 Molecular analysis of the degeneration of the vagal motoneurons in the DMV after right vagotomy.……… 55

7.2 Molecular analysis of the interactions of chemkines and chemokine receptors in mediating the migration of rMSCs to the impaired site in the brain after hypoglossal nerve avulsion.………58

7.3 Investigation of expression of chemokine receptors by neural stem or progenitor cells isolated from the SVZ of the adult rat brain as the potential mechanism of their migration ………60

CHAPTER 2: MATERIALS AND METHODS……… 62

1 Animals……….63

2 Right vagotomy………63

3 Avulsion of the left hypoglossal nerve……….64

4 Histology 64

4.1 Perfusion……….64

4.2 Tissue preparations………65

4.3 Histochemistry……… 67

4.3.1 Nissl staining with cresyl fast violet (CFV)……….67

4.3.2 NADPH-d histochemistry………68

4.3.3 Lectin histochemistry……… 68

5 TUNEL method………69

6 In situ hybridisation……… 69

6.1 Labelling probes with digoxigenin………70

6.2 In situ hybridisation.……… 71

7 Primary culture of rat MSCs (rMSCs)……… 74

7.1 Fluorescent activated cell sorting (FACS) analysis……… 76

7.2 In vitro differentiation assays……….78

7.3 In vitro chemotaxis assay……… 81

7.4 MTT (3-(4,5-dimethylthiazoyl-2-yl)2,5-diphenyltetrazolium-bromide) assay….83

7.5 Labelling of rMSCs………84

7.5.1 Labelling of rMSCs with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE)….……… 84

7.5.2 Transfection of rMSCs with enhanced green fluorescent protein-N1 (EGFP-N1) vector ……….85

7.6 In vivo rMSCs chemotaxis assay……… 88

8 Primary culture of neural stem or progenitor cells……… 89

8.1 In vitro differentiation assay……… 91

9 Primary culture of rat microglia……… 91

10 Propidium iodide (PI) staining.………92

11 Immunohistochemistry/immunocytochemistry.……… 93

12 Reverse transcription polymerase chain reaction (RT-PCR).……… 98

12.1 RNA isolation 98

12.2 One step RT-PCR.……… 99

12.3 Real time RT-PCR 102

13 Transplantation of rMSCs into lateral ventricles of the rat brain after left hypoglossal nerve avulsion ……… 104

CHAPTER 3: RESULTS……… 106

1 Molecular analysis of vagal motoneuronal degeneration after right vagotomy…….107

1.1 Morphological studies of vagal motoneurons in the DMV.………107

1.2 Expressions of cytokines TNF-α, IL-1β, IL-6, and TGF-β1 at mRNA and/or protein levels in the DMV.… ……….109

1.2.1 mRNA expressions of TNF-α, IL-1β, and TGF-β1 in the right brainstem ………… 109

1.2.2 Immunoreactivities of TNF-α, IL-1β, TGF-β1 and IL-6 in the DMV ….111

1.3 Expression of iNOS at mRNA and proteins levels in the DMV……… 115

1.4 Activation of apoptotic pathway in the DMV after right vagotomy………116

1.4.1 Expressions of Bcl-2 and Bax at both mRNA and protein levels……… 117

1.4.2 mRNA expression of Caspase-3………118

1.4.3 TUNEL labeling in vagal motoneurons……….118

1.5 Activation of NMDAR-Calcium-nNOS pathway in the DMV after right vagotomy ……… 119

1.5.1 Expression of nNOS in the DMV ………119

1.5.2 Colocalization of nNOS with NMDAR1 and CB in the DMV ……… 120

1.6 Expressions of chemokines MCP-1, fractalkine and SDF-1 at mRNA and/or protein levels in the DMV after right vagotomy ……… 121

1.6.1 mRNA expressions of MCP-1 and fractalkine ………121

1.6.2 Immunoreactivities of MCP-1, fractalkine and SDF-1 in the DMV … 122

1.6.3 Colocalization of lectin with CX3CR1 staining ……… 124

2 Involvement of the interactions of chemkines and chemokine receptors in the migration of rMSCs into the HN after nerve avulsion ……… 125

2.1 Immunoreactivities of chemokines SDF-1, fractalkine and MCP-1 in the HN after nerve avulsion……… 125

2.2 In vitro characterization of rMSCs.……….128

2.2.1 Morphology of rMSCs in vitro.……….128

2.2.2 Epitope analysis of rMSCs.………128

2.2.3 The differentiation of rMSCs in vitro ……… 129

2.3 Expressions of chemokine receptors CCR2, CCR5, CXCR4 and CX3CR1 in rMSCs at mRNA and protein levels ……….129

2.3.1 mRNA expressions of CCR2, CCR5, CXCR4 and CX3CR1 in rMSCs 130

2.3.2 Expressions of CCR2, CCR5, CXCR4 and CX3CR1 in rMSCs at the protein level….……… 131

2.4 rMSCs migration to fractalkine and SDF-1α in heterotrimeric G protein -dependent manner in vitro ………132

2.5 Labelling of rMSCs in vitro.………133

2.6 rMSCs migration to SDF-1 α in vivo ……… 133

2.7 Selective migration of transplanted rMSCs into the avulsed left HN ………….134

3 Expressions of chemokine receptors CXCR4, CCR2, CCR5 and CX3CR1 at mRNA and protein levels in neural stem or progenitor cells isolated from the SVZ of the adult rat brain ……….135

3.1 In vitro characterization of the cells isolated from the SVZ of the adult rat brain 136

3.1.1 Morphology of the cells ………136

3.1.2 The differentiation of the cells in vitro ……….136

3.2 Neural stem or progenitor cells isolated from the SVZ of the adult rat brain express CXCR4, CCR2, CCR5 and CX3CR1 ……… 137

3.2.1 mRNA expressions of CXCR4, CCR2, CCR5 and CX3CR1 in the cells… 137

3.2.2 Colocalization of nestin with CXCR4, CCR2, CCR5 and CX3CR1 in the cells……… 138

CHAPTER 4: DISCUSSION………139

1 Axotomy as model to study neurodegeneration……… 140

2 Factors and pathways involved in the vagal motorneuronal death……….141

2.1 TNF-α and IL-1β……… 141

2.2 iNOS-derived NO………143

2.3 Apoptotic pathway……… 144

2.4 NMDAR-calcium-nNOS pathway……… 148

2.5 Chemokines……… 150

2.5.1 MCP-1.……… 150

2.5.2 Fractalkine.……….152

2.5.3 SDF-1.………153

2.5.4 TGF-β1.……… 154

2.5.5 IL-6.……… 155

3 Interactions between chemokines and their receptors in the directed migration of rMSCs into axotomized HN.……… 158

3.1 SDF-1 and CXCR4……… 160

3.2 Fractalkine and CX3CR1……… 162

3.3 MCP-1/CCR2 and other chemokines/receptors……… 163

4 Expression of chemokine receptors in neural stem or progenitor cells isolated from the SVZ of the adult rat brain ……… 165

4.1 CXCR4……….167

4.2 CX3CR1……… 167

4.3 CCR2 and CCR5……… 168

CHAPTER 5: CONCLUSION……….171

REFERENCES……… 175

Figures and Figure Legends

ABBREVIATIONS

ABC avidin-biotin complex

α-MEM alpha minimal essential medium

CFV cresyl fast violet

CFDA-SE 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester

CNS central nervous system

DAB 3,3’-diaminobenzidine tetrahydrochloride

DMV dorsal motor nucleus of vagus

DEPC diethyl pyrocarbonate

DMEM Dulbecco’s Modified Eagle’s Medium

EAE experimental autoimmune encephalomyelitis

eNOS endothelial nitric oxide synthase

EGF epidermal growth factor

FACS fluorescent activated cell sorting

GAPDH glyceraldehyde-3-phosphate dehydrogenase homolog

GFAP glial fibrillary acidic protein

GluRs glutamate receptors

HCl hydrochloric acid

HN hypoglossal nucleus

hMSCs human bone marrow stromal cells or human mesenchymal stem cells HRP horseradish peroxidase

IEGs immediate early genes

IL-1 β interleukin-1 beta

IL-6 interleukin-6

IL-8 interleukin-8

IMDM Iscove’s Modified Dulbecco’s Medium

iNOS inducible nitric oxide synthase

MCP-1 monocyte chemoattractant protein 1

MIP-1 α macrophage inflammatory protein 1 alpha

mRNA messenger ribonucleic acid

MS multiple sclerosis

MSCs bone marrow stromal cells or mesenchymal stem cells

MTT 3-(4,5-dimethylthiazoyl-2-yl)2,5-diphenyltetrazolium-bromide

NA nucleus ambiguus

NADPH nicotinamide adenine dinucleotide phosphate

NCAM neural cell adhesion molecule

NeuN neuronal nuclei

NMDAR n-methyl-D-aspartatereceptor

nNOS neuronal nitric oxide synthase

NO nitric oxide

NOS nitric oxide synthase

NSCs neural stem cells

NUMI National University Medical Institute, Singapore

OB olfactory bulb

PBS phosphate buffered saline

PCR polymerase chain reaction

PI propidium iodide

PNS peripheral nervous system

PSA-NCAM polysialylated form of neural cell adhesion molecule

RGC retinal ganglion cells

rER rough endoplasmic reticulum

rhSDF-1α recombinant human stromal cell-derived factor 1 alpha

RMS rostral migratory stream

rMSCs rat bone marrow stromal cells or rat mesenchymal stem cells

rrfractalkine rat recombinant fractalkine

RT-PCR reverse transcription polymerase chain reaction

SDF-1 stromal cell-derived factor 1

SVZ subventricular zone

TBS tris buffered saline

TdT terminal deoxynucleotidyl transferse

TGF-β transforming growth factor-beta

TNF tumor necrosis factor

TNF-α tumor necrosis factor alpha

TUNEL terminal transferase-mediated deoxyuridine triphosphate-biotin nick end labeling

WD Wallerian degeneration

of a degenerative morphology and significant reduction in the number of neurons in the

vagal motor nuclei after right vagotomy Upregulated immunoreactivities of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), interleukin-6 (IL-6), and transforming growth factor-beta 1 (TGF-β1), significant increase in the numbers of their immunopositive cells, and enhanced expression of TNF-α, IL-1 β and TGF-β1 mRNA in

the right dorsal motor nucleus of the vagus nerve (DMV) after operation were shown by immunohistochemical, quantitative, and real-time PCR analysis, respectively The expression of iNOS protein and mRNA was induced in the DMV of vagotomized rats as

shown by immunohistochemistry, in situ hybridization and real-time PCR analysis The

enhanced bcl-2 and reduced bax mRNA levels and subsequent upregulation of both bcl-2 and bax mRNA as well as protein level in the DMV of vagotomized rat were observed In addition, the increase of caspase-3 mRNA level was detected in ipsilateral vagal motor

nuclei after right vagotomy Double immunofluorescence analysis showed complete localization of nNOS with NMDAR1 and with Calbindin D28K (CB) in ipsilateral DMV

co-at 10 days following right vagotomy The increased immunoreactivities of stromal derived factor 1 (SDF-1) and the upregulated expressions of monocyte chemoattractant

cell-protein 1 (MCP-1) and fractalkine at both the cell-protein and mRNA levels in the right DMV were shown by immunohistochemistry and/or real-time PCR analysis

The roles of interactions between chemokines and their receptors in the trafficking of rMSCs in the model of the left hypoglossal nerve avulsion were

investigated in the present study We demonstrated the expressions of chemokine

receptors CXCR4, CCR2, CCR5, and CX3CR1 by rMSCs at the mRNA and protein levels Recombinant human SDF-1α (rhSDF-1α), the ligand for CXCR4 and recombinant rat fractalkine (rrfractalkine), the ligand for CX3CR1 induce the migration of rMSCs in a

heterotrimeric G protein-dependent manner in vitro Furthermore, rhSDF-1α injected

intracerebrally acts as a potent stimulus for the homing of transplanted rMSCs to the site

of injection in the brain rMSCs, transplanted into the lateral ventricles of the rat brain, migrated to the avulsed nuclei at 1 and 2 weeks after operation The expressions of chemokines SDF-1 and fractalkine were observed to be increased in the avulsed nuclei at

1 and 2 weeks after the operations

In addition, the expressions of chemokine receptors CXCR4, CCR2, CCR5 and

CX3CR1 were demonstrated at the protein and mRNA levels by neural stem or progenitor cells isolated from the SVZ of the adult rat brain

These studies suggest that the cytokines signalling, apoptotic and calcium-nNOS pathways could be activated in the vagal motor nuclei after right vagotomy, which could play important roles in the vagal motoneuronal degeneration The present study suggested that increased expressions of chemokines such as MCP-1,

NMDAR1-fractalkine and SDF-1 could not only be involved in the motoneuronal degeneration after peripheral nerve injuries, but also the interaction between fractalkine with CX3CR1 and

the interaction between SDF-1 with CXCR4 could mediate the trafficking of transplanted rMSCs into the HN after avulsion of the left hypoglossal nerve In addition, chemokine

receptors expressed on neural stem or progenitor cells might play roles in the regulation

of adult neural stem or progenitor cells in the physiological or pathological conditions Further investigations may be necessary to understand whether chemokine receptors

could also play important roles in the selective migration of neural stem or progenitor cells

CHAPTER 1 INTRODUCTION

1 General introduction: Animal model of axotomy to study neurodegeneration

Neurodegeneration as a consequence of various clinical diseases (including

stroke, traumatic brain injuries and neurodegenerative diseases) seriously impairs functions in the brain Understanding the mechanisms which govern neurodegeneration will provide crucial clues to develop therapeutic strategies to inhibit the processes of

neurodegeneration To achieve the aim, a number of animal models like axotomy, ischemia, brain trauma, and neurodegenerative diseases have been developed to examine the mechanisms underlying neurodegeneration

Animal models of axotomy in different systems have been studied for many years

to gain insight into the mechanisms of progressive neuronal injury and degeneration (Lieberman, 1971; Torvik and Skjorten, 1971; Matthews, 1973; Decker, 1978; Al

Abdulla et al., 1998; Ginsberg and Martin, 1998) It is appropriate to review the neuronal

and glial cell responses to axotomy and mechanisms underlying neurodegeneration

2 Neuronal and glial responses to axotomy

The responses to axotomy are complex and described under the following three headings: i) axonal reaction; ii) perikaryal alteration; and iii) glial cell response

2.1 Axonal reaction

2.1.1 Morphological changes in nerve fibres

Axotomy of a peripheral nerve results in the nerve being separated into two parts: the segments distal and proximal to the lesion site Both of them undergo a succession of morphological changes The distal segment, separated from its neuronal cell body, will

degenerate in the way described by Waller in 1850, now termed Wallerian degeneration (WD) WD entails a sequence of breakdown and removal of the axon and myelin of the

nerve stump distal to an injury site following axotomy (see review by Stoll et al., 2002)

It begins with disintegration and degeneration of the axoplasma and axolemma which is

completed within 24 hours in small and 48 hours in larger nerve fibres (Stoll et al., 1989)

In response to axonal loss, Schwann cells proliferate and dedifferentiate, downregulate myelin protein synthesis, sequester myelin debris, and fragment their own myelin sheaths

into ovoids (Stoll et al., 2002) At a later stage, axons are no longer seen or have become vacuolated Schwann cell bands of Büngner with occasional leukocytes appear Then the bands wither and Schwann cells survive, leaving Schwann cell fingers to guide new nerve fibres sprouting from the proximal segment of the sectioned axon

The proximal segment which remained attached to the cell body following axotomy also undergoes degeneration similar to the distal part within a short distance from the lesion site However, sprouting of fibres starts within 4-6 hours after axotomy The definitive sprouting of the majority of the axons occurs 24 hours following axotomy

(Grafstein and Mcquarrie, 1978)

2.1.2 Changes in axonal transport

Axonal transport is defined by the movement of macromolecules and organelles through an axon Most materials like tubulin, actin and neurofilamentous proteins are

transported anterogradely from the perikaryon to the axon terminal at a fast and a slow rate In mammals, fast transport advances at 200-400 mm a day while slow transport at 0.2-6 mm a day (Lasek et al., 1984; McQuarrie et al., 1986) Some materials such as neurotrophic factors are also transported retrogradely from the axon terminal to the

perikaryon The rate of retrograde transport is thought to be about 100-200 mm a day

(Grafstein, 1977)

Axotomy does not appear to have any immediate effect on either the amount of materials that continue to move along the length of axon remaining attached to the cell

body, or the rate at which these materials move (Grafstein and Murray, 1969; Grafstein,

1971) After 1-2 days, there may be a decrease in the amount of transported

transmitter-associated materials (Boyle and Gillespie, 1970; Kirk, 1974), which may reflect their

decreased content in the neuron Still later, after axonal outgrowth has been initiated, there may be an increase in the overall amount of proteins that are axonally transported or

in their rate of transport or both (Grafstein, 1971; Kreutzberg and Schubert, 1971; Frizell and Sjostrand, 1974ab)

2.2 Perikaryal alteration

2.2.1 Morphological changes

The typical morphological changes in the cell body, first recognized by Nissl in

1892, include swelling of the cell body, migration of the nucleus to an eccentric position

in the perikaryon, and the apparent disappearance of basophilic material (“Nissl substance”) from the cytoplasm (reviewed by Grafstein, 1975) The phenomena of Nissl substance disintegration, redistribution and disappearance in the cytoplasm of the axotomized neuron was termed chromatolysis by Marinesco in 1896 The chromatolysis

has been revealed by electron microscopy to be due to the disorganization of the rough endoplasmic reticulum (rER) which constitutes the Nissl substance In the normal state, rER consists of concentrations of flattened elongate cisternae arranged in parallel arrays and the outer surfaces of the cisternae are lined with membrane-attached ribosomes

where numerous free polyribosomal elements lie (Palay and Palade, 1955) Upon axotomy, the regularity of the cisternal arrays is lost, the individual cisterns become

shorter, the total amount of membrane-associated ribosomes may be reduced, and the proportion of free polyribosomes concomitantly increases (Kirpatrik, 1968) This

accounts for the observation that the dispersion of the Nissl substance is often accompanied by an increase in diffuse cytoplasmic basophilia which, in some cases, is so intense that the cell body becomes frankly hyperchromatic (Murray and Goldberger,

1969)

Morphological alterations of the axotomized neurons are often not restricted to their cell bodies Sectioning of one axon branch may result in axonal sprouting at the terminals of the collateral branches (Pickel et al., 1974), and a significant retraction of the

dendritic field of the axotomized neuron has been observed (Sumner and Watson, 1971)

synthesis leads to an increased cytoplasmic RNA content Additionally, there are also changes in the configuration of the RNA, which involves disorganization of the ordered arrays of rER (Nissl substance) The change in the form of the RNA is the basis for the histologically detectable features of chromatolysis

ii) Protein metabolism:

The increased incorporation of radioactive protein precursors has been shown in

the axotomized neurons, indicating an increase in protein synthesis (Lieberman, 1971) However, certain specific proteins, many of which are associated with synaptic transmission, are decreased in the axotomized neurons (Lieberman, 1971; Reis and Ross,

1973; Cheah and Geffen, 1973) There is a change in the relative proportions of different

types of materials synthesized by the neuron In general, the production of structural component required for transmitter function shifts toward the production of structural component required for restitution of the axon

iii) Lipid synthesis:

A significant increase in lipid synthesis may begin very soon after axotomy (Miani, 1962; Harkonen and Kauffman, 1974) This presumably corresponds to an increased synthesis of membranous constituents of the axotomized neuron

2.3 Glial cell reaction

In the central nervous system (CNS), the glial cells consist of microglia, astrocytes, and oligodendrocytes In parallel with neuronal alterations after axotomy, the surrounding non-neuronal cells also respond with marked alterations in their numbers

and/or structures and molecular expressions

Microglia and astrocytes close to the axotomized neurons are activated rapidly following axon injury Microglial cells proliferate and migrate towards the axotomized neuronal perikarya and the activated microglial cells express a number of inflammatory

and immune mediators Astrocytes also proliferate and upregulate glial fibrillary acidic protein (GFAP), a specific marker for astrocyte (Aldskogius and Kozlova, 1998)

2.4 Fate of axotomized neurons

There are three possible fates of neurons in response to axotomy: (i) complete

recovery; (ii) atrophy or (iii) degeneration and death When the injured axon is rebuilt and has made contact with a functionally adequate target, the neuron has a possibility to regain normal structure and function If target contact is not made, but the neuron

survives, it will end up with an atrophic state The size of its perikaryon and the diameter

of its axon will be reduced compared to the normal state and many of the biochemical responses to axon injury will become permanent or at least longstanding

Several factors influence the progression of axotomy-induced neuronal injury and

the likelihood of subsequent neuronal death or survival (reviewed by Lieberman, 1971; Fry and Cowan, 1972):

i) Location of the axotomized neuronal body within peripheral nervous system (PNS) or CNS The axotomized neurons in the CNS show a high probability of

degeneration, whereas neurons in the PNS are more likely to recover or regenerate after axotomy

ii) Age of the animal at the time of axotomy While the axotomized neurons in the immature nervous system show more prominent changes and often die quickly, they are

more likely to recover or persist in some altered form like atrophy (Lavelle and Lavelle, 1958; Lieberman, 1971; Pilar and Landmesser, 1976) It has been shown that transection

of the facial cranial nerve or sciatic nerve in newborn rodents causes loss of motor neurons in the facial nucleus or in the lumbar cord, respectively In contrast, a similar

lesion in adults produces no discernible loss of motor neurons and neurons appear to atrophy and survive rather than die (Decker, 1978)

iii) Location of axonal trauma in relation to its cell body The axotomized neurons are more prone to death if the axonal trauma is closer to the neuronal bodies

3 Neuronal death after axotomy

3.1 Apoptosis

3.1.1 Discovery of apoptosis

Apoptosis has been first recognized during vertebrate development as part of a

natural process to remove superfluous or used-up cells (Saunders, Jr., 1966) It has since

become evident that death morphologies closely resembling ontogenic deaths occur in post-developmental cells in response to various physiological, pathological or

pharmacological agents (Thompson, 1995) Crucial roles of apoptotic death have thus

been extended to morphogenesis, tissue homeostasis, immune regulation and the elimination of infected, mutated or damaged cells (Thompson, 1995; Jacobson et al., 1997) In all these cases, an afflicted cell senses that its environment or physical state has

been compromised and by consequence undergoes a suicide process, using an intrinsic molecular death machine

3.1.2 Morphological characteristics of apoptotic cell death

Apoptosis is a major form of cell death, characterized by a series of distinct

morphological and biochemical alterations (see review by Arends and Wyllie, 1991) It occurs in two phases: first a commitment to cell death, followed by an execution phase characterized by dramatic stereotypic morphological changes in cell structure (Jacobson

et al., 1994; Takahashi and Earnshaw, 1996) Morphologically, apoptotic cell death is

defined by condensation and fragmentation of nuclear chromatin, compaction of cytoplasmic organelles, dilatation of the endoplasmic reticulum (frequently in a

subplasmalemmal distribution), a decrease in cell volume, and alterations to the plasma membrane resulting in the recognition and phagocytosis of apoptotic cells, thereby

preventing an inflammatory response

3.1.3 Apoptosis in the model of axotomy

Inappropriate apoptosis is implicated in many human diseases, including

neurodegenerative diseases such as Alzheimer’s disease and Huntington’s disease, ischemic damage, autoimmune disorders, and several forms of cancers (Thompson, 1995; Nicholson, 1996) Apoptotic cell death has been reported in various neural tissues i.e., retinal ganglion cells (RGC) (Rabacchi et al., 1994; Quigley et al., 1995), spinal motor

neurons (Gu et al., 1997), sensory neurons (Groves et al., 1997; Oliveira et al., 1997), and facial nerve cells (de Bilbao and Dubois-Dauphin, 1996) in neonatal and adult rodents after axotomy of the optic, sciatic and facial nerves, respectively

3.2 Necrosis

In contrast to the active form of apoptotic cell death, necrosis is a passive form of cell death caused by gross physical or chemical insults Necrotic cell death is characterized by the early loss of plasma membrane integrity The leaky membrane leads

to increase in the content of intracellular fluid and subsequent swelling The organelles of

the necrotic cells also become swollen and finally disintegrated The DNA becomes randomly fragmented and the entire cell undergoes lysis with the resultant spillage of intracellular contents to the surrounding tissues The spillage of lytic enzymes can cause damage to the surrounding tissues and may lead to inflammation

4 Mechanisms of neurodegeneration

4.1 Involvement of Cytokines

Cytokines are soluble extracellular proteins and glycoproteins that are generated

in response to various injurious stimuli by virtually every nucleated cell (Oppenheim, 2001) Functionally, cytokines have been classified as either pro-inflammatory (Th1-type,

stimulatory) mediators including tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) or anti-inflammatory (Th2-type, inhibitory) cytokines such as transforming growth factor β (TGF-β) depending on the final balance of their effects on the immune system (Mosmann et al., 1986) Usually the pro-inflammatory cytokines are first

upregulated in response to various stimuli, which induce the synthesis of inflammatory cytokines, creating an autoregulatory feedback loop that re-establishes a resting immunological status However, cytokines are involved not only in the immune response but also in a variety of physiological and pathological processes In general,

anti-they are crucial participants in receptor-mediated intercellular signaling that regulate cells engaged in innate and adaptive immunity, inflammation, cell growth and differentiation, cell death, angiogenesis and repair processes (Hallenbeck, 2002)

In the brain, there is ample evidence for low-level constitutive expression of

numerous pro-inflammatory and anti-inflammatory cytokines as well as their active receptors This supports the role for cytokines in normal functioning and maintenance of homeostasis (Hallenbeck, 2002) However, in pathological conditions, it has been widely recognized during the last decade that neurotoxic and neuroprotective mechanisms are

both closely associated with the balance between the pro-inflammatory and inflammatory cytokines The process of neurodegeneration is closely related to the shift

anti-of cytokine balance towards the pro-inflammatory cytokines like IL-1β or TNF-α

Evidence, in support of the role of cytokines in driving the inflammatory response and

that this process is causally related to the degree of brain injury, includes: (1) the capacity

of inflammatory cytokines to exacerbate brain damage; (2) the capacity of inflammatory cytokine blockade to reduce ischemic brain damage; and (3) depletion of

pro-circulating neutrophils reduces ischemic brain injury (Feuerstein et al., 1998) On the other hand, anti-inflammatory cytokines in the CNS maintain homeostasis and protect cell viability by inhibiting inflammatory responses (Feuerstein et al., 1998; Rothwell, 1999) Since there is an inhibitory cross regulation between two groups of cytokines that

indirectly suppress each other’s synthesis, this provides a further fine-tuning of the

balance of their neurodegenerative and neuroprotective effects

4.1.1 Pro-inflammatory cytokines TNF-α and IL-1β

Tumor necrosis factor (TNF) exists in α and β forms TNF-α is produced as a 17

kD secreted polypeptide by the cleavage of TNF-α converting enzyme There are two types of TNF receptors ie 55 kD (TNFR1, p55) and 75 kD (TNFR2, p75) receptors While the released TNF-α reacts with both p55 and p75, TNF-induced effects are mediated by TNFR1 in most cells (Goodman et al., 1990; Wang and Shuaib, 2002)

Interleukin-1 (1) is a 17-kDa polypeptide that also exits as two isoforms, α and 1β (Rothwell and Luheshi, 2000) There are two types of IL-1 receptors, i.e types I and II IL-1β binds to both receptors, whereas signal transductions of IL-1 is mediated by type I receptors (Loddick et al., 1998)

IL-Receptor bindings of the cytokines result in the initiation and propagation of cytoplasmic signals usually leading to transcriptional activation Although TNF-α and IL-

1β interact with receptors that are structurally unrelated, there is significant overlap in functional and post-receptor events including phospholipid hydrolysis, protein tyrosine

phosphorylation, and subsequent transcriptional activation of nuclear factor-kappa B (NF-κB) (Dinarello, 1991; Ariga et al., 1998; Perry and Hannun, 1998; Mathias et al., 1998)

The expression of IL-1β has been described in healthy mouse, rat, pig, and human

In rats, IL-1β and its mRNA are detected in neurons from several brain structures including the cerebellum, hypothalamus, and hippocampus (Bandtlow et al., 1990; Lechan et al., 1990) IL-1β mRNA has also been observed in glial cells of the white

matter in different brain regions in rats (Bandtlow et al., 1990) TNF-α is also expressed

in the brain of healthy adult mice and rats (Taupin et al., 1993) In pathological conditions, increases in both TNF-α and IL-1β production have been observed after damage due to traumatic brain injury, ischemia, infections, or diseases that are associated

with degeneration of the CNS parenchyma Moreover, in the model of axotomy, it has been shown that TNF-α or IL-1β was increased in the axotomized facial motor nucleus or axotomized superior cervical ganglia (Carlson et al., 1996; Streit et al., 1998; Raivich et al., 2003)

The functional roles of TNF-α and IL-1β after CNS injury are controversial Depending on the type of injury and experimental conditions, TNF-α and IL-1β may produce detrimental or beneficial effects in CNS injury It has been shown that TNF-α

and IL-1β rescue RGC from retrograde cell death in vivo after axotomy of the optic nerve

(Diem et al., 2001; Diem et al., 2003) However, in the model of axotomy of the facial nerve in the transgenic mice, neutralization of endogenous TNF-α by means of

overexpression of its soluble receptor (sTNFR1) decreases cell death of the injured facial motoneurons, suggesting that TNF-α may play an important role in neuronal

degeneration in the CNS following a lesion (Terrado et al., 2000)

4.1.2 Interleukin-6 (IL-6)

IL-6 is a member of the neuropoietic cytokine family which binds to specific cell

surface receptors Interaction of IL-6 with its specific receptors and an additional common receptor component, the gp130 molecule, triggers a cascade of intracellular events including activation of JAKs-STAT pathway or Raf-MEK-MAPK pathway, leading to the activation of transcriptional factors (Darnell, Jr et al., 1994; Hirano et al.,

1994; Kishimoto et al., 1994; Ihle et al., 1995)

In the normal rat CNS, IL-6 transcripts are expressed in specific neuronal subpopulations in various brain areas (Schobitz et al., 1992; Yan et al., 1992; Schobitz et al., 1993) Although IL-6 has been reported to be involved in pathophysiological events

of neurodegenerative diseases (Bauer et al., 1991; Hull et al., 1996), a variety of studies demonstrate the neuroprotective properties of IL-6 (Hama et al., 1989; Kushima and Hatanaka, 1992; Toulmond et al., 1992; Ikeda et al., 1996; Matsuda et al., 1996) It has been shown that IL-6 supports the survival of cholinergic, catecholaminergic and sensory

neurons against detrimental insults (Hama et al., 1989; Toulmond et al., 1992; Kushima and Hatanaka, 1992; Ikeda et al., 1996; Matsuda et al., 1996)

4.1.3 TGF-β1

The TGF-β isoforms are closely related polypeptides which belong to a larger

group of molecules, the TGF-β superfamily, with whom they share both sequence homologies and a similar three-dimensional structure They are potent regulators of

cellular growth and differentiation, inflammatory events, extracellular matrix formation and wound healing (Sporn and Roberts, 1992) Biological actions of TGF-β are mediated

by its binding to a heteromeric transmembrane receptor complex of two subunits designated type I (RI) and type II (RII) It has been proposed that binding of TGF-β to RII induces assembly of RII-RI heterodimer and transphorylation of RI by RII and this

then activates signal transduction pathways eliciting a biological response (Massague, 1996)

In the adult rat brain, while TGF-β2- and TGF-β3-like immunoreactivities were widely found in subpopulations of neurons throughout the brain, TGF-β1 mRNA is only

detected at very low levels, and TGF-β1 immunoreactivities appear to be entirely absent from the parenchyma (Unsicker et al., 1991) However, TGF-β1 has been shown to be upregulated in diverse experimental lesions to the rat brain to promote the survival of impaired neurons (Lindholm et al., 1992; Kiefer et al., 1995)

4.2 Role of NO in neurodegeneration

4.2.1 Historical perspective of NO

In mammals, the synthesis of NO is derived from the oxidation of the amino acid L-arginine catalyzed by nitric oxide synthase (NOS) in the presence of oxygen and

nicotinamide adenine dinucleotide phosphate (NADPH) (Dawson and Dawson, 1998)

For many decades, NO did not gain the interests of biologists and was used as a preservative Moreover, it has also been noted as an atmosphere pollutant, being a by-

product of jet and motor engines (Leong, 1999)

Great interest was aroused when NO was found to be the active metabolite responsible for blood vessel relaxation induced by nitroglycerin and the organic nitrates

used in the treatment of cardiac angina (Murad et al., 1978) Furchgott and Zawadzki first

described endothelial-derived relaxing factor (EDRF) in 1980 (Furchgott and Zawadzki,

1980), which was later identified as NO (Palmer et al., 1987), Since then, the study of

NO signaling has become one of the fastest growing areas in biomedical research Its importance has been recognized by the award of the Nobel Prize in Physiology or

Medicine to Furchgott, Ignarro and Murad in 1998

TNF-α NOS III (NOS3, eNOS) is expressed mainly in endothelial cells (Moncada et al.,

1991; Nathan and Xie, 1994)

The nNOS and eNOS isoforms are constitutively expressed, but are subjected to expressional regulation (Forstermann et al., 1998) Their catalytic activities are dependent

on Ca2+ and calmodulin (Bredt and Snyder, 1990) However, iNOS is Ca2+ and calmodulin independent and is a transcriptionally regulated enzyme (Lowenstein et al.,

1992) The expression of iNOS requires protein synthesis, which is mediated by specific

combination of cytokines

4.2.3 Biological functions of NO

NO is a key informational substance, the effects of which can either contribute to

or counteract pathophysiology in blood vessels, heart, lung, kidney, brain, pancreas, gut

and the immune system (Murad, 1998) Unlike other signalling molecules, NO tends to form covalent bonds with its targets, rather than mediating its effects through typical

receptor-activated mechanisms Although NO does not appear to have receptors in the traditional sense, heme-containing macromolecules have been implicated as NO carriers (Murad, 1998) NO is lipophilic and can passively concentrate in cell membranes Its

biological availability is therefore based on transcellular diffusion to intracellular target

Biological activity of NO is classified by dependent and independent pathways, both attributed to physiology and pathology (Stamler, 1994; Schmidt and Walter, 1994) Activation of soluble guanylyl cyclase, formation of cGMP,

cGMP-and concomitant protein phosphorylation is considered the main physiological signalling pathway of NO However, cGMP-independent reactions appear to be of greater importance for cytostatic or cytotoxic effects of NO NO is a key transducer of the vasodilator message from the endothelium to vascular cells, a constituent in central and

peripheral neuronal transmission, and a participant in non-specific immune defence

4.2.4 Roles of NO in the nervous system

The roles of NO in the nervous system have been characterized on the following three aspects: i) neuronal messenger; ii) neurotoxicity; and iii) neuroprotection

i) Neuronal messenger:

The highest levels of NO throughout the body are in neurons, where NO functions

as a unique messenger molecule In the autonomic nervous system, NO functions as a major noncholinergic nonadrenergic (NANC) neurotransmitter This NANC pathway

plays a particularly important role in producing relaxation of smooth muscles in the cerebral circulation, and the gastrointestinal, urogenital and respiratory tracts

(Grozdanovic et al., 1994) In the brain, NO functions as a neuromodulator and appears to mediate aspects of learning and memory (Bredt, 1999) While NO normally functions as

a physiological neuronal mediator, excess production of NO may cause brain injury

ii) Neurotoxicity:

In the brain, iNOS expression has been well characterized in astrocytes,

microglia, and to a lesser extent in endothelial cells (Murphy et al., 1993) Glial iNOS expression has been described after an injury or trauma (Loihl and Murphy, 1998), and in several pathologies, including demyelinating diseases (Willenborg et al., 1999), cerebral ischemia (del Zoppo et al., 2000), and Alzheimer’s disease (Weldon et al., 1998) iNOS,

once induced, is active for hours to days and produces NO in 1000-fold larger quantities than the constitutive enzymes eNOS and nNOS Pathophysiological concentration of NO can induce direct DNA damage and disruption of the mitochondrial membrane potential The inhibition of the mitochondrial function is suggested to lead to the formation of

superoxide anion (O2.-) NO reacts with superoxide to form an even more potent oxidant, peroxynitrite (ONOO-), which also causes direct DNA damage DNA damage induces the activation of p53 followed by upregulation of the pro-apoptotic bax gene, downregulation

of the anti-apoptotic bcl-2 gene (Nishio and Watanabe, 1997), and activation of caspases,

the final death pathway, to mediate apoptosis (Nishio and Watanabe, 1998)

Through these mechanisms, NO appears to play a major role in the pathophysiology of various neurological diseases, including stroke (Samdani et al., 1997), Parkinson's disease (Hirsch et al., 2003), Huntington's disease (Ferrante et al.,

1985), amyotrophic lateral sclerosis (Hirsch et al., 2003), and spinal cord injury (Wu et al., 1994)