MECHANISMS OF NEURODEGENERATION AND STEM CELL
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 i

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.

iv
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 CX
3
CR1 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-


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.
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS…………………………………………………………… i
DEDICATIONS…………………………………………………………………………iii
PUBLICATIONS……………………………………………………………………… iv
TABLE OF CONTENTS……………………………………………………………….vi

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

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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

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

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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

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

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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

3. Expressions of chemokine receptors CXCR4, CCR2, CCR5 and CX
3
CR1 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 CX
3
CR1 ………………………… 137
3.2.1. mRNA expressions of CXCR4, CCR2, CCR5 and CX
3
CR1 in the
cells… 137
3.2.2. Colocalization of nestin with CXCR4, CCR2, CCR5 and CX
3
CR1 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

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ABBREVIATIONS

ABC avidin-biotin complex
α-MEM alpha minimal essential medium
AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate
AP alkaline phosphate
BBB blood-brain-barrier

CaBPs Ca
2+
binding proteins
CB calbindin D28K
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

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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

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TNF-α tumor necrosis factor alpha
TUNEL terminal transferase-mediated deoxyuridine triphosphate-biotin nick end
labeling
WD Wallerian degeneration

SUMMARY xx
Summary
Cytokines signalling, inducible nitric oxide synthase (iNOS) expression,

3
CR1 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 CX
3
CR1 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
CX
3
CR1 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 NMDAR1-
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,
fractalkine and SDF-1 could not only be involved in the motoneuronal degeneration after
peripheral nerve injuries, but also the interaction between fractalkine with CX
3
CR1 and
SUMMARY xxii
the interaction between SDF-1 with CXCR4 could mediate the trafficking of transplanted

INTRODUCTION
2
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