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THE INVOLVEMENT OF NEUROPEPTIDES IN
NERVE REGENERATION
byNeil W Bindemann
A thesis presented for the degree of
Doctor of Philosophy in Neurobiology
at London University
Department of Biology
Medawar Building
University College London
ProQuest Number: 10017353
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2
ABSTRACT
The studies presented in this thesis are concerned with
the role of peptides in regeneration of the peripheral
nervous system of the adult rat, and the presence of
macrophages expressing different antigenic markers in the
peripheral nervous system.
The major section examines the time course of
accumulation of two neuropeptides, calcitonin gene-related
peptide (CGRP) and vasoactive intestinal peptide (VIP), as
well as the lymphokine, gamma interferon (IFNy), following
lesion to the sciatic nerve. Using radioimmunoassay, elevated levels of CGRP and VIP-like immunoreactivity
expressed in the sciatic nerve after injury are quantified.
The time course for IFNy accumulation is documented by
immunohi stochemi stry.
Data are also presented demonstrating that addition of
synthetic human CGRP causes an accumulation of cyclic
adenosine monophosphate (cyclic AMP) within macrophages
isolated from the sciatic nerve, peritoneum and spleen.
However, additional experiments demonstrate that the
ability of CGRP to alter such cyclic AMP levels is
influenced by the state of macrophage activation. This
raises the possibility that CGRP is important for
regulating the response of macrophages which infiltrate
injured sciatic nerve.
3
In the second section of the thesis, the distribution of
macrophage subpopulations/phenotypes in normal versus
lesioned sciatic nerve are described. A series of
monoclonal antibodies to macrophage markers including,
ED1,2,3, Leukocyte Common Antigen (LCA) and 0X42
demonstrate how numbers and distribution of macrophage
phenotypes alters in response to peripheral nerve injury.
4
ACKNOWLEDGEMENTS
I thank all my friends in the Medawar building for their
help and advice. I especially like to thank my supervisor,
Anne Mudge, from whom I received support and excellent
guidance on how to approach scientific research. I also
thank Martin Raff for his support, Elaine Clarke for her
excellent technical assistance, and Anne Lawrence and Jim
Voyvodic for many useful suggestions on the manuscript.
Finally, I would like to thank my father for his great
support and encouragement.
5
CONTENTS
ABSTRACT 2
ACKNOWLEDGEMENTS 4
LIST OF TABLES AND FIGURES 7
LIST OF ABBREVIATIONS 12
CHAPTER ONE. INTRODUCTION 14
Introduction 15
The peripheral nerve: 16
The Neuron
Glial cells of the peripheral nervous system Fibroblasts and macrophages
The damaged axon: 20
Inflammatory changes
The cell body response
The degenerating distal stump
The dual role of the macrophage 22
(i) macrophages and degeneration
(ii) macrophages and regeneration
Rebuilding the axon 27
The distal stump and regeneration 29
Proliferation of Schwann cells
Schwann cells and axon guidance
Interstump gap
6
Nerve Growth Factor: A role in regeneration? 34
Neuropeptides: a trophic role? 38
Neuropeptides and the inflammatory response. 41
Thesis objectives 44
CHAPTER TWO. CGRP and VIP IN THE PERIPHERAL
NERVOUS SYSTEM: A ROLE FOR CGRP IN
PERIPHERAL NERVE INJURY
Introduction 46
Materials and methods 51
Results 64
Discussion 96
CHAPTER THREE. DISTINCT MACROPHAGE PHENOTYPES IN LESIONED
VERSUS UNLESIONED SCIATIC NERVE
Introduction 107
Materials and methods 111
Results 115
Discussion 141
CONCLUSIONS 14 9
REFERENCES 155
7
LISTS OF TABLES AND FIGURES
CHAPTER 1
Figure 1.1 General structure of a peripheral nerve 18
trunk.
CHAPTER 2
Figure 2.1 Diagram of a lesioned sciatic nerve. 52
Figure 2.2 Displacement curves for synthetic human 65
CGRP and CGRP present in lesioned sciatic
nerve.
Figure 2.3 Displacement curves for synthetic human 66
VIP and VIP-like immunoreactivity present
in lesioned sciatic nerve.
Figure 2.4 Elution profile of CGRP-immunoreactivity 68
from (a) lesioned sciatic nerve and (b)
synthetic '”I-CGRP.
Figure 2.5 CGRP immunohistochemistry in sciatic 69
nerve, distal to the crush site, 6 days post
crush nerve.
Figure 2.6 CGRP levels in 2 days post-sham operated 70
Figure 2.7 CGRP levels in regenerating sciatic nerves 73
crushed with forceps
Figure 2.8 CGRP levels in regenerating sciatic nerves 74
crushed with a suture.
8
Figure 2.9 CGRP levels in nonregenerating sciatic 76
nerves crushed with a suture.
Figure 2.10 CGRP levels in transected sciatic nerves. 77
Figure 2.11 The effect of different types of lesions 79
on CGRP levels in the sciatic nerve.
Figure 2.12 VIP levels in crushed sciatic nerve. 80
Figure 2.13 IFNy staining in sciatic nerve, distal to 82
the crush site, (a) 2 hours post-crush and
(b) 2 days post-crush.
Figure 2.14 IFNy staining in sciatic nerve, distal to 83
the crush site (a) 6 days post-crush and
(b) 18 days post-crush.
Figure 2.15 Time course of cAMP accumulation by 85
activated and resident peritoneal macrophages.
Figure 2.16 Effect of CGRP on cAMP accumulation by 86activated and resident peritoneal
macrophages.
Figure 2.17 Effect of CGRP on cAMP accumulation by 88
peritoneal macrophages after IFN-y
preincubation.
Figure 2.18 Time course of cAMP accumulation by spleen 89
macrophages.
Figure 2.19 Effect of CGRP on cAMP accumulation by 90
spleen macrophages.
Figure 2.20 Effect of CGRP preincubation on the cAMP 91
response by spleen macrophages to CGRP.
Figure 2.21 Effect of CGRP on cAMP accumulation by 92
spleen macrophages after IFN-y preincubation.
9
Figure 2.22 Effect of CGRP on cAMP accumulation by 94
macrophages isolated from crushed sciatic
nerve.
Figure 2.23 Effect of CGRP on cAMP accumulation by 95
macrophages isolated from unlesioned sciatic
nerve.
CHAPTER 3
Table 3.1 Antibodies used for immunohistochemistry. 113
Table 3.2 Summary of distribution of macrophage 119
phenotypes in normal and lesioned sciatic
nerve.
Figure 3.1 Staining of myeloperoxidase-positive cells 116
in lesioned sciatic nerve of (a) mouse and
(b) rat.
Figure 3.2 Staining of (a) ED3* and (b) 0X42* 117
macrophages in the endoneurium of an
unlesioned rat sciatic nerve.
Figure 3.3 Staining of (a) ED2* and (b) EDI* 118
macrophages in the endoneurium of an
unlesioned rat sciatic nerve.
Figure 3.4 Higher magnification of (a) EDI* 121
macrophages in an unlesioned rat sciatic
nerve, close to a blood vessel, with (b) the
corresponding phase photograph of the
endoneurial blood vessel.
10
Figure 3.5 Staining of (a) ED7* and (b) 0X1/LCA* 122leukocytes found in the endoneurium within 1.2mm of the crush site of a 2 hour postcrush rat sciatic nerve.
Figure 3.6 Staining of (a) ED7* and (b) 0X1/LCA* 123leukocytes found in the sheath of a 2 hour post-crush rat sciatic nerve.
Figure 3.7 Staining of EDI* macrophages in the 124endoneurium (a) within 1.2mm of the crush site and (b) 6mm distal to the crush site of a 3 day post-crush rat sciatic nerve.
Figure 3.8 Staining of EDI* macrophages found in the 125endoneurium, 3mm proximal to the crush site of a 3 day post-crush rat sciatic nerve.
Figure 3.9 Staining of (a) ED3* and (b) ED2* 126macrophages in the endoneurium, within 1.2mm of the crush site of a 3 day post-crush rat sciatic nerve.
Figure 3.10 Staining of ED2* macrophages in the 128endoneurium, 3mm proximal to the crush site of a 3 day post-crush sciatic nerve.
Figure 3.11 Staining of (a) 0X42 and (b) 0X1/LCA* 129macrophages in the endoneurium, within 1.2mm of the crush site of a 3 day post-crush rat sciatic nerve.
Figure 3.12 Staining of 0X4/MHC class II* macrophages 130in the endoneurium, within 1.2mm of the crushsite of a 3 day post-crush rat sciatic nerve.
11
Figure 3.13 Staining of (a) EDI* and (b) ED3* 132macrophages in the endoneurium of an 18 day post-crush rat sciatic nerve.
Figure 3.14 Staining of ED2* macrophages in the 133endoneurium of an 18 day post-crush rat sciatic nerve.
Figure 3.15 Staining of (a) ED3* and (b) ED2* 134macrophages in the sheath of an 18 day postcrush rat sciatic nerve.
Figure 3.16 Staining of EDI* macrophages in the sheath 135 of an 18 day post-crush rat sciatic nerve.
Figure 3.17 Staining of (a) EDI* macrophages, with (b) 137the corresponding SlOO staining, in the endoneurium within 1.2mm of the crush site of 6 day post-crush rat sciatic nerve.
Figure 3.18 Staining of (A) EDI* macrophages with (B) 138the corresponding SlOO staining 6mm distal to the crush site of a 6 day post-crush rat sciatic nerve.
Figure 3.19 Staining of EDI* macrophages, with (B) the 139 corresponding SlOO staining, in the endoneurium of an 18 day post-crush rat sciatic nerve.
Figure 3.20 Staining of (A) BrdU* cells and (B) EDI* 140 and BrdU* cells 2mm distal to the crush site of a 4 day post-crush rat sciatic nerve.
12
LISTS OF ABBREVIATIONS
ACh acetylcholine
APC antigen presenting cell
bFGF basic fibroblast growth factor
BrdU bromodeoxyuridine
cAMP 3',5'-cyclic adenosine monophosphate
CGRP calcitonin gene-related peptide
CNS central nervous system
DAB 3,3'-diaminobenzidine tetrahydrochloride
DRG dorsal.root ganglion
FCS foetal calf serum
GFAP glial fibrillary acidic protein
GGF glial growth factor
HCl hydrochloric acid
IBMX isobutyl-methylanthine
I-CAM intercellular adhesion molecule
IFNy gamma interferon
IHC immunohistochemistry
IL-1 interleukin-1
IL-4 interleukin-4
KCl potassium chloride
LCA leucocyte common antigen
LFA-1 leucocyte function-associated antigen-1
LPS 1ipopolysaccharide
MAC-1 monocyte/myeloid adhesion complex
MBP myelin basic protein
mcAb monoclonal antibody
MHC major histocompatibility antigens
13
NaCl sodium chloride
N-CAM neural cell adhesion molecule
Ng-CAM neural-glia cell adhesion molecule
NF-H high molecular weight neurofilament
NF-L low molecular weight neurofilament
NF-M middle size molecular weight neurofilament
NGF nerve growth factor
NKA neurokinin A
NKB neurokinin B
NP-Y neuropeptide Y
PALS periarteriolar lymphatic sheath
PBS phosphate buffered saline
PDGF platelet-derived growth factor
PNS peripheral nervous system
RIA radioimmunoassay
RNA messenger ribonucleic acid
SCa slow component a
SCb slow component b
s.e.m. standard error of the mean
SK substance K
SP substance P
TNF tumour necrosis factor
VIP vasoactive intestinal peptide
14
CHAPTER 1
INTRODUCTION
15
INTRODUCTION
The ability of peripheral nervous system (PNS) neurons
in mammals to regenerate following injury, while those of
the central nervous system in mammals do not, is a
fundamental difference between the two systems which has an
important bearing on clinical medicine. Although, the
proximal stumps of axons transected within the central
nervous system begin to regenerate, this growth ceases
after approximately 2 weeks (Cajal, 1928). By contrast,
peripheral nerves are able to continue to regenerate beyond
2 weeks and may successfully reinnervate their targets.
Early studies by Augustus Volney Waller and Santiago
Ramon Y Cajal pioneered research in peripheral nerve
degeneration and regeneration. Waller, using the frog
glossopharyngeal nerve, showed that damaged nerve fibres
degenerate from the site of transection all the way down to
the nerve's target (reviewed by Cajal, 1928). Such
degeneration, distal to the site of injury is termed
Wallerian degeneration. Waller also observed that the
proximal part of the damaged nerve fibre, connected to the
neuronal cell body retains its normal form except for a
small portion of nerve adjacent to the injury site which
undergoes retrograde degeneration. Further to Waller's
description of peripheral nerve degeneration, Cajal
demonstrated that nerve regeneration occurs by outgrowth
from the proximal stump and not by autoregeneration of the
16
degenerated distal nerve (Cajal, 1928).
With the development of immunohistochemistry and the
powerful tools of molecular genetics and molecular biology,
more recent studies have re-explored in detail, the events
of peripheral nerve regeneration. In this thesis,
immunochemical techniques were used to investigate further
the cellular and molecular changes that take place during
peripheral nerve injury and repair.
The peripheral nerve.
The peripheral nervous system (PNS) consists of all
nervous tissue outside the bony structures of the vertebral
column and skull. There are two major divisions; the
voluntary and the involuntary (or autonomic) nervous
systems. The autonomic nervous system, composed of both
sympathetic and parasympathetic nerves, is involved in
activities such as blood pressure and heartbeat regulation,
gastrointestinal motility and exocrine gland secretion. The
major functions of the voluntary nervous system are to
carry sensory information into the central nervous system
(CNS), by sensory axons and to carry motor commands out to
muscle or autonomic ganglia, via motor axons. The sensory
and motor axons can be found together in mixed peripheral
nerves or they can be found in separate sensory or motor
nerves respectively; autonomic axons are also found
together with sensory and/or motor axons. The organisation
17
of all but the smallest peripheral nerves consist of axon
bundles or fasciculi, involving three connective tissue
sheaths. The entire nerve is surrounded by the epineurium.
Within the epineurium each bundle of fibres is enclosed by
a perineurium sheath, and individual axons with their
Schwann cells are embedded in connective tissue termed the
endoneurium (figure 1.1).
The neuron.The functional unit of the nervous system is the neuron,
consisting of a cell body, with or without dendrites, and
the axon. In the PNS the cell body (perikaryon) is found
within the dorsal root ganglion (DRG) for sensory neurons,
in the ventral horn for motoneurons innervating muscle.
Short branching dendrites associated with the cell body
form a major part of the receptive area of the cells. The
axon varies greatly in length from one type of neuron to
another and conducts impulses away from the cell body.
Glial cells of the peripheral nervous system.Surrounding the axons are the supporting glial cells.
These neural crest-derived glial cells are known as Schwann
cells, of which there are two kinds: myelinating and
nonmyelinating Schwann cells. In nonmyelinated nerves, one
Schwann cell envelopes several (up to 15) thin axons
(Terzis and Smith, 1990). In contrast, myelinating Schwann
cells envelope only one axon, laying down many layers of
specialised cell membrane by spiralling around the axon
Perineurium
Epineurium ÀxonhBsk
NIssI bodMBEnd Plate
Endoneurium Dendittas
Figure 1.1: General structure of a peripheral nerve trunk. Endoneurial connective tissue surrounds each axon,- and bundles of axons or fasicles surrounded by multinucleated perineurium are embedded in a loose connective tissue, the epineurium. The inserted box shows a schematic diagram of a neuron, showing the cell body, dendrites, and myelinated axon with basal lamina.
19
perimeter. This myelin sheath is a proteophospholipid
multilayered spiral of compacted apposed cell membranes.
The myelin sheath has an important physiological function,
in that it insulates the electrical signal passing down the
axon. The myelin sheath formed by individual Schwann cells
along the length of the axon has gaps at regular intervals,
known as nodes of Ranvier, with one Schwann cell
ensheathing the distance between each node (internode). The
node allows extracellular ions to reach the axon, which is
essential for the propagation of action potentials. These
regularly spaced gaps in the myelin sheath result in
saltatory propagation of impulses from node to node.
Fibroblasts and macrophages.
A peripheral nerve also consists of other cell types:
fibroblasts, important for connective tissue formation such
as the perineurium, and a third cell type, the macrophage.
The role of the resident macrophage population in a normal
peripheral nerve remains unclear. By contrast, the
population of macrophages recruited into the peripheral
nerve after injury plays a major part in the events
following injury and is discussed in detail later.
Significant advances have been made over the last few
years in understanding how each component of a peripheral
nerve contributes to the regenerative response of the PNS
following injury. The following discussion of peripheral
nerve response to injury will review recent data which has
20
provided a more detailed picture of cellular events
following injury.
The damaged axon.
Inflammatory changes.
Traumatic nerve injury, such as crush lesion or
transection produces, as in other well vascularised
tissues, an inflammatory reaction. The integrity of the
specialised blood-nerve barrier maintained by the
perineurium and endoneurial vessels is lost, with a
resulting increase in vascular permeability and leakage of
proteins from the blood into the interstitium. Vascular
permeability is also altered by mast cell vasoactive amines
released in response to the injury (reviewed by Terzis and
Smith, 1990). These changes to a peripheral nerve move in
a distal direction from the site of injury, with the entire
nerve distal to an injury showing increased vascular
permeability by four days after the injury. There is also
a small spread in the proximal direction.
The cell body response.
Lesioning of the axon also produces characteristic
structural and functional changes in the nerve cell body
(for review see Lieberman, 1971). The classical
chromatolysis response, first described by Nissl in 1892 is
now known to be associated with morphological and
biochemical alterations within the cell body. Nissl
21
described an increase in cell body volume, displacement of
the nucleus to the periphery, and a disappearance of
basophilic material from the cytoplasm (see Cajal, 1928).
The reaction reflects an alteration in the arrangement and
concentration of RNA-containing material (Nissl substance)
in the cell, leading to changes in protein synthesis.
Ultrastructurally, chromatolysis represents a
disorganisation of the structure of the rough endoplasmic
reticulum and release of associated ribonucleoprotein particles (ribosomes, the site of protein synthesis in the
cell). Changes in the cell's metabolic activity, such as
increases in glycogen phosphorylase activity and iron
uptake in axotomised motoneurons (Woolf et al., 1984;
Graeber et al., 1989), are some of the other lesion-induced
biochemical alterations. Peripheral nerve injury also
causes a general increase in RNA and protein synthesis
which may be dependent upon the type of lesion (reviewed by
Leiberman, 1971).
The degenerating distal stump.Several structural changes take place during Wallerian
degeneration of the distal segment of a severed peripheral
nerve, (i) Within the first few minutes of injuring the
nerve, myelin retracts from the nodes of Ranvier close to
the site of injury and the retraction spreads distally over
the next six hours (Cajal, 1928 ). By twenty eight hours
after the lesion nearly all the nodes, along the entire
length are unrecognisable. (ii) Axonal disintegration
22
usually commences with a varicose appearance of the axon
approximately two to four hours after the lesion. Within
twenty four hours, there is a general disintegration of the
neurofilaments, extending along the entire distal portion
of the nerve by four days (Lunn et al., 1990). (ill)
Fragmentation of myelin leading to the formation of
ellipsoid bodies, occurs four to five days post-injury, and
by fourteen days the myelin has almost completely
disappeared from fine and medium size nerve fibres (Cajal,
1928 ) .
The general pattern and rate of axonal degeneration in
the distal stump is known to be affected by different types
of lesions; a cut nerve degenerates faster than a crushed
nerve (Lunn et al., 1990). Even where the outer sheath, the epineurium, is cut and the blood supply damaged at the site
of injury, degeneration after a severe crush still proceeds
at a similar rate to that of a simple crush. However, when
a suture remains tightly tied after crushing, the nerve
behaves as though it had been severed (Lunn et al, 1990),
suggesting that interruption of axonal flow, either by
transection or suture, influences the speed at which
degeneration occurs.
The dual role of the macrophage.
(i) Macrophages in degenerationResearch in the last five years has uncovered some of
23
the most convincing evidence for the importance of
macrophages in * both Wallerian degeneration and
regeneration of the PNS following injury. Macrophages are
"professional" phagocytic cells in addition to being a
major component of the immune system. They are found in
most tissues, especially in haematopoietic and lymphoid
organs (bone marrow, liver, spleen, lymph nodes) and in
connective tissues. Cajal noted that a large number of
macrophages were present within the PNS following axotomy
(Cajal, 1928). Although Cajal held the view that
macrophages were important for the phagocytosis and removal
of myelin debris, it was difficult to provide clear
evidence at that time for their ability to phagocytose and remove such debris. The opposing view of Bungner, Zalla and
Stroebe, (see Cajal, 1928) amongst others, was that the
peripheral nerve phagocytic cells were derived from the
Schwann cells. There is now considerable data to support
the hypothesis that the removal of debris formed by nerve
degeneration is dependent upon the recruitment of
macrophages from the vasculature (Beuche and Friede, 1984;
Bonnnekoh et al., 1989; Lunn et al., 1989). A reduction in
myelin debris removal and decreased removal of myelin
proteins are some of the consequences of either preventing
the entry of macrophages into a severed sciatic nerve or
blocking phagocytosis (Beuche and Freide, 1984; Scheldt et
al, 1986).
New light was shed upon the importance of macrophages in
24
peripheral nerve degeneration following the discovery of an
unusual mouse strain, C57Bl/6/01ac (Lunn et al., 1989).
Whilst examining nerve degeneration, Lunn et al. (1989)
discovered that sciatic nerves in C57Bl/6/01ac mice
degenerated at a slower rate when compared to a number of
other strains; the rate of degeneration measured by
neurofilament breakdown, endplate degeneration, and the
disappearance of myelinated axons was dramatically reduced
in C57Bl/6/01ac mice (Lunn et al., 1989). Furthermore,
action potentials recorded from the distal segment of the
sciatic nerve of the C57Bl/6/01ac mice were maintained for
a significantly longer period after injury, compared to
other strains. When the injured nerves were stained for
macrophages, the normal recruitment of macrophages into the
distal nerve segment following injury was not observed in
C57Bl/6/01ac mice. (Perry et al., 1987; Lunn et al., 1989).
Subsequent experiments on this mouse strain have also provided evidence for the existence of a factor, unique to
the nervous system, which attracts macrophages into the
lesioned nerve. Perry et al. (1990a) showed that macrophage
recruitment in the C57Bl/6/01ac still occurs in
inflammatory sites other than the nerve. Furthermore, it
was shown, using bone marrow chimaeric experiments, that
the haemopoietic cells of the C57Bl/6/01ac still invade the
sciatic nerve of a different mouse strain (Perry et al.,
1990a). This suggests that the failure to recruit these
cells into the nerve results from a defect within the
C57Bl/6/01ac nerve. Lack of degeneration is also observed
25
in the central nervous system and is controlled by a single
autosomal dominant gene (Perry et al., 1990b; Perry et al.,
1991a).
The expression of adhesion molecules, such as macrophage
antigen-1 (MAC-1) or lymphocyte function-associated
antigen-1 (LFA-1), on the macrophage cell surface, in
addition to the expression of similar structures such as
intercellular adhesion molecule (I-CAM) on the endothelial
cells, is important for migration of macrophages into
inflamed tissue (Wawryk et al., 1989). Therefore, the
defect in the C57Bl/6/01ac mouse strain may be due to
either an absence of appropriate adhesion molecules on the
endothelial cells, or lack of a neurally derived
chemotactic factor.
(ii) Macrophages and regeneration.
The absence of macrophages in the nerves of C57Bl/6/01ac
mice during Wallerian degeneration does not seem to hinder
regeneration of motoneurons (Brown et al., 1991), whereas
peripheral sensory nerve regeneration is dependent upon
their presence (Brown et al., 1991). The ability of
macrophages to modify the normally nonpermissive state of
the optic nerve to one which aids neurite outgrowth from
embryonic DRGs also supports the importance of macrophages
in regeneration (David et al., 1990).
Although macrophages were originally noted for their
26
powerful phagocytic properties, it has now been well
established that they also secrete a large number of
bioactive products (Nathan, 1987; Rappolee and Werb, 1988).
Secretion of the enzyme collagenase, along with a number of
other proteases, suggests a mechanism by which macrophages
may contribute to the degenerative events following nerve
injury (Welgus et al., 1985). However, the ability of
macrophages to produce collagenase inhibitor, fibronectin
and apol ipoprotein E, may also be an important role of
these cells in nerve regeneration (Welgus et al., 1985;
Alitalo et al., 1980; Stoll and Muller, 1986). Recent
studies on the macrophage cytokine, interleukin-1 (IL-1),
have provided data in support of their role in nerve
regeneration. IL-1 regulates the production of nerve growth
factor (NGF) (Lindholm et al., 1987) and activity blocking antibodies raised against IL-1 inhibit cytokine-induced
Schwann cell proliferation (Lisak and Bealmear, 1991).
Furthermore, IL-1 is a mitogen for Schwann cells in
vitro (M.Khan, pers. comm.). Since macrophages express
properties important for both degeneration and regeneration
of peripheral nerves, it is possible that different
macrophage subpopulation, previously described by Dijkstra
et al. (1985) perform different functions in response to
peripheral nerve injury. Alternatively, the changing
environment of the nerve following axotomy may influence
macrophage function, regulating their phagocytic properties
in addition to its secretory properties.
27
Rebuilding of the axon.
Regeneration of a peripheral nerve usually commences
after a latent period of one to two days, at which point
formation of a growth cone occurs; the growth cone is a
swelling at the tip of the proximal disrupted axon.
Reconstruction of lesioned axons requires the presence of
essential building materials such as extracellular matrix
and cytoskeletal elements. The cell body responds to injury
by increasing the manufacture of a number of cytoskeletal
elements for example act in, perpherin and tubulin are known
to be up regulated in DRGs in response to peripheral nerve injury (Tetzlaff et al., 1988; Troy et al., 1990; Wong and
Oblinger, 1990a and b). The cytoskeletal elements are then
transported from the cell body down to the growth cone.
Transportation of proteins from the nerve cell body,
along the axon to the newly formed growth cone occurs at
either fast or slow rates (reviewed by Alberts et al,
1989). Neuronal cytoskeletal elements (actin, spectrin,
tubulin, and the three neurofilament (NF) subunits, NF-H,
NF-M, NF-L) are all transported via the slow rate transport
which is subdivided into two separate components. The slow
component 'a’ (SCa), is mainly composed of microtubules and
neurofilaments and does not appear to be affected by
axotomy (Jacob and McQuarrie, 1991), whereas the slow
component 'b ' (SCb), comprised of actin and spectrin in
addition to clathrin and calmodulin, has recently been
28
shown to accelerate after axotomy (Jacob and McQuarrie,
1991). The importance of SCb transport during regeneration
is also reflected by the fact that the rate of SCb
transport is further increased, along with the regeneration
rate, following a second nerve injury inflicted 14 days
after the first "conditioning" lesion (McQuarrie and
Grafstein, 1973; Bisby and Pollok, 1983; McQuarrie and
Jacob, 1991).
Structural support for peripheral nerve regeneration is
also provided by a number of extracellular matrix
substances. Fibronectin, tenascin, collagen (produced by
endoneurial fibroblasts), and laminin (produced by Schwann cells), provide the substratum for neurite outgrowth during
regeneration (Longo et al., 1984; Kuecherer-Ehret et al.,
1990; Wehrle and Chiquet, 1990). Further support for axon
regeneration is gained by the cell adhesion molecules
(CAMS). Two CAMS which have been widely studied in
peripheral nerve regeneration are neural cel 1-adhesion
molecule (N-CAM) and the LI glycoprotein (also known as the
neuron-glia cel 1-adhesion molecule or Ng-CAM). Recent
studies in rats showed that mRNA and protein levels for N-
CAM and LI increase as a result of sciatic nerve
transection (Tacke et al., 1990; Martini and Schachner,
1988; Daniloff et al., 1986). Also, when antibodies
directed against N-CAM are applied to a transected sciatic
nerve, muscle reinnervation is delayed for up to 30 days
(Remsen et al., 1990) and abnormal regeneration and
29
reinnervation processes occur (Reiger et al., 1988). In the
presence of anti-N-CAM antibodies, ectopic synapses occur
and the axon terminals lack a terminal Schwann cell capping
the nerve-basal lamina contact area (Reiger et al., 1988).
It appears that, if regeneration of a peripheral nerve
is to be successful then the expression of many different
protein structures within the PNS microenviroment are
required.
The distal stump and regeneration.
Cajal recognised and documented in 1928 the importance
of the distal stump for effective regeneration.
Subsequently, with the aid of modern techniques, more
recent studies have uncovered some of the properties of the
distal stump, including Schwann cells with its basal lamina
and a number of diffusible trophic and tropic factors,
which promote axonal regeneration (Politis et al., 1982;
Ide et al., 1983 ;).
Proliferation of Schwann cells.Schwann cell proliferation is a major event in response
to peripheral nerve injury. Early studies on the Schwann
cell proliferative response describe mitosis commencing
some 4 days after transection of the peripheral nerve and
peaking between 6 to 9 days (Cajal, 1928). The numbers of
total cell nuclei increase 3 days after injury and reach a
30
maximum of 8 times the initial population after 25 days
(Joseph, 1950; Abercrombie and Johnson, 1946). More
advanced techniques, for example, the incorporation of
tritiated thymidine into cells, have facilitated more
accurate measurement of mitosis. There are know known to be
two periods of Schwann cell proliferation following axotomy
in vivo. The first period commences almost immediately
following injury within the immediate vicinity of the crush
(Pellegrino et al., 1986 ; Clemence et al., 1989). It is
independent of blood monocyte/macrophage recruitment, with
the peak of proliferation occurring three to four days
after injury (Clemence et al, 1989; Brown et al., 1991). In
contrast the second period of Schwann cell proliferation
which occurs during the regenerative phase also coincides
with the recruitment of macrophages (Brown et al., 1991).
The importance of Schwann cell proliferation during
nerve regeneration has lead to an extensive search to
identify Schwann cell mitogens, produced as a result of
peripheral nerve injury. This has proved to be a complex
task. Potential Schwann cell mitogens include axon derived
factors (Bunge, 1987; Salzer et al., 1980), products of
activated lymphocytes and macrophages (Lisak et al., 1985),
in addition to a myelin membrane fraction (Bigbee et al.,
1987). A number of growth factors including platelet-
derived growth factor (PDGF), fibroblast growth factor
(FGF) and transforming growth factor B (TGF) are mitogenic
for Schwann cells in vitro (Ridley et al., 1989; Davis and
31
Stroobant, 1990). However, none of these agents are
mitogenic when applied alone, in serum free culture medium
(Weimaster and Lemke, 1990 ). Work by Weimaster and Lemke
(1990) suggests that there is an absolute requirement for
elevation of the second messenger, cyclic adenosine
monophosphate (cAMP), by a second factor, in order for
Schwann cells to respond to the above growth factors
(Weimaster and Lemke, 1990). Additional data has revealed
that voltage-gated ion channels, such as potassium
channels, expressed on the membrane of Schwann cells are
also important for their proliferative response (Chiu and
Wilson, 1989). Chui and Wilson ( 1989) showed that when
potassium currents in Schwann cells are blocked by using
potassium channel blockers, Schwann cell proliferation was
inhibited in a dose dependent manner. It would seem that
one factor alone is not an adequate signal for
proliferation but that the combination of factors present
in an injured nerve contributes to the control of Schwann
cell proliferation.
Schwann cells and axon guidance.A role for Schwann cells in the guidance of axon
regrowth to their target, and as trophic support providing
nutrients, was suggested early in the 20th century, (Cajal,
1928). However, recent studies have cast doubt over whether
Schwann cells are an essential requirement for such growth.
If, for example, acellular nerve explants are sutured to
the proximal stump of a transected nerve, then they will
32
support the growth of regenerating axons (Anderson and
Turmaine, 1986; Tohyama et al, 1990). Moreover, if segments
of sciatic nerve distal to a crush are repeatedly frozen
and thawed, killing the Schwann cells, only a 30% reduction
in the rate of elongation of sensory and motor axons is
observed (Sketelji et al., 1989). As the nerve was crushed
rather than transected, the extracellular matrix and basal
lamina remains intact. However, if the Schwann cell basal
laminae is denatured, by scalding the segments with moist
heat, then there is a greater reduction in the rate of axon
elongation (Sketelji et al., 1989). These findings question
the importance of Schwann cells themselves in the distal
stump for regeneration, although the products of Schwann cells, such as laminin, are clearly important.
However, there are several studies which provide
conflicting data, supporting the necessity of Schwann cells
in regeneration. When Schwann cell mitosis is inhibited in
the distal stump of a severed nerve, using mitomycin C,
neurite outgrowth is virtually inhibited (Hall, 1986). It
is also the case that Schwann cell surfaces and conditioned
medium provide the best substrate to be identified which
promotes neurite outgrowth in culture and peripheral
motoneuron outgrowth in vitro (Kleitman et al., 1988;
Assouline et al., 1987; Bixby et al., 1988 ). A role for
Schwann cells in regeneration is also reflected by the
ability of sciatic nerve grafts to support regeneration of
a severed optic nerve which normally fails to regenerate
33
(Berry et al., 1988; Benfey and Aguayo, 1982). Although
axonal regeneration has been described in the absence of
Schwann cells, it would appear that optimum regeneration
does require their presence.
The interstump gap.The extent to which peripheral nerve regeneration takes
place following transection is dependent upon the distance
separating the distal stump from the regenerating proximal
stump. A gap of 10mm is thought to be the maximum distance
that sciatic nerves are able to grow across unaided
(Lundborg et al., 1989). However, transected nerves can
traverse a distance greater than 10mm providing a guidance
channel or implanted chamber is employed (Aebischer et al.,
1989). The use of such chambers has also provided a means
of testing different factors which enhance the regenerative
response. For example, the release of basic fibroblast
growth factor (bFGF) from synthetic guidance channels
provides sufficient support for the reconstruction of a
transected rat sciatic nerve across a 15mm gap (Aebischer
et al., 1989). Furthermore, basic FGF can also accelerate
the growth of new fibres through similar chambers
(Danielsen et al, 1988). Although several studies have
shown the distal stump to be essential for successful nerve
regeneration (Scaravilli et al., 1984; Williams et al.,
1984; Jenq and Coggeshall, 1986), especially where
nonmyelinated axons are involved (Jenq and Coggleshall,
1986), its absence does not prevent regeneration when a
34
semipermeable guidance channel is used (Aebischer et al.,
1988). Structural support from the channel and trophic
factors secreted from the surrounding enviroment, which
includes the distal stump, is sufficient for regeneration
to occur.
Nerve Growth Factor (NGF); A role in regeneration ?
Nerve growth factor (NGF), the best characterised
target-derived trophic factor in the nervous system, was
discovered in the early fifties by Levi-Montalcini and
Hamburger (Levi-Montalcini and Hamburger, 1951). It was
found to enhance the outgrowth of neurites selectively from
neurons of sympathetic and embryonic dorsal root ganglia both in vivo during foetal development and in vitro (Levi-
Montalcini and Hamburger, 1951; Levi-Montalcini and
Angeletti, 1963; Levi-Montalcini et al., 1954). NGF is
produced by the targets of NGF-sensitive fibres, taken up
by the presynaptic terminals and retrogradely transported
to the perikaryon (Hendry et al., 1974; Stoeckel et al,
1975 ; Stoeckel et al, 1976). The importance of NGF for
neuronal survival during development has been demonstrated
conclusively by immunosympathectomy. Administration of NGF
antiserum to newborn rats results in a significant
reduction of neuronal number in the lumbar DRG (Yip et al.,
1984). Moreover, Gorin et al. (1979) observes that
peripheral sympathetic neurons in offspring of NGF-
immunized female rats are destroyed. A similar result is
35
observed for rat and guinea pig DRG after exposure in utero
to maternal antibody to NGF (Johnson et al., 1980; Johnson
et al., 1983). The importance of NGF for neuronal survival
during development is strengthened by studies which show
that the addition of exogenous NGF during development
reduces the amount of naturally occurring cell death
(Hamburger et al., 1981). This ability to reduce naturally
occurring cell death is thought to be via the ability of
NGF to suppress an endogenous, active cell death program
(Martin et al., 1988).
A considerable amount of neuronal cell death also occurs
in sympathetic and dorsal root ganglia following peripheral
axotomy in both newborn and adult rats (Hendry, 1975; Yip
and Johnson, 1984; Tessler et al., 1985; Arvidsson et al.,
1986). The importance and function of NGF in peripheral
nerve regeneration has not been defined to the same extent
as its role in the development of the nervous system.
Deprivation of NGF does not increase the extent of cell
death in the lumbar DRG or the extent of regeneration after
sciatic nerve transection (Rich et al., 1984). However, NGF
partially reverses the central changes in the cell body
which occur after nerve transection, if it is applied
locally to the lesioned nerve (Fitzgerald et al., 1985;
Rich et al., 1987). Furthermore, NGF will also prevent the
decrease in neuronal number which normally follows combined
central and peripheral lesions (Yip and Johnson, 1984). The
importance of NGF after axotomy is also supported by the
36
recent discovery of its involvement in collateral sprouting
of sensory axons in the skin (Diamond et al., 1992) and the
observation that NGF mRNA increases distal to the injury
site following axotomy (Neumann et al., 1987a). This
les ion-induced increase in NGF mRNA is biphasic, with an
initial increase 6 hours after transection, followed by a
second peak occurring 3 days after transection (Neumann et
al., 1987a; Neumann et al., 1987b ). When the nerve is
prevented from regenerating, i.e. cut rather than crushed,
the increase in NGF mRNA remains elevated (Neumann et al.
1987b).
In culture, only a single rapid and transient increase
in NGF mRNA is observed following axon injury (Lindholm et
al, 1987). Nowever, failure to observe the second, more persistent increase is rectified by the addition of either
macrophage conditioned medium or the macrophage cytokine,
interleukin-1 (IL-1) (Lindholm et al., 1987), indicating a
role for macrophages in regulation of NGF expression.
Although the role of NGF in regeneration is unclear, its
increased expression following nerve injury may enable the
distal stump to act as a surrogate target, providing
support to regenerating sympathetic and sensory neurons. If
NGF is important for such regeneration, then its receptor
should also be expressed after injury. The re-expression
and redistribution of the NGF receptor was shown to occur
after nerve transection (Raivich and Kreutzberg, 1987). NGF
37
receptors accumulate transiently on both sides of crushed
or transected sciatic and brachial nerves, due to
retrograde and anterograde axonal transport of the receptor
(Johnson et al., 1987; Raivich and Kreutzberg, 1987).
However, a rapid decrease in uptake and both retrograde and
anterograde axonal transport of NGF receptors starts one
day after injury (Raivich and Kreutzberg, 1987; Raivich et
al., 1991), thus calling into question the relevance of NGF
for axon regeneration.
NGF receptors expressed on Schwann cells throughout the
distal part of axotomised nerves demonstrate a different time course. Receptors are not detected until 4 days after
injury, becoming maximal 6 days after injury (Raivich and
Kreutzberg, 1987). Subsequently, as the axons regenerate,
Schwann cell NGF receptor expression decreases (Taniuchi et
al., 1988). In contrast to NGF, expression of the receptor
does not appear to be regulated by the recruited
macrophages (Neumann et al., 1987b; Brown et al., 1991) but
it may be controlled by axonal contact.
The precise role of the Schwann cell NGF receptor in
nerve regeneration is unclear, since NGF has no discernable
effect on these cells. It was thought that, together with
the release of NGF from the Schwann cells and fibroblasts
(Neumann et al., 1987a; Matsuoka et al., 1991) in the
distal stump, NGF receptors may support the regeneration of
sensory neurons via cell adhesion-like activity, similar to
38
the role of N-CAM (Taniuchi et al., 1988). NGF may act as
’bridge' between the axon and the surrounding Schwann cells
via its ability to bind to receptors on both the Schwann
cell and the axon. (Taniuchi et al., 1988). However, with
the disappearance of axonal expression and retrograde
transport of the NGF receptor following axotomy (Raivich et
al., 1991), the regenerating axons may not themselves be
the targets for the increased levels of NGF present in the
distal stump. The NGF produced by the Schwann cells and
fibroblasts after axotomy may interact locally with Schwann
cells distal to the injury site which are known to
expressing NGF receptors (Raivich et al., 1991). Clearly
the role of NGF in the damaged nerve is unresolved,
however, its up-regulation in the distal stump indicates a
role in nerve regeneration.
Neuropeptides : A trophic role following nerve injury?
The identification and distribution of biologically-
active peptides throughout the nervous system, so called
neuropeptides, has rapidly expanded over the last twenty
years with the aid of immunohistochemistry. This technique
has produced some interesting findings concerning the
effect of peripheral nerve injury on neuropeptide
expression.
For a number of years, neuropeptides had been regarded
as simple chemical transmitters, manufactured in the cell
39
body and transported to the presynaptic membrane, where
they are released as signalling molecules. Most effects of
neuropeptides on their targets seem to be relatively short
term, acting over a range of seconds or at most minutes.
Neuropeptides can be divided into two groups according to
whether they have a direct effect on a postsynaptic
membrane (neurotransmitter) or modulates the effects of
other molecules (neuromodulator). The distinction between
neurotransmitters and neuromodulators is frequently
unclear, since the same neuropeptide acts at some synapses
as a transmitter and at others as a modulator. One such
peptide is substance P (SP), which has been proposed to be
a sensory neurotransmitter. It is present in small-
diameter, primary afferent neurons that project to the
superficial laminae of the dorsal horn (Hbkfelt et al., 1976). It induces slow excitation in the dorsal horn cells
that respond to noxious stimuli (Henry, 1976). However it
also depresses the nicotinic acetylcholine (ACh)-induced
excitation of various cell types, including adrenal
chromaffin cells (Mizobe et al., 1979).
Although many peptides have relatively short-term
effects on their target cells, there is now considerable
evidence to suggest a trophic role for neuropeptides acting
over a time scale of many minutes or even longer. The
regulation by calcitonin gene-related peptide (CGRP), of
muscle ACh receptor expression and suppression of disuse-
induced terminal sprouting after chronic block of nerve-
40
muscle activity are examples of neuropeptide involvement in
a trophic response (New and Mudge, 1986; Tsujimoto and
Kuno, 1988).
The characterisation of the neuropeptide receptors,
localisation and regulation has been made possible by the
use of gene cloning, one of the many molecular biology
techniques developed in the last ten to fifteen years. All
of the neuropeptide receptors discovered, including the
receptors for SP and neurotensin, belong to the family of
G protein-coupled receptors with seven membrane-spanning
segments (reviewed by Hokfelt, 1991).
Interest in a possible trophic role for neuropeptides
during peripheral nerve regeneration has developed from the
observations that vasoactive intestinal (VIP) and
neuropeptide Y (NPY) increase in the DRG neurons, and CGRP
expression increases in the motoneurons, after peripheral
nerve injury (Shehab and Atkinson, 1986; Wakisaka et al.,
1991; Jessell et al., 1979; Streit et al., 1989; Arvidsson
et al., 1990; Noguchi et al., 1990). In contrast to the
expression of CGRP in motoneurons, its expression in DRGs
decreases following injury, suggesting that the regulation
of CGRP in the PNS following injury is complex. However,
since peptides with transmitter-like functions would be
expected to be redundant after injury, because they could
not be released at the terminal synapse, these increases in
certain peptides were intriguing. They suggested a role for
‘ 41
neuropeptides in nerve regeneration, since only those
peptides and proteins involved in rebuilding the axon and
supporting the cells survival are thought to increase after
nerve injury. The hypothesis that certain neuropeptides
expressed in the PNS are involved in peripheral nerve
regeneration is strengthened by the finding that CGRP acts
in vitro as a permissive signal for Schwann proliferation
via its ability to stimulate Schwann cell cyclic adenosine
monophosphate (cAMP) production (M.Khan, personal
communication); Schwann cell proliferation is a major event
following peripheral nerve injury.
Neuropeptides and the inflammatory response.
A lesion within a peripheral nerve, as in other tissue,
causes inflammation. This inflammatory response encompasses a vast array of events, including vascular changes, the
release of histamine and the migration of leucocytes,
neutrophils and macrophages into the inflammatory site. The
ability of the nervous system to influence certain aspects
of inflammation is recognised by the capacity of
neuropeptides, such as SP, to induce histamine release and
vascular changes following skin lesions (Foreman and
Jordan, 1983). This is supported by the demonstration that
afferent nerve stimulation produces vasodilation and plasma
extravasation into the skin, a process termed neurogenic
inflammation (reviewed by Foreman and Jordan, 1984). The
absence of these events after blockage of nerve
42
transmission either by mechanical injury, or application of
capsaicin, producing a loss of smal1-diameter primary
afferent neurons, supports the hypothesis that peripheral
nerves modulate the inflammatory response (Helme and
Andrews, 1985). In addition to the effects of SP in
neurogenic inflammation, additional neuropeptides such as
neurokinin A (NKA) and B (NKB), induce plasma protein
extravasation, and CGRP augments extravasation induced by
histamine. (Gamse et al., 1987). The possibility arises
that certain neuropeptides expressed following peripheral
nerve injury have a role in the ensuing inflammatory
response. The observation that SP, substance K (SK) and the
carboxyl-terminal peptide SP(4-11) increase the release of
the inflammatory cytokines IL-1, tumour necrosis factor-a
(TNF-a) and interleukin-6 from monocytes (Lotz et al.,
1988), supports the hypothesis that neuropeptides regulate
certain events of the inflammatory response following nerve
injury, in particular the macrophage response. Further
evidence for such regulation is provided by studies
demonstrating that VIP up regulates cAMP levels in blood
monocytes, and CGRP inhibits peritoneal macrophage
activation (Wiik, 1989; Nong et al., 1989).
If certain neuropeptides, present following peripheral
nerve injury, regulate the macrophage response then they
may indirectly influence regeneration of the peripheral
nervous system. Therefore, it is important to determine
what changes occur, if any, in levels of expression of
43
certain neuropeptides around the injury site as well as
distal to the crush during regeneration.
44
Thesis objectives.
This thesis aims to clarify the importance of
neuropeptides to PNS regeneration by examining in detail
the expression of CGRP and VIP as well as the lymphokine,
gamma interferon in the damaged rat sciatic nerve. It also
investigates whether CGRP has a role to play in regulating
the function of macrophages recruited into an injured
sciatic nerve.
The second section investigates whether the multiple functions of macrophages, which may be important in
peripheral nerve degeneration and regeneration, are related
to the presence of different macrophage phenotypes in the sciatic nerve, before and after injury. It aims to document
the time course and spatial distribution of these
macrophages.
45
CHAPTER 2
CGRP AND VIP IN THE PERIPHERAL
NERVOUS SYSTEM; A ROLE FOR CGRP IN
PERIPHERAL NERVE INJURY.
46
Introduction.
Calcitonin gene-related peptide (CGRP) is a 37 amino acid
neuropeptide which exists in two forms, a-CGRP and &-CGRP.
a-CGRP is generated by alternative splicing of the primary
transcript of the calcitonin gene (Amara et al.,1982).
However, 13-CGRP, which was discovered in brain and thyroid
(Amara et al., 1985), is generated from a different gene.
When the peptide sequence of rat a-CGRP is compared with
rat &-CGRP, there is only a single substitution, a lysine
in &-CGRP for a glutamate in a-CGRP at position 35 (Amara
et al., 1985). A similar comparison of a and fi human CGRP
shows that they differ by 3 amino acids (Steenbergh et al.,1985).
CGRP is widely distributed within the central and
peripheral nervous system (Skofitsch and Jacobowitz, 1985;
reviewed by Yamamoto and Tohyama, 1989). It is found in a
large population of primary sensory neurons, and it was the
first peptide to be localised in motoneurons (Gibson et
al., 1984 ; New and Mudge, 1986; Fontaine et al., 1986).
Furthermore, when the distribution of the mRNA for a and &-
CGRP is analysed, it is a-CGRP which is more abundant in
sensory ganglia and motoneurons (Gibson et al., 1988).
However, only B-CGRP is present in enteric autonomic
neurons (Mulderry et al., 1988).
The localisation of CGRP within chick spinal motoneurons
47
during development and enhanced biosynthesis of the AChR in
primary cultures of chick muscle by CGRP (New and Mudge,
1986; Fontaine et al., 1986) raises the possibility that
CGRP may be important at the neuromuscular junction. This
is supported by the observation of CGRP-like
immunoreactivity within synaptosomal vesicles and that
CGRP-like immunoreactivity is also found at motor end
plates (Takami et al., 1985; Gulbenkian et al., 1986; Freid
et al., 1989). Furthermore, either electrical or high K*
stimulation of the phrenic nerve, in vitro, causes release
of a CGRP-like immunoreactive substance (Uchida et al.,
1990).
The ability of CGRP to regulate AChR expression appears to
be via stimulation of adenylate cyclase and cyclic
adenosine monophosphate (cAMP) production (Laufer and
Changeux, 1987). The addition of CGRP to skeletal muscle in
vitro causes the activation of adenylate cyclase and
elevates intracellular cAMP of skeletal muscle (Laufer and
Changeux, 1987; Roa and Changeux, 1991). Consistent with
this observation is that electrical stimulation of the
phrenic nerve, in addition to causing the release of CGRP-
like immunoreactivity, also causes an increase in cAMP
content in the diaphragm (Uchida et al, 1990).
Although CGRP is expressed at a high level in spinal
motoneurons during development, CGRP expression in the
adult stage is at a low level (Gibson et al., 1988).
48
Interestingly, recent studies have shown that peripheral
axotomy induces an increased immunostaining for CGRP in
injured motoneurons, as well as up-regulation of mRNA
encoding a-CGRP but not &-CGRP (Streit et al., 1989;
Arvidsson et al., 1990; Noguchi et al., 1990). This
contrasts with decreased a and 13-CGRP mRNA in dorsal root
ganglia following peripheral nerve injury (Noguchi et al.,
1990). Since levels of transmitter-related enzymes usually
decrease in response to axotomy, it was surprising that
CGRP in motoneurons increases after injury. This raises the
question as to whether CGRP has role in peripheral nerve
régénérât ion.
This thesis examines whether the initial increase of CGRP
at the injury site remained elevated as the axons
regenerate. The technique of radioimmunoassay was employed to measure levels of CGRP as far distal as fifteen
millimetres and up to fourteen days following crush injury.
Two of the main events following peripheral nerve injury
are macrophage recruitment and Schwann cell proliferation,
as discussed in chapter one. Substantial evidence has
recently been provided to support the ability of CGRP to
interact with Schwann cells in vitro (M.Khan, pers. comm.).
As with a number of other cell types, including astrocytes,
endothelial cells and skeletal muscle cells (Lazar et al.,
1991; Hægerstrand et al., 1990; Laufer and Changeux, 1989),
CGRP activates adenylate cyclase, thus elevating cAMP
49
within Schwann cells as well as fibroblasts (M.Khan,
pers.comm.). This thesis will examine whether CGRP
interacts with the third major cell type of peripheral
nerve regeneration, the macrophages, by regulating their
cAMP production.
This thesis will also investigate the expression of
another neuropeptide, VIP, following peripheral nerve
injury. VIP, first isolated from porcine duodenum (Said and
Mutt, 1970), is a 28-amino acid peptide and a member of the
glucagon-secret in family of gastrointestinal peptide
hormones. It has been identified in neurons of the CNS,
including neurons in the cerebral cortex and hypothalamic
regions (see Hokfelt et al., 1980), in addition to its
localisation within primary sensory neurons, intrinsic
spinal neurons and autonomic neurons innervating various
exocrine glands (Fuji et al., 1983; see Hokfelt et al.,
1980). A large number of VIP expressing nerves have also
been observed within the gastrointestinal tract (Dimaline
and Dockray, 1982; Hokfelt et al 1980).
Interestingly, although VIP is not normally detectable in
sensory neurons in lumbar 4/5 DRG, both mRNA and peptide
for VIP appear in these sensory ganglia following sciatic
nerve injury (Nielsch and Keen, 1988). VIP mRNA appears 3
days following injury with increasing levels with time,
measured up to 9 days following injury. Similar studies
have shown that VIP peptide also increases in the dorsal
50
horn following sciatic nerve injury (McGregor et al., 1984;
Shehab and Atkinson, 1986). In order to postulate a role
for VIP in peripheral nerve regeneration, one needs to
show, like CGRP, that VIP is present and maintained at
elevated levels in the sciatic nerve during regeneration.
Therefore, this thesis also investigated levels and
determined the time course for VIP in the sciatic nerve
following injury.
51
MATERIALS AND METHODS.
Animal Surgery.
Male adult Sprague-Dawley rats were anaethesized with
halothane and the sciatic nerve exposed at mid-thigh level.
The nerves were subjected to two types of injury. The first
(i) allowing regeneration and the second (ii) where
regeneration was prevented.
(1) crushing the nerves with either watchmaker forceps
(No.5) left in position for approximately 1 minute or with
a silk suture (2/0 Mersilk) for approximately 1 minute, (ii) transecting the nerve or leaving a silk suture
attached to the nerves until the nerves were removed from the animals.
In sham operated animals the nerve was expbsed and
manipulated but not damaged.
The animals were killed between 2 hours to 18 days
following^nerve lesioning. Nerve segments measuring 5mm
were removed proximal and distal to the lesion site (see
figure 2.1). CGRP or VIP levels in these 5mm nerve segments
were determined by radioimmunoassay (RIA). Nerve segments,
12mm in length, were removed 6 days post-suture crush;
these segments included regions proximal and distal to the
crush, and were used for immunohistochemical detection of
CGRP. Similar lengths of nerve were removed 2 hours to 18
days post-crush for IFNy immunohistochemistry.
DIAGRAM OF LESIONED SCIATIC NERVE.
SPINAL__________________ "I "I"
CsJL D
CORD MUSCLE
Crush site
Figure 2.1: Diagram of lesioned sciatic nerve with an area around the lesion site divided into a number of 5mm segments. Levels of VIP and CGRP in the segments were quantified by RIA.
53
Tissue preparation for immunohistochemistry.
After dissection, the nerves were immediately immersed in
4% (w/v) paraformaldehyde in phosphate buffered saline,
pH7.4 (PBS) and fixed overnight at 4'C. The following day
they were placed in 30% (w/v) sucrose in PBS for up to 2
hours to prevent crystal formation within the tissue. The
tissue was mounted in OCT embedding medium (Tissue-tek,
Miles) and stored in liquid nitrogen until sectioned on a
freezing microtome (Bright).
CGRP immunohistochemistry.
Longitudinal sections of rat sciatic nerves, 10-15 um
thick, removed 2 hours and 6 days post-suture crush, were
labelled with a rabbit anti-human CGRP polyclonal antibody
(Peninsula Labs). This antibody was applied at a 1/700
dilution overnight at 4"C in 4% (v/v) horse serum (Gibco),
1% (v/v) Triton-XlOO (Sigma), 0.01% (v/v) sodium azide and
20mM L-lysine in PBS (" immunohistochemistry buffer"). The
next day, sections were washed 3 times for 5 minutes each
in the same buffer. The second antibody, a goat anti-rabbit
fluoroscein-conjugated antibody (Amersham) was diluted
1/100 in immunohistochemistry (IHC) buffer and preadsorbed
with 15-20% (v/v) rat serum for 30 minutes. It was then
centrifuged in a micro-centrifuge for 10 minutes to remove
precipitate. The sections were incubated with the second
antibody for 45 minutes at room temperature. The sections
were washed a further 3 times in IHC buffer and then
mounted in Citifluor (City University, U.K). Sections were
54
viewed with a fluorescence microscope (Zeiss Universal).
IFNy immunohistochemistry.Longitudinal sections, 12um thick, of rat sciatic nerves
removed 2 hours, 2, 6 and 18 days post-crush were labelled
with the monoclonal antibody, DB-1 (1/1000), which
recognises IFNy (a gift from P van der Meide). The staining
protocol was the same as that for detection of CGRP except
the second antibody was a goat anti-mouse biotinylated
antibody (1/100) (Amersham). After a 45 minutes incubation
at room temperature and 3 washes for 5 minutes each in the
ICH buffer, a third layer of fluoroscein-conjugated
streptavidin (1/100) (Amersham) was added for a further 45
minutes to visualise the IFNy staining. All sections were
washed a further 3 times in ICH buffer, mounted in
Citif lour (City University) and viewed with a fluorescence
microscope (Zeiss Universal).
Tissue Extraction for Radioimmunoassay.Each 5mm segment of sciatic nerve was placed in a 2ml
Eppendorf microtube, and then frozen immediately in liquid
nitrogen. Frozen tissue was stored for up to one month
before assay. To extract CGRP and VIP, each 5mm segment of
nerve was placed in 1ml of ice cold 2M acetic acid and
homogenised using an electric homogeniser (Ultra-Turrax
with UT-dispersing tool lOG; Janke and Kunkel). The
homogenates were placed in a boiling water bath for 5
minutes to inactivate proteases and were then lyophilised
55
overnight.
Radioimmunoassays.Amount of CGRP or VIP in each nerve segment were measured
on the basis that CGRP or VIP in the tissue extract would
compete with '”I -CGRP (Amersham) or '”I-VIP (Amersham) for
binding to the CGRP or VIP antibodies respectively. The
buffer used for diluting all RIA components was lOmM
ethylene diamine tetra acetic acid (EDTA), 0.01% (w/v)
sodium azide, 0.05% (w/v) Polybrene (Sigma), 0.1% bovine
serum albumen (BSA) (w/v) (Sigma; RIA grade) and 0.1% (w/v)
gelatin in PBS. The RIAs were carried out in borosilicate glass tubes (Corning) using the CGRP antibody (Peninsula
Labs) at 1/1000 and VIP antiserum L25, supplied by
R.Dimaline (Liverpool University) (pers.comm. A.Mudge), at
1/22,000 final dilution respectively. Radiolabelled
peptides '^I-CGRP or '”I-VIP were used at ca.5,000
counts/min (cpm) per tube (Amersham; specific activity
2,000 Ci/mmo1).
The lyophilised samples were resuspended in 1ml of RIA
buffer and both lOOul and 200ul aliquots were assayed in
duplicate. The final incubation volume was 400ul which
includes lOOul of antibody and lOOul of '“I-CGRP or ‘“-VIP
(the trace); controls for separation were included and
contained only trace without antibody or sample. The tubes
were incubated at 4’C for 24-48 hours. Trace that was bound
to antibody was separated from free trace by adsorbing free
56
trace with activated charcoal. At the end of the initial
incubation period, 400ul of charcoal mixture, 3.2% (w/v)
activated charcoal (Sigma), 0.32% (w/v) Dextran T70
(Pharmacia) and 0.01% (w/v) sodium azide in PBS, was added
to each tube and the tubes vortexed. The tubes were
centrifuged at 2,500g for 15 minutes and the supernatants
were decanted and counted for 1 minute in a gamma counter
(Nuclear Enterprises, NE1600). The counts obtained
represent the trace bound to antibody (the bound fraction).
CGRP radioimmunoassay.
In each RIA a standard curve for synthetic human CGRP
(Bachem) was obtained in the range of 8-100 fmol CGRP/tube
and two dilutions of nerve extract, both in duplicate, were
assayed to allow displacement slope comparisons to be made
between sample and standard. This provided a means to
compare the nature of the immunoreactive samples compared
to standard CGRP. Standard and sample curves were
calculated using the logit-log linearisation method
(Rodbard et al.,1969). The following terminology is used
for each RIA: 'D ' represents the counts/minute in the bound
fraction in the absence of antibody used at 1/400,000
dilution, 'B, ' is the maximum binding (counts/minute in the
bound fraction in the absence of peptide). ' B ' is the
binding with sample (counts/minute in the bound fraction in
the presence of antibody and either CGRP, VIP or nerve
extract). The percentage binding was calculated from the
equation (B-D) / (B*-D) x 100. The logit of the percentage
57
binding was plotted against the log of the sample dose so
that a curve closely approximating linearity was obtained,
over the percentage binding range of 12-88%.
VIP radioimmunoassay.The VIP RIA was performed as described for the CGRP assay
except that the standard curve for synthetic human VIP
(Bachem) was obtained in the range of 3-50 fmol VIP/tube.
The trace was ‘ ^I-VIP (Amersham) and the anti-VIP antibody
(R.Dimaline, Liverpool University) used at 1/22,000. The
percentage binding was calculated as above and the logit of
the percentage binding was plotted against the log of the
sample dose.
Interassay variability.Each RIA incorporated an aliquot of the same spinal cord
sample which contained a quantity of CGRP. This provided a
means of measuring the variation between each RIA. Also
sciatic nerve segments from control unoperated animals were
assayed in most RIAs.
Gel permeation chromatography.
Materials and equipment.A Superose-12 column (Pharmacia) was used together with
a 2111 Multirac fraction collector, 2238 Uvicord SII UV
recorder, and 2210 2-channel chart recorder, all from LKB.
Fractions were collected into borosilicate glass tubes
58
(Corning). Bovine thyroglobulin, Cytochrome C (Type VI) and
BSA (RIA grade) were from Sigma.
Gel permeation methods.CGRP-immunoreactive material obtained from nerve segments
was characterised by gel filtration chromatography. The
tissue extract was obtained as described above and SOOul
was applied to a Superose-12 column (Pharmacia). The
peptide was eluted with PBS at a flow rate of Iml/minute.
Aliquots of each fraction were assayed for CGRP content
using the RIA. The elution profile was compared with that
obtained by gel filtration of synthetic '“I-CGRP.
59
MACROPHAGE TISSUE CULTURE.
Peritoneal macrophage isolation (resident or activated).Sprague-Dawley rats were killed and injected
intraperitoneally with 20ml of Hams F12 medium (Flow). The
peritoneum was gently massaged to obtain the maximum
possible number of macrophages. The medium was drawn out of
the cavity and placed into a conical tube (Falcon) and
centrifuged at 1600rpm for ten minutes. The cell pellet was
resuspended in 1-2 mls of F12 medium and the cell
concentration determined using a haemocytometer. Exclusion
of Trypan blue stain was used as a measure of cell
viability. The cells were plated into 96 well plates
(Falcon).
Activation of peritoneal macrophages in vivo.Lipopolysaccharide (LPS) (Sigma), SOug, in Hams F12
medium (Flow) was injected into the peritoneal cavity of
Sprague-Dawley rats under halothane anaesthetic. The rats
were left for 4 days after which the animals were killed
and the peritoneal cells removed as described above.
Spleen macrophage isolation.Sprague-Dawley rats were killed and their spleens removed
and placed into Hams F12. Up to eight spleens were crushed
through a sheet of gauze and the resulting suspension
containing rat spleen macrophages was incubated for 2 hours
in FI2 medium containing 10% (v/v) foetal calf serum (FCS)
(Gibco) in tissue culture grade 100mm dishes (Falcon) and
60
placed into a 3 7"C, 5% CO^ incubator (LEEC). The macrophages
were observed to adhere to the plastic dishes after 2
hours. Nonadherent cells were removed by extensive washing
with a defined salt solution, and the remaining adherent
cells were cultured overnight in the above incubator. Cells
which detached from the dish overnight were collected and
used without further purification as a source of
macrophages. The cells were centrifuged at 1600rpm for 10
minutes, counted as above and plated into 96 well plates
{Primaria grade,Falcon).
Preparation of panning dishes.Petri dishes (100mm) (Falcon) were coated with a
nonspecific rabbit anti-mouse immunoglobulin (Dakopatts) at lOug/ml in 15mM Trisma base buffered to pH9.7 with
hydrochloric acid (HCl). After an overnight incubation at
4'C the buffer was aspirated and the dishes washed 2-3 times
with a balanced salt solution to remove excess antibody.
Hams F12 medium with 0.2% (w/v) BSA (Sigma) was added for
45 minutes to block nonspecific binding. Panning dishes
used for the isolation of macrophages from unlesioned
sciatic nerves were then incubated a further 45 minutes
with a second antibody. The second antibody used was either
anti-leucocyte common antigen (Seralab) or 0X42 (Seralab),
both of which are known to label macrophage surface
antigens. They were added at a 1/400 dilution. The dishes
were then washed as before.
61
Macrophage isolation from injured sciatic nerve.A total of 4 sciatic nerves, 3 days after crushing, were
removed from 4 Sprague-Dawley adult rats. Two sets of two
nerves were washed in dissociation buffer (136mM NaCl, 5mM
KCl, 5mM Na phosphate, 33mM glucose, pH 7.4) and chopped to
small pieces in 1ml of 0.6% (w/v) collagenase (Sigma) in
dissociation buffer. The chopped nerves were placed into a
15ml conical tube (Falcon) and 1ml of 0.0175% (w/v) trypsin
(Sigma), in dissociation buffer, added. After a
dissociation period of 40-50 minutes in a water bath (37‘C),
growth medium with 10% (v/v) FCS was added to the solution
in order to stop further trypsin activity. The resulting
suspension was centrifuged at 1600rpm for 10 minutes and
the pellet resuspended in 2-3ml of medium containing 10%
FCS and passed through gauze to remove excess debri. The cells were distributed evenly into two 100mm Petri dishes
which had been coated with a nonspecific rabbit anti-mouse
immunoglogulin (Dakopatts). The dishes were shaken briefly
every 5 minutes for 15 minutes (37'C). The nonadherent cells
were then aspirated, and the remaining cells were removed
from the dishes by adding 0.125% trypsin for ten minutes at
37‘C. The enzyme solution, with the cell population was
placed into a 15ml conical tube (Falcon) containing Hams
F12 medium with 10% FCS. After the cells had been
centrifuged for 10 minutes they were resuspended in 1ml of
Hams F12 medium, counted and plated out into 96 well plates
(Falcon, Primaria).
62
Preparation of macrophages from uninjured sciatic nerve.Sciatic nerves were removed from 3 or 4 day old Sprague-
Dawley rat pups (P3/4) immediately following decapitation.
The nerves were digested with 0.025% (w/v) trypsin (Sigma)
and 0.15% (w/v) collagenase (Sigma) in dissociation buffer
for 30 minutes. Hams FI2 medium containing 10% (v/v) FCS
was added to the enzyme mixture preventing further trypsin
activity. The dissociate was centrifuged at 1600rpm for 10
minutes and the supernatant discarded. The pellet was
resuspended in 2ml of Hams F12 medium and the cell
suspension was passed through gauze removing excess tissue
debris. The cells were added to Petri dishes (Falcon)
coated with either leucocyte common antigen (LCA) or 0X42
antibody, and incubated at 37'C, 5% CO; for 20 minutes
following which nonadherent cells were removed by washing
the Petri dishes extensively with dissociation buffer. The
cells were removed from the Petri dish by adding 0.125%
trypsin for 10 minutes. The trypsini sat ion was terminated
as before and the suspension was centrifuged at 1600rpm for
10 minutes. The pellet of cells was resuspended in Hams F12
medium and plated into 96 well tissue culture plates
(Primaria grade. Falcon) at a density of 5000 cells/well .
Cell culture techniques.The macrophages plated into 96 well plates were cultured
in Hams F12 medium. If the cells were incubated overnight
or longer, then 2mM glutamine, 50mg/ml streptomycin
(Gibco), 50U/ml penicillin (Gibco) and 10% FCS (v/v) were
63
added to the medium. The cells were cultured at 37'C in the
presence of 5% CO,.
Macrophage activation in vitroPeritoneal and spleen macrophage cultures are treated
with gamma interferon (IFNy) at 300 Units/ml (a gift from
P. van der Meide) for 2 hours before the addition of CGRP.
Cyclic adenosine monophosphate (cAMP) assay.Culture medium was replaced by fresh medium without FCS
2 hours before adding the phosphodiesterase inhibitor,
isobuty1-methylanthine (IBMX) (Sigma). After a period of 30
minutes, CGRP (Bachem) was added to the cells. Cyclic AMP
was extracted from the cells by removing the medium and
adding ice cold 70% (v/v) ethanol in water. The cells were
left overnight at -20'C. Ethanol from each well was
transferred to an Eppendorf tube and dried down using a
Speed Vac SClOO (Savant). The cAMP in each sample was
measured in duplicate using the more sensitive acétylation
method of a dual range cAMP kit (Amersham). The radioactive
counts present in each pellet, containing cAMP bound to
antibody, was measured using a gamma counter (Nuclear
enterprises NE1600) gamma counter.
64
RESULTS.
Standard curves for the CGRP and VIP RIA.An example of an RIA standard slope for human synthetic
CGRP RIA is shown in figure 2.2. The mean of twelve
standard slopes for the human synthetic CGRP was calculated
and a value of -3.0325 + 0.16 was obtained. Similarly, a
mean of twelve slopes was obtained for CGRP extracted from
lesioned sciatic nerves. Values of -2.90 + 0.28, -3.05 +
0.36 and - 2.64 ± 0.33 were calculated for proximal, distal and contralateral segments of sciatic nerve, respectively
(mean + s.e.m. n=12 for all segments). The small
differences between the slopes including the standard slope
were not statistically significant, measured by students t-
test (p=0 . 1 ) .
A similar example of an RIA standard curve for synthetic
human VIP is shown in figure 2.3. The mean of four standard
slopes for VIP extracted from lesioned sciatic nerve was
calculated and a value of -3.12 + 0.09 was obtained. Values
of -3.12 + 0.24, -3.05 + 0.36 and -2.81 + 0.44 were
calculated for proximal, distal and contralateral segments
of sciatic nerve, respectively (mean + s.e.m. n=4 for all
the segments). The small differences between the slopes
including the standard slope were not statistically
significant, measured by students t-test (p=0.1).
65
DISPLACEMENT CURVES FOR SYNTHETIC HUiMAN CGRP AND CGRP PRESENT IN SCIATIC NERVE.
10
N e rve e x tr a c t , fi\ 10^ 10
95
90
ODd80
70
60dOJoucu
CL
50
40
30
o — O S ynthetic CGRP B— ■ Distal segm ent # — # P ro x im al segm ent
□ — □ C o n tra la te ra l segm ent
20
10
CGRP, fm o l ( lo g )
F ig u re 2.2 : A s ta n d a rd d is p la c e m e n t s lo p e o f log dose a g a in s t
lo g i t p e rc e n ta g e b in d in g is p lo t te d fo r h u m a n s y n th e t ic
CGRP ( O ). The s lo p e sh o w n re p re s e n ts an e x a m p le o f th e
s lo p e s o b ta in e d fo r e a c h RIA. E x a m p le s o f s lo p es o b ta in e d fo r
CGRP e x tra c te d f r o m s e g m e n ts o f s c ia t ic n e rv e a re a lso
p lo t te d a n d re g re s s io n lin e s f i t t e d
66
DISPLACEMENT CURVES FOR SYNTHETIC HUMAN VIP AND V IP -L IK E IMMUNQREACTIVITY PRESENT IN SCIATIC NERVE.
N e rve e x t r a c t , /i,l10 100
95
90
00
70 -
60
50
40
30
200 —0 S ynthetic VIP ■ — ■ D ista l segm ent # — # P ro x im al segm ent □ — □ C o n tra la te ra l segm ent10
100101Im m iin o re a c t iv e VIP, fm o l ( lo g )
F ig u re 2.3: A s ta n d a rd d is p la c e m e n t c u rv e o f log dose a g a in s t
lo g it p e rc e n ta g e b in d in g is p lo t te d fo r h u m a n s y n th e t ic
VIP ( O ). The s lo p e sh o w n is a n e x a m p le o f th e s lo p es
o b ta in e d fo r e a c h RIA. E x a m p le s o f s lo p e s o b ta in e d f o r V IP - l ik e
im m u n o r e a c t iv i t y e x t r a c te d f r o m s e g m e n ts o f s c ia t ic n e rv e a re
a lso p lo t te d a n d re g re s s io n lin e s f i t te d .
67
Interassay standard for RIA.The mean + s.e.m. for the interasssay spinal cord sample
was 25 + 1.5 pmolf n=12.
Elution profile for nerve extract and synthetic '"I-CGRP.The elution profile of CGRP immunoreactive material
derived from either pooled distal segments or pooled
proximal segments from injured sciatic nerve (figure 2.4a)
was virtually identical to that of synthetic '"I-CGRP
(figure 2.4b).
CGRP immunohistochemistry in lesioned sciatic nerve.CGRP immunoreactivity was clearly observed within a
subset of axons found approximately 6mm distal to the crush
site (figure 2.5).
CGRP expression after sham lesion versus control unlesioned sciatic nerve.
There was no significant variation (p=0.1) in the amount
of CGRP expressed between any of the 5mm segments of
sciatic nerve analysed following a sham operation, measured
by RIA (figure 2.6). However, there was a significant
elevation (p=0.001) in CGRP content in the nerves both
ipsilateral and contralateral after sham operation compared
to the amount of CGRP in the sciatic nerve of an unoperated
animal (figure 2.6).
coü
CIhP:ou
500
400
3 00
200
100
100
80
60
40
20
68(a ) N e rve e x t r a c t
O
O Ô O O O — d D -010 20
-0-
30
(b )125
I-C G R P
40
10 20 30E lu t io n v o lu m e , m l
40
F ig u re 2.4: E lu t io n p r o f i le o f C G R P - im m u n o re a c t iv ity
(a ) e x t r a c te d f r o m p r o x im a l a nd d is ta l s e g m e n ts o f125
c ru s h e d s c ia t ic n e rv e a n d (b ) s y n th e t ic " I-C G R P .
F ra c t io n s 8 —30 ( I m l / t u b e ) o f n e rv e e x t r a c t w e re
m e a s u re d fo r CGRP c o n te n t by RIA. The e lu t io n p r o f i le 125
o f I-C G R P w as d e te rm in e d b y m e a s u r in g th e le v e l o f
r a d io a c t iv i t y in e a c h f r a c t io n e lu te d a n d c o n v e r te d to fm o l.
V^, v o id v o lu m e ; t o t a l v o lu m e .
69
Figure 2.5: Staining of CGRP-like immunoreactivity in the axons 6mm distal to the crush site of a 6 days post-crush rat sciatic nerve. Scale bar = 45vun
70
CGRP LEVELS IN 2 DAY POST-SHAM OPERATED NERVE
oUUU p
2000
È 1000oo
X
1 L » .SCIATIC NERVE SEGMENTS
F ig u re 2 .6 : A m o u n t o f CGRP in 5 m m n e rv e s e g m e n ts
s h a m o p e ra te d (P X ^D ^D ^ ), c o n t r a la te r a l (C^) a n d f r o m
u n o p e ra te d (C) r a ts d e te rm in e d b y RIA. In th is a nd
fo l lo w in g h is to g ra m s ( f ig u re s 2.6 to 2 .12 ) e a ch b a r
re p re s e n ts th e m e a n + / — s .e .m fo r 4 a n im a ls ..
* The am ou n t of CGRP in segm ent C is s ignifican tly
below th e am ou n t CGRP m easured in any of the
nerve segm ents of sham operated rats, m easured
by students t-te s t . (p = 0 .0 01 ).
- sensitiv ltv of the a.ssav
71
CGRP expression after sciatic nerve lesion.Changes in the level and distribution of CGRP in a
defined length of sciatic nerve (see figure 2.1) at several
time points following lesion were quantified using the RIA.
Results are presented in the following order:
1) Contralateral nerves examined 1 to 14 days after
lesioning;
2) Regenerating nerves examined either 1, 2 and 6 days
following crush with forceps or 6, 10 and 14 days
following crush with suture;
3) Nonregenerating nerves examined either 1, 2 and 6 days
following transection or 1, 6 and 14 days following
crush with suture left transection.
Amount of CGRP was measured in 5mm segments of the sciatic
nerve both proximal and distal to the lesion site (see
figure 2.1 for definition of the different segments
analysed).
Contralateral nerve.Lesioning the sciatic nerve produced a significant
increase (p=0.001) in CGRP expression in the contralateral
nerve at all time points monitored (figures 2.6-2.10). CGRP
levels in the contralateral nerve remained above control,
unoperated levels, for up to 14 days after crush and 6 days
after transection.
CGRP levels in regenerating sciatic nerves.1 and 2 days post-crush: When the level of CGRP observed in
72
nerve segment P, was compared to that observed in a similar
length of nerve from the contralateral sciatic nerve (C,),
there was a 3 fold increase in CGRP content 1 day post
crush (figure 2.7a). A further increase was seen in P, 2
days after crushing (figure 2.7b). A smaller accumulation
was observed distal to the lesion (D,) at both time points
which became significant (p=0.001) 2 days following lesion
when compared to contralateral levels (figures 2.7a and b) .
Although CGRP was detectable in P% 1 day after crush, it was
below that found in the contralateral nerve. However, an
increase in CGRP significantly above the level in the
contralateral nerve segment was seen in P%Zdays after crush
(figure 2.7b).
6 days post-crush: A shift in the location of the peak of
CGRP going from proximal to distal to the lesion site was
observed (figure 2.7c) after the nerve had been crushed
with forceps. The level of CGRP was significantly higher in
D] than in P, (p=0.001). When a suture was used to crush the
nerve, the shift in CGRP from proximal to distal was not as
great as in forceps-crushed nerves (figure 2.8a). This
result indicated that the suture caused greater damage to
the axons and therefore, sutures were used to crush the
nerves in subsequent experiments. When the level of CGRP in
nerve segments P% to Dj was totalled, a reduction from a
peak of 6342 + 355fmol 2 days after crush to 3544 + 92fmol
6 days after crush was observed.
coü
eua:oo
73CGRP LEVELS IN REGENERATING SCIATIC NERVES
CRUSHED WITH FORCEPS.4 0 0 0
(a) 1 Day post-crush.
3 0 0 0
2000
1000
0 L_ 4 0 0 0 p
( b ) 2 Days p o s t -c ru s h .
3 0 0 0
2000
1000
04 0 0 0
( c ) 6 Days p o s t -c r u s h
300 0
2000
1000 r ...T
c c D- U P i - 1SCIATIC NERVE SEGMENT.
F ig u re 2 .7 ; A m o u n t o f CGRP fo u n d in 5 m m s c ia t ic
n e rv e s e g m e n ts a t (a ) 1 d a y , (b ) 2 d a y s a n d (c )
6 d a vs a f t e r c r u s h i n iu r v w i th w a tc h m a k e r fo r c e n s .
74
CGRP LEVELS IN REGENERATING SCIATIC NERVESCRUSHED WITH A SUTURE.
0)ooPuoa
300 0
6 Days p o s t-c ru s h .
2000
1000
03 0 0 0
) 1 0 Days p o s t -c ru s h .
2000
1000
0 - 3 00 0 r
(c) 14 Days p o s t -c ru s h
2000
1000
0 c c.SCIATIC NERVE SEGMENTS
F ig u re 2 .8 : A m o u n t o f CGRP in 5 m m n e rv e s e g m e n ts f r o m
r e g e n e r a t in g s c ia t ic n e rv e (a ) 6 d a y s , (b ) 10 d a y s a n d
(c ) 14 d a y s a f t e r c r u s h w i t h s u tu r e .
* A m ount of CGRP in is s ig n ifican tly above th a t found m any of the o ther segments 1Ü days post—crusli, m easured by students t - t e s t (p =0 001)
75
10 and 14 days post-crush; There was an overall reduction
in the level of CGRP measured in the segments of sciatic
nerve P; to D, 10 and 14 days post-crush (figures 2.8b and
c). However, the amount of CGRP present in the segment
furthest distal to the crush (D, ) was significantly above
the amount measured in any of the other segments, 10 days
after lesion (p=0.005) (figure 2.8b). However, by day 14,
CGRP levels had returned to contralateral levels in all
segments examined, with no significant difference (p=0.05)
between any of the segments from the crushed nerve (figure
2.8c).
CGRP levels in nonreaeneratina sciatic nerves.
When the suture was used to block nerve regeneration, a
significantly lower level (p=0.01) of peptide was found in
Di, 2 days post-crush (figure 2.9a) compared to 2 days post
crush with forceps (figure 2.7b). Moreover, CGRP could not
be detected in the distal segments at the later time points
of 6 and 14 days post-crush. However, a high level of CGRP
was still observed in the proximal segment P, at the latter
time points (figures 2.9b and c).
Cutting the sciatic nerve produced a 3 fold elevation in
CGRP proximal to the lesion site 1 day after transection,
which increased to a 4 fold elevation 2 days post
transection (figures 2.10a and b). The amount of CGRP found
distally, 1 day after transection, was only slightly below
that expressed in the contralateral nerve and not
76
CGRP LEVELS IN NONREGENERATING SCIATIC NERVESCRUSHED WITH A SUTURE
aooÛHù:o
( a ) 2 days p o s t-c ru s h4 0 0 0
3000
2000
1000
04 0 0 0
( b ) 6 days p o s t-c ru s h .3 00 0
2000
1000
04 00 0
(c) 14 days p o s t-c ru s h300 0
2000
1000
0" *
SCIATIC NERVE SEGMENTS
F ig u re 2.9 A m o u n t o f CGRP m 5 m m n e rv e s e g m e n ts
f r o m n o n re g e n e ra t in g s c ia t ic n e rv e s (a ) 2 days
(b ) 6 days a n d 14 days a f t e r c ru s h w ith a s u tu re .
CGRP was n o t d e te c te d in o r a t th e la t t e r t im e
p o in ts as th e le v e ls w e re b e lo w th e s e n s i t iv i ty o f th e
RIA. s e n s i t iv i t y o f th e assay)
77
CGRP LEVELS IN TRANSECTED SCIATIC NERVES
5 0 0 0
Day post— cut.4 0 0 0
3 0 0 0
2000
1000
05 0 0 0
( b ) 2 Days p o s t -c u t .
o4 0 0 0
3 0 0 0
coÜ 2000CuKOO 1000
5 0 0 0
(c ) 6 Days p o s t - c u t4 0 0 0
3 0 0 0
2000
1000
SCIATIC NERVE SEGMENT.
F ig u re 2 .10; A m o u n t o f CGRP in 5 m m s c ia t ic n e rv e
s e g m e n ts (a ) 1 day, (b ) 2 days a nd (c ) 6 days a f t e r
t ra n s e c t io n . CGRP w as n o t d e te c te d in 6 days
p o s t - c r u s h as th e le v e l w as b e lo w th e s e n s i t iv i t y o f
th e RIA. f - s e n s i t iv i t y o f th e a ssay )
78
significant (p=0.1). However, the level of CGRP in D,, 2
days after transection, had declined to levels
significantly below that found in the contralateral nerve
(p=0.001) and it became undetectable distal to the cut, 6
days following the injury (figure 2.10c). Although the
amount of CGRP in P, 6 days after transection was
significantly above that in the contralateral nerve, it was
significantly lower (p=0.001) than the amount of CGRP found
in P, of nonregenerating sciatic nerves 6 days after crush
with suture (figure 2.9b).
Total CGRP levels in lesioned nerve.The total amount of CGRP expressed over the area of P; to
Di declined at a much faster rate in transected nerves
compared to all other types of lesions (figure 2.11). The
total level of CGRP throughout this area of sciatic nerve,
6 days after lesion, was significantly greater (p=0.001) in
nerves which had the suture left attached after crushing
compared to transected nerves (figure 2.11). When a suture
was left attached for 14 days then the levels of CGRP in
the sciatic nerve (Pj to DJ declined to similar levels
found in a 6 day post-transected nerve.
VIP-like immunoreactivitv in lesioned sciatic nerve.Sciatic nerve lesion caused an accumulation of VIP-like
immunoreactivity proximal and distal to the crush site
measured 2 days post-crushed (figure 2.12a). This is
compared to levels in the contralateral nerve and
79
THE EFFECT OF DIFFERENT TYPES OF LESIONS
ON CGRP LEVELS IN THE SCIATIC NERVE.
7 0 0 0
6 0 0 0
5 0 0 0
o 4 0 0 0ÜOh0:g 3 0 0 0
2000
1000
\ / / I - c ru sh
ly y i - t r a n s e c t i o n
H H - s u tu re b lo c k
2 6 14
t im e (d a y s p o s t le s io n )
F ig u re 2 .1 1 : T o ta l a m o u n t o f CGRP fo u n d in s c ia t ic
n e rv e s e g m e n ts P^ - (see f ig u r e 2 .1 ) fo l lo w in g
s c ia t ic n e rv e le s io n in g .
n = 4 f o r e a c h m e th o d o f le s io n .
80VIP LEVELS IN CRUSHED SCIATIC NERVES
oÜ
>
5 00
4 0 0
300
200
100
05 00
4 0 0
300
200
100
05 00
4 0 0
(a ) 2 days p o s t -c ru s h .
( d ) 6 days p o s t -c ru s h .
( c ) 14 days p o s t -c ru s h
300
200
100
0SCIATIC NERVE SEGMENTS
F ig u re 2.12: A m o u n t o f VIP im m u n o re a c t iv ty in 5 m mn e rv e s e g m e n ts f r o m re g e n e ra t in g s c ia t ic n e rv e s
(a ) 2 days, (b ) 6 d a ys a n d (c ) 14 days a f t e r c ru s h
w ith s u tu re .The am ou n t of VIP in segm ents C, C . P , D . and in (a ) and P„ and C in (b) and C in (c) was below the sens itiv ity of the RIA. No value is given fo r D„ in (b) since th e sam ple slopes were not p a ra lle l w ith the standard s Io d r ( - sensitiv ity of assav)
81unoperated nerve, which were below the sensitivity of the
assay. The level found proximal to the crush (P,) was
significantly higher than the level distal to the crush (D, )
(p=0.001). A shift in the peak of VIP-immunoreactivity from
proximal to distal to the crush was observed 6 days after
crushing (figure 2.12b). However, this was not significant
until 14 days after crushing (p=0.001) (figure 2.12c).
Gcunma interferon-like immunoreactivity in the sciatic nerve.
Gamma interferon (IFNY)-like immunoreactivity was clearly
visible in a subset of axons in the sciatic nerve 2 hours
post-crush (figure 2.13a). Furthermore, the staining
intensity, distal to the crush site was observed a much
higher level 2 days after crush (figure 2.13b). No IFNy-
like immunoreact ivi ty was, however, visible in the axons
distal to the crush site, 3-6 days post-crush. However, a
low level of IFNy-immunoreactivity was seen distal to the
crush site, 18 days post-crush (figures 2.14a and b).
82
Figure 2.13; Staining of DB-1 immunoreactive axons in the sciatic nerve (a) 2hrs and (b) 2 days post-crush. The
sections are 2mm distal to the crush. Scale bar = 45um
83
v f
Figure 2.14: Staining of DB-1 immunoreactive axons in the sciatic nerve (a) 6 days and (b) 18 days post-crush. Thesection is 2mm distal to the crush. Scale bar = 45um
84CGRP AND CAMP ACCUMULATION IN MACROPHAGES.
The following experiments were performed in order to
determine whether CGRP accumulated after sciatic nerve
crush has the ability to interact with macrophages
recruited into the nerve after injury. CGRP was also added
to cultures of spleen and peritoneal macrophages and its
affect on intracellular cAMP was measured.
Effect of CGRP on cAMP accumulation in peritoneal
macrophages ; Resident vs Activated.
The time course of cAMP accumulation by peritoneal
macrophages in response to CGRP stimulation is shown in
figure 2.15. The time course was not significantly altered
by the state of macrophage activation. The peak of cAMP
production was reached after approximately 1 minute after
CGRP addition and dropped to around 40% of the maximum at
approximately 5 minutes. A significant lower level
(p=0.005) of cAMP was seen in macrophages activated in vivo
with LPS compared to resident macrophages, 15 minutes after
the addition of CGRP.
Resident peritoneal macrophage cAMP accumulation was
stimulated by CGRP in a dose-response manner, in the
presence or absence of IBMX (figure 2.16). A significant
increase in cAMP accumulation by peritoneal macrophages,
occurred with the lowest dose (InM) of CGRP. Furthermore,
the presence of IBMX enhanced the accumulation of cAMP
85
TIME COURSE OF cAMP ACCUMULATION BYACTIVATED AND RESIDENT PERITONEAL MACROPHAGES
w(D00(CapÜcC
oooLO
Ph<o
—W a c t i v a te d m a c ro p h a g e s 7— V r e s id e n t m a c ro p h a g e s .
20 30T im e ( m in u t e s ) .
F ig u r e 2 .15 ; T im e c o u r s e o f CGRP s t i m u l a t e d cAMP
a c c u m u l a t i o n b y p e r i t o n e a l m a c r o p h a g e s . M a c r o p h a g e s
w e r e i n c u b a t e d w i t h IBMX (1 0 ^M, 37°C) f o r 3 0 m in p r i o r
t o t h e a d d i t i o n o f CGRP (1 0 ^M). C u l t u r e m e d i u m w as
r e m o v e d a n d 70% e t h a n o l a d d e d to t h e c e l l s a t d i f f e r e n t
t i m e p o in t s a f t e r CGRP a d d i t i o n . cAMP le v e ls w e re
d e t e r m i n e d b y RIA. E a c h t i m e p o i n t r e p r e s e n t s t h e m e a n
a n d S.D. f o r d u p l i c a t e w e l ls o f m a c r o p h a g e s .
D = 0 .0 0 5
86
EFFECT OF CGRP ON cAMP ACCUMULATION BYACTIVATED AND RESIDENT PERITONEAL MACROPHAGES
res ide n t, +IBMX
20ui0.)aoCOrgOfHDCOa
ooo
CuSCo
15
10
▲-------▲ a c t iv a te d , + IB M X
□ --------□ re s id e n t , - IB M X
A ------- A a c t iv a te d , - IB M X
-6- 9 -8 - 7
CGRP (M)
F ig u re 2.16: D o s e - re s p o n s e c u rv e s o f CGRP s t im u la t e d
cAMP a c c u m u la t io n b y a c t i v a te d a n d r e s id e n t p e r i t o n e a l
m a c ro p h a g e s , in th e p re s e n c e o r a bs e n c e o f IBXEX (10
E x p e r im e n ts w e re p e r fo r m e d as e x p la in e d in t h e le g e n d
to f ig u r e 2.15, e x c e p t th e m e d iu m was re m o v e d a n d th e
c e l ls e x t r a c te d 2 m in u t e s a f t e r CGRP a d d i t io n . E ach p o in t
r e p r e s e n ts th e m e a n a n d S.D. f o r d u p l ic a te w e lls o f
m a c ro p h a g e s . The e x p e r im e n t wwas re p e a te d tw ic e , w i t h
s im i l a r r e s u l t s ( d a ta n o t s h ow n ).
87after 2 minutes of stimulation (figure 2.16). Activation of
the macrophages in vivo, with LPS (50ug), decreased their
ability to accumulate cAMP in response to the addition of
CGRP; even in the presence of IBMX the response was
significantly reduced (p=0.005) when CGRP at a
concentration of 10‘*M or greater was added (figure 2.16).
In vitro treatment of resident peritoneal macrophages with
300U/ml of IFNY/ 2 hours before addition of CGRP, caused a
significant reduction (p=0.005, p=0.001) in accumulation of
cAMP in response to the addition of 10'’ and 10'“ CGRP
respectively (figure 2.17).
Spleen macrophage production of cAMP in response to CGRP.
The peak production of cAMP in spleen macrophages
occurred 5 minutes after addition of CGRP (10‘‘), which was
slightly later than that of peritoneal macrophages (figure
2.18). The presence of IBMX did not alter the time course
of the peak. However, the levels of cAMP produced after the
peak were maintained at a higher level in the presence of
IBMX. Spleen macrophages demonstrated a more acute dose
dependent response to CGRP (figure 2.19) compared to
peritoneal macrophages (figure 2.16). However, this
response was blocked if these cells were pretreated for 2
hours with CGRP (figure 2.20). Similarly, the spleen
macrophage cAMP response to CGRP was slightly depressed if
they were pretreated with IFNy for 2 hours (figure 2.21).
However, the level of inhibition was much less compared to
that obtained with peritoneal macrophages.
EFFECT OF CGRP ON cAMP ACCUMULATION BYPERITONEAL MACROPHAGES AFTER IFN- 7 PREINCUBATION
wQ)%0CO-aAoL,Oin
ooolO
CLco
12
10
2 h r p r e in c u b a t io n w i t h IF N - 7 No p r e in c u b a t io n .
# *
10-9 -8
10 ' 10 CGRP (W)
10-c
F ig u r e 2 .17 ; D o s e - r e s p o n s e c u r v e s o f CGRP s t i m u l a t e d
cAMP a c c u m u l a t i o n b y p e r i t o n e a l m a c r o p h a g e s , in t h e
p r e s e n c e o f IBMX (1 0 ^M), w i t h o r w i t h o u t a 2 h o u r
p r e i n c u b a t i o n w i t h g a m m a i n t e r f e r o n ( 3 0 0 U / m l ) .
E x p e r i m e n t s w e re p e r f o r m e d as e x p l a in e d in t h e
le g e n d to f i g u r e 2 .1 5 , e x c e p t c e l ls w e re e x t r a c t e d
2 m i n u t e s a f t e r CGRP a d d i t i o n . E a c h p o i n t r e p r e s e n t s
t h e m e a n a n d S.D. f o r d u p l i c a t e w e l ls o f m a c r o p h a g e s
T h e e x p e r i m e n t w as r e p e a t e d tw ic e , w i t h s i m i l a r
r e s u l t s ( d a ta n o t s h o w n ) .
p = 0 .0 0 5 , p = 0.001 ( m e a s u r e d b y s t u d e n t s t - t e s t )
89
TIME COURSE OF cAMP ACCUMULATION BYSPLEEN MACROPHAGES.
40 r
M0)00rCaoÎ-HÜcc
ooo
cus<o
-▼ + IB M X - IB M X
^0T im e ( m in u t e s )
F ig u re S. 18: T im e c o u rs e o f CGRP (10 ^M) s t im u la t e d
cAMP a c c u m u la t io n b y s p le e n m a c ro p h a g e in th e
p re s e n c e o r a b se n ce o f IBMX (10 \ l ) . E x p e r m e n ts
w e re p e r fo r m e d as e x p la in e d in t h e le g e n d to
f ig u r e 2.15. E ach t im e p o in t r e p r e s e n ts d u p l ic a te
w e l ls o f m a c ro p h a g e s a n d shows th e m e a n a n d S.D.
The e x p e r im e n t was r e p e a te d w i t h s im i l a r r e s u l t s
(d a ta n o t sh ow n )
90EFFECT OF CGRP ON cAMP ACCUMULATION BY
SPLEEN MACROPHAGES
mOPCÜ)cdAOL,ÜCDeoooLOosCL2<Ü
50
V V + IB M X
V V - IB M X40
30y
20
T /
10y #
i i-9 — 8 -710CGRP (M ).
F ig u re 2.19 D o s e - re s p o n s e c u rv e s o f CGRP s t im u la t e d
cAMP a c c u m u la t io n b y s p le e n m a c ro p h a g e s , in
p re s e n c e o r a bse n ce o f IBMX (10 ^M). E x p e r im e n ts
w e re p e r fo r m e d as e x p la in e d in th e le g e n d to
f ig u r e 2.15, e x c e p t c e l ls w e re e x t r a c te d 5 m in u t e s a f t e r
CGRP a d d i t io n . E a ch p o in t r e p r e s e n ts t h e m e a n a n d S.D.
f o r d u p l ic a te w e l ls o f m a c ro p h a g e s . The e x p e r im e n t
w as re p e a te d , w i t h s im i l a r r e s u l t s ( d a ta n o t sh o ivn ).
* p = 0 .001, # p - 0 .0 0 5
91
EFFECT OF CGRP PREINCUBATION ON THE cAMP
RESPONSE BY SPLEEN MACROPHAGES TO CGRP
w(DOjOCOaoÜCOgoooIT)
CIh<u
30
□ □ n o p r e in c u b a t io n■ ■ 2 h r p r e in c u b a t io n w i t h CGRP
20
* **
-6- 9 - 7-8
CGRP (M)
F ig u r e 2 .20 ; D o s e - r e s p o n s e c u r v e s o f CGRP s t i m u l a t e d
cAMP a c c u m u l a t i o n b y s p le e n m a c r o p h a g e s , in t h e
p r e s e n c e o f IBMX (1 0 ^M) a n d w i t h o r w i t h o u t a 2 h o u r-6
p r e i n c u b a t i o n w i t h CGRP (1 0 M). E x p e r i m e n t s w e re
p e r f o r m e d as e x p la in e d in t h e le g e n d to f i g u r e 2 .15 ,
e x c e p t c e l ls w e re e x t r a c t e d 2 m i n u t e s a f t e r CGRP
a d d i t i o n . E a c h p o i n t r e p r e s e n t s t h e m e a n a n d S.D. f o r
d u p l i c a t e w e l ls o f m a c r o p h a g e s . T h e e x p e r i m e n t w as
r e p e a t e d tw ic e , w i t h s i m i l a r r e s u l t s ( d a ta n o t s h o w n ) .
* p = 0.001 ( m e a s u r e d b y s t u d e n t s t - t e s t . )
92EFFECT OF CGRP ON cAMP ACCUMULATION BY
SPLEEN MACROPHAGES AFTER IFN- 7 PREINCUBATION
60 r
S 50
a2 40ocdn
§ 30lOoB
a.20
/X/ /
10 - 9 —810 ' 10CGRP (M)
- 7 10— 6
F ig u re 2.21: D o s e - re s p o n s e c u rv e s o f CGRP s t im u la t e d
cAMP a c c u m u la t io n b y s p le e n m a c ro p h a g e s , in t h e
p re s e n c e of IBMX (10 ^M), w i t h o r w i t h o u t a 2 h o u r
p r e in c u b a t io n w i t h g a m m a i n t e r f e r o n ( 3 0 0 U /m I ) .
E x p e r im e n ts w e re p e r f o r m e d as e x p la in e d in t h e le g en d
t o f ig u r e 2.15, e x c e p t c e l ls w e re e x t r a c t e d 5 m in u te s a f t e r
CGRP a d d i t io n . E ach p o in t r e p r e s e n ts th e m e a n a n d S.D.
f o r d u p l ic a te w e lls o f m a c ro p h a g e s . The e x p e r im e n t was
was r e p e a te d tw ic e , w i t h s im i l a r r e s u l t s ( d a ta n o t sh ow n ).
+ p = 0 ,0 0 1
93The response of peripheral nerve macrophages.
Incubation of macrophages isolated from lesioned sciatic
nerves with CGRP stimulated a accumulation of cAMP, in a
dose dependent manner (figure 2.22). However, this was only
seen in the presence of IBMX (figure 2.22). Macrophages
isolated from unlesioned sciatic nerves also demonstrated
a dose-dependent accumulation of cAMP when stimulated with
CGRP (figure 2.23). Furthermore, the level of cAMP
accumulated by macrophages from unlesioned nerve, after the
addition of CGRP (10’‘), was greater than the level
accumulated by macrophages from lesioned sciatic nerve
(figures 2.22 and 2.23).
94
EFFECT OF CGRP ON cAMP ACCUMULATION BY
MACROPHAGES ISOLATED FROM CRUSHED SCIATIC NERVE
COoCÜjCAoucCsooo
8 r
V
+IBMX -a7 —I3MN
10- 9 -8
10 10
CGRP (M)
- 7
/ !
10-6
F ig u re 2 .22: D o s e - re s p o n s e c u rv e s o f CGRP s t im u la t e d
cAMP a c c u m u la t io n b y m a c ro p h a g e s is o la te d f r o m
c r u s h e d s c ia t ic n e rv e , i n t h e p re s e n c e o r a b se n ce
o f IBMX (10 ^M). E x p e r im e n ts w e re p e r f o r m e d as
e x p la in e d in th e le g e n d to f ig u r e 9, e x c e p t c e l ls w e re
e x t r a c t e d 5 m in u t e s a f t e r CGRP a d d i t io n . R e s u lts a re
m e a n s w i t h s .e .m , n = 4 w e l ls o f m a c ro p h a g e s .
95
EFFECT OF CGRP ON cAMP ACCUMULATION BY
MACROPHAGES ISOLATED FROM UNLESIONED SCIATIC NERVE
14
& 12COOho u ocd
<du
10
o ooccn3 6
2
0
-T
1
10-9 -8
10 10 CGRP (M)
-710
-6
F ig u re 2 .23: D o s e - re s p o n s e c u rv e s o f CGRP s t im u la t e d
cAMP a c c u m u la t io n b y m a c ro p h a g e s is o la te d
u n le s io n e d s c ia t ic n e rv e , i n th e p re s e n c e of
IBMX (10 ^M). E x p e r im e n ts w e re p e r fo r m e d as
e x p la in e d in th e le g e n d to f ig u r e 2.15, e x c e p t t h e
ce l ls w e re e x t r a c te d 5 m in u te s a f t e r CGRP a d d i t i o n
E a c h p o i n t r e p re s e n ts t h e m e a n a n d S.D. f o r
d u p l i c a te w e l ls o f m a c ro p h a g e s . The e x p e r im e n t was
r e p e a te d w i t h s im i l a r r e s u l t s ( d a ta n o t s h o w n ) .
96DISCUSSION.
This study has provided a detailed time course for the
expression of both CGRP and VIP following sciatic nerve
injury. It has shown that the rise in CGRP and VIP-like
immunoreactivity around the injury site, previously
observed immediately following injury (Kashihara et
al,1989; Lundberg et al, 1981;) remains above control
levels for up to 14 days. CGRP produced in motoneurons in
response to injury (Streit et al., 1989; Arvidsson et al.,
1990; Noguchi et al., 1990) is anterogradely transported at
about 1mm/hr (Kashihara et al., 1989). The movement of the
peak of CGRP, observed by RIA, corresponded with the rate
of axon regeneration of 2-4mm/day (reviewed by Terzis and
Smith, 1990). Furthermore, the axonal-like distribution of
CGRP, shown by the immunohistochemical study, suggests that
CGRP is transported down the axons and accumulates at the
tips of the regenerating axons. Also, the disappearance of
CGRP in the distal stump soon after injury, if regeneration
was prevented, suggests that CGRP is not produced by non
neuronal cells in response to injury. The initial
accumulation of CGRP seen in the distal stump was probably
due to a small amount of retrograde axonal transport.
The accumulation of CGRP observed immediately proximal to
the lesion site 1 to 2 days following a crush injury, is
consistent with the temporal changes in CGRP-like
immunoreact ivi ty and CGRP mRNA in the ventral horn
97following injury (Arvidsson et al, 1990; Noguchi et al,
1990). Furthermore, the gradual decline in CGRP peptide
levels in the sciatic nerve, back to control levels after
it had peaked at 2 days, is also observed for both mRNA and
peptide for CGRP in the ventral horn (Dumoulin et al, 1991;
Arvidsson et al, 1990).
The level of CGRP observed in a length of 15mm which
includes the lesion site, appears to be influenced by the
direct contact of the distal stump with the proximal stump.
Removal of the distal segment of sciatic nerve by
transection led to a more rapid decrease in the amount of
CGRP measured over the 15mm length of nerve, compared to
when the distal segment remained attached. Since the
presence of the distal stump is thought to enhance
regeneration (Williams et al., 1984; Scaravilli, 1984), it
is possible that this influence of the distal stump upon
regeneration is connected to its ability to maintain CGRP
expression. Alternatively, it is possible that transmission
of action potentials down the sciatic nerve triggers the
release of CGRP from the newly formed terminals, and since
the distal stump is not attached to the proximal stump
after the nerve is severed, CGRP released from the
terminals would not be contained within the epineurium.
Thus, contact with the distal stump in the case of crush,
even with the suture left attached, would prevent such
release.
98The increase in CGRP in contralateral sciatic nerve
compared to unoperated sciatic nerve may reflect increased
usage of the contralateral leg and hence increased muscle
activity. It is possible that this rise in CGRP is
important in modulating the increased muscle activity,
since early studies show that CGRP modulates the
contraction of striated muscle (Takami et al., 1985). The
small increase observed in the ipsilateral nerve after sham
operation may reflect the manipulation of muscle whilst
exposing the nerve.
Although VIP-like immunoreactivity within the sciatic
nerve is also maintained above control levels several days
after crush, it is not expressed at such a high level as
CGRP. This may reflect a smaller number of VIP expressing
neurons in comparison to CGRP expressing neurons. The
change in distribution of VIP-like immunoreactivity with
time also appears to correlate with the advance of
regenerating axons, but at a slower rate than CGRP positive
axons. Furthermore, Giachetti and Said (1979) have shown
that, if the sciatic nerve is prevented from regenerating,
then VIP continues to accumulate just proximal to the
lesion site for at least 72 hours. This is slightly later
than that found for CGRP which peaked at 2 days, under
nonregenerating condition. A further difference between the
production of CGRP and VIP following peripheral nerve
injury is that mRNA and peptide for VIP increases in the
DRG (Neilsch and Keen, 1989), in addition to an increase in
99peptide for VIP in the dorsal horn (McGregor et al., 1984;
Shehab and Atkinson, 1986). The mRNA and peptide for CGRP
does not increase in the DRG, although it does increase in
the motoneurons, as discussed above. The different time
course for VIP expression in the sciatic nerve compared to
the CGRP time course, is consistent with a slower response
in VIP mRNA production in the DRG compared to the
production of CGRP mRNA in the motoneurons (Nielsch an
Keen, 1989; Dumoulin et al., 1991).
Since both CGRP and VIP are normally thought to be
released at the nerve terminals, and have neurotransmitterlike properties, it was intriguing to find that both
peptides are up-regulated and maintained at elevated levels
for a period of time following injury. This raises the
possibility that they have a role in peripheral nerve
regeneration.
There is evidence to suggest that the initial rise in
CGRP expression, shown in this study to have remained
elevated in the distal stump for at least 14 days after
crush, is an important permissive signal for the wave of
Schwann cell proliferation in the distal segment following
injury. This is thought to be via CGRP mediated elevation
of cAMP within Schwann cells (M.Khan, pers.comm.). CGRP may
be released from the regenerating axon tips, triggered by
action potentials travelling down the nerve. The CGRP would
then be able to interact with the Schwann cells in the
100distal stump and, therefore, influence their mitotic
response.
A recent study describing a biphasic time course for CGRP
expression following nerve injury supports a role for CGRP
in both regeneration and at the reinnervated target
(Dumoulin et al., 1991). They describe an early peak in
CGRP mRNA within motoneuron cell bodies following facial
nerve transection, similar to the results of previous
studies (Streit et al., 1989; Noguchi et al., 1990;
Arvidsson et al., 1990). However, Dumoulin et al. (1991)
also observe a second peak 21 days after injury but only if
the nerve is allowed to reinnervate the target (Dumoulin et
al., 1991). This second rise in CGRP, which coincides with
renewed contact of the regenerating axons at the
neuromuscular junction, supports a role for CGRP in
peripheral nerve regeneration. Furthermore, the ability of
CGRP to prevent disuse-induced sprouting of the motor nerve
terminals (Tsujimoto and Kuno, 1988) and to regulate the
expression of chick AChR in vitro (Fontaine et al., 1986;
New and Mudge, 1986) supports the importance of CGRP for
successful reconnection to the target tissue.
This thesis has provided evidence for the ability of CGRP
to regulate macrophage function, as well as Schwann cell
proliferation (M.Khan, pers.comm.), via activation of
adenylate cyclase and production of cAMP. Macrophage
recruitment is one of main events which occurs after
101sciatic nerve injury and is important for regeneration, as
discussed in chapter one. The data presented in this
chapter showed that CGRP produced an accumulation of cAMP
within macrophages isolated from both normal and injured
sciatic nerve. This effect on macrophage cAMP levels,
recently shown in peritoneal macrophages (Vignery et al.,
1991), has important implications for the regulation of the
macrophage response following peripheral nerve injury; such
elevation of cAMP inhibits phagocytosis and IFNy-induced
major histocompatibility (MHO) class II gene product
expression (Figueiredo et al, 1990; Newman et al, 1991).
Furthermore, since regulation of IL-1 expression by
macrophages is influenced by their cAMP levels (Ohmori et
al, 1990), it is possible that CGRP may be important during
the IL-1 dependent stage of NGF production after peripheral
nerve injury (Lindholm et al, 1987).
Although CGRP inhibits macrophage function in vitro (Nong
et al, 1989), its ability to do so may depend upon their
state of activation. It is clear that CGRP did not elevate
cAMP levels within activated peritoneal macrophages to the
same extent as in resident peritoneal macrophages.
Similarly, macrophages isolated from injured vs normal
sciatic nerve are possibly in an activated state and this,
therefore, may be the reason why CGRP failed to produce the
level of cAMP accumulation observed in resident macrophage
populations. Alternatively, macrophages isolated from
injured sciatic nerve may not respond so well to exogenous
102CGRP since they could have previously encountered CGRP
secreted from damaged axons. This is supported by the
observation that preincubation of spleen macrophages with
CGRP inhibited elevation of cAMP levels when CGRP was added
to the cell cultures.
The ability of IFNy to reduce the level of cAMP
accumulated in peritoneal and spleen macrophages in
response to CGRP may reflect the in vivo situation of
lesioned peripheral nerve. The IFNy present in the axons of
the sciatic nerve may be released from the damaged axons
and may influence the state of macrophage activation.
Whilst in such an activated state, they phagocytose and
break down myelin, removing debris which would otherwise
hinder regeneration. During the first two to three days
following injury, CGRP present in the peripheral nerve may
not be able to influence macrophage function, as the
macrophage activating factor, IFNy, is still present, and
may maintain the macrophages in an activated state.
However, the disappearance of IFNy» as early as 3 days
after crush, may allow CGRP released from the regenerating
axons to influence macrophage function via elevation of
cAMP.
VIP-like immunoreactivity in lesioned peripheral nerve
may have a similar role to CGRP since it has been shown to
increase cAMP within blood monocytes (Wiik, 1989). The
secretion of both CGRP and VIP from the axons, as they
103
regenerate back to the target, may be important in
preventing the inflammatory response progressing into an
autoimmune type peripheral neuritis.
Macrophages play a central role in the immune response,
acting as antigen presenting cells (APC) during the
induction stage of an immune response. They are also an
important component of the nonspecific arm of the immune
system, due to their powerful phagocytic and secretory
activities. Their production of an extraordinary variety of
biologically active substances, such as the cytokines IL-1
and tumour necrosis factor (TNF), and acute phase proteins
provides a means by which they can contribute and regulate
many aspects of the immune system. Therefore, the ability
of CGRP in addition to other neuropeptides e.g. VIP, to
regulate cAMP levels of macrophages has important
implications for the regulation of both an inflammatory and
immune response. The observation of CGRP immunoreactive
nerve-like profiles within certain lymphoid organs
including the spleen (Bellinger et al., 1990), together
with the finding of this thesis that CGRP regulated cAMP
levels of spleen macrophages, provides further evidence for
neuroimmune interactions.
Innervation of both primary and secondary lymphoid
tissues and the close association of nerve terminals with
cells of the immune system e.g lymphocytes, suggests that
released neurotransmitters may interact with surrounding
104
cells (Felten et al., 1985; Felten et al., 1987; Felten and
Olschowska, 1987; Felten and Felten, 1990). The presence of
numerous neuropeptides including neurotensin SP, VIP and
CGRP, in nerve-like profiles in primary and secondary
lymphoid organs may also indicate sensory and sympathetic
innervation and neurotransmission by neuropeptides (Felten
et al., 1985; Weihe et al, 1989; Lorton et al, 1990; Lorton
et al, 1991; Bellinger et al, 1990). Substance P positive
fibres have been described to enter the spleen in the hilar
region and arborize along the venous sinuses. Such fibres
are found in the white pulp, in the marginal zone, and in
the outer regions of the periarteriolar lymphatic sheath
(PALS) (Lorton et al., 1991). A similar pattern for
noradrenergic innervation is also observed in the spleen
(Ackerman et al., 1989). Nerve fibres showing positive
immunoreactivity for CGRP have been observed along the
marginal zone of the white pulp of the spleen, and in the
thymus (Bellinger et al., 1990; Weihe et al., 1989). Such
fibres are found in the capsule and intralobular septa of
the thymus.
Neuropeptide or neurotransmitter regulation of the immune
cells may be via direct communication at the cell surface,
if the appropriate receptors are expressed. Receptors for
both VIP and CGRP have previously been described on T
lymphocytes (Ottaway et al., 1984; Umeda and Arisawa,
1989). Furthermore, both neuropeptides inhibit the
proliferation of T-lymphocytes in response to concanavalin
105
A (Boudard and Bastide, 1991). Alternatively, neuropeptides
or neurotransmitters may be able to regulate the immune
response by influencing other cells in the environment in
which the immune cells are situated, thus exerting their
effects indirectly.
The data presented here supports a role for CGRP in nerve
regeneration, regulating one of the most important cells of
the inflammatory and immune response i.e macrophages, which
enter following injury. The ability of CGRP to regulate
macrophage properties, via elevation of cAMP has important
consequences for nerve regeneration. If the macrophage
response is not appropriately controlled then any attempt
to form new myelin could be continuously disrupted due to
its removal via phagocytosis by the recruited macrophages.
Moreover, if class II MHC antigen expression, important for
presentation of exogenous antigens, is not regulated then
myelin phagocytosis could lead to the inappropriate
presentation of myelin proteins in association with class
II MHC antigens. Such presentation of self antigens could
ultimately produce an autoimmune type response, as in
experimental allergic neuritis. This would obviously be
detrimental to any attempt made by the peripheral nervous
system to regenerate.
106
CHAPTER 3
DISTINCT MACROPHAGE PHENOTYPES
IN LESIONED VERSUS UNLESIONED SCIATIC NERVE
107
Introduction.
There is now convincing evidence that the infiltration of
macrophages into a peripheral nerve, in response to injury,
is essential for the normal degeneration and regeneration
of peripheral nerves. The removal of myelin debris by
phagocytosis, and the release of cytokines such as IL-1 are
two of the known roles that macrophages play in
degeneration and regeneration, respectively (Beuche and
Freide, 1984; Lindholm et al., 1987). Beuche and Freide
(1984) demonstrated that if peritoneal macrophages are
prevented from entering a sciatic nerve segment implanted
into the peritoneal cavity, then one fails to see the usual
breakdown and phagocytosis of myelin. Furthermore, the
Schwann cells fail to proliferate in response to the
lesion. Subsequently it was shown that, following
transection of a peripheral nerve, there is a rapid
infiltration of monocytes to the distal nerve segment
(Perry et al., 1987).
Some of the most recent data supporting the importance of
macrophages in peripheral nerve degeneration has been
provided by the discovery of an unusual mouse strain,
C57B1/6/Olac, (Lunn et al., 1989; Perry et al., 1990a and
1990b). It was found that following injury, the sciatic
nerve of C57Bl/6/01ac mice degenerate at a slow rate
compared to other mouse strains (Lunn et al., 1989). This
was subsequently found to correlated with the absence of
108
the normal recruitment of macrophages into peripheral
nerves. Further studies with the C57Bl/6/01ac mouse strain
have shown that a property of the nerve is important in
macrophage recruitment (Perry et al., 1990a).
It would appear that macrophages are not only important
for nerve degeneration but that they also contribute to
certain events of peripheral nerve regeneration. In
particular, macrophages appear to influence the production
of NGF in non-neuronal cells via an IL-1 mediated pathway
(Lindholm et al., 1987). More recently, Brown et al (1991)
found that mRNA levels for both NGF and its receptor only
increased slightly after axotomy of the sciatic nerve in
the C57Bl/01ac mouse when compared to normal and this
correlated well with an impaired sensory nerve
regeneration.
Further support for the importance of macrophages in
peripheral nerve regeneration is provided by Baichwal et
al. (1988). They showed that conditioned medium from
peritoneal macrophages treated with a myelin membrane
fraction could produce a mitogenic signal for cultured
Schwann cells. Furthermore, Schwann cells in the nerves of
C57Bl/6/01ac mice failed to proliferate to the same extent
as in normal mice (Lunn et al., 1989).
The majority of the published studies which have analysed
macrophage recruitment into peripheral nerves after injury
109have used the mouse. Furthermore, macrophages expressing
the phenotypic marker myeloperoxidase (myelomonocytic
cells) have often been analysed in the mouse. However,
there has been, to my knowledge, no documented data
describing similar macrophages in the peripheral nerve of
the rat following peripheral nerve injury.
The discovery in the rat of different macrophage
phenotype markers, the ED protein series, has provided a
method of carrying out more detailed studies of the
distribution of macrophage phenotypes in many rat tissues
including the peripheral nervous system.
The existence of macrophage subpopulations, defined by
the monoclonal antibodies EDI, ED2 and ED3, was originally
described by Dijkstra et al. (1985). They observed that
macrophages recognised by the ED monoclonal antibodies had
a preference for definite compartments within the lymphoid
organs, for example, the cortex of the thymus stained
conspicuously with ED2, whereas the medulla was completely
negative for ED2.
Although EDl-positive macrophages have previously been
visualised during Wallerian degeneration (Stoll et al.,
1989) there is no data, to date, describing the different
subpopulations within the peripheral nervous system before
and/or after injury. Since macrophage phenotypes have been
found to vary in different environments it is important to
110
characterise macrophage phenotypes which may be present in
injured vs uninjured peripheral nerves.
Several monoclonal antibodies which recognise
monocyte/macrophage markers and phenotypes such as the ED
monoclonal antibody series (EDl-3) were used to
characterise macrophage phenotypes in both normal and
lesioned peripheral nerves. This study also used the
macrophage markers to follow the time course and extent of
recruitment into the damaged nerve. Furthermore,
incorporation of bromodeoxyuridine (BrdU) into dividing
cells, in vivo, was used to study whether macrophages found
in lesioned peripheral nerves contribute to the population
of cells which proliferate following injury to peripheral
nerves. Finally, using a polyclonal antibody to SlOO, a
calcium binding protein found in Schwann cells and
frequently used as a phenotypic marker for these cells, the
distribution of SlOO positive cells was correlated with the
distribution of macrophages before and after lesion.
Ill
MATERIALS AND METHODS.
Animal Surgery.Male adult Sprague-Dawley rats were anaethesized with
halothane and the sciatic nerve exposed at mid-thigh level.
The nerve was crushed with a silk suture (2/0 Mersilk). At
various times ranging from 3-18 days postoperative, the
animals were killed and a segment of sciatic nerve,
approximately 12-15mm in length, was removed. Sciatic
nerves from BIO strain mice were also crushed as described
for rats and the nerves removed after 2 or 6 days.
Tissue preparation.After dissection, the nerves were immediately immersed in
4% paraformaldehyde (w/v) in PBS, pH7.4 and fixed overnight
at 4*C. The following day they were transferred to 30%
sucrose (w/v) in PBS for up to 2 hours to prevent crystal
formation within the tissue. The tissue was mounted in OCT
embedding medium (Tissue-tek, Miles) and stored in liquid
nitrogen until sectioned on a freezing microtome (Bright).
BrdU labelling.Sprague-Dawley rats were injected intraperitoneally with
30mg of bromodeoxyuridine (BrdU) (Sigma) 4 days post-crush.
After 1 hour the animals were killed and the sciatic nerve
removed and placed in ice-cold 60% (v/v) ethanol in
112
water, fixing for 20 minutes. The nerve segment was then
transferred to 30% sucrose (w/v) in PBS at 4*C and prepared
as above.
Immunohi stochemi stry.Longitudinal sections of rat sciatic nerve, 10-15um
thick, removed 2 hours, 3, 6 and 18 days post-crush were
treated with immunohistochemistry buffer (IHC) for 1 hour.
The buffer consisted of 4% horse serum (v/v) (Gibco), 1%
Triton-XlOO (v/v), 0.01% sodium azide (v/v) and 20mM L-
lysine in PBS. The sections labelled with BrdU were treated
first with 2M hydrochloric acid (HCl) for 10 minutes and
then O.IM sodium borate (NaB^O? ) , pH8.5, for 5 minutes
before adding the IHC buffer. The sections were then
labelled with one of a selection of mouse monoclonal
antibodies (mcAbs) raised against macrophage antigens
and/or BrdU, or rabbit antiserum to SlOO protein or a mcAb
to the class II MHC antigen (table 1). The primary
antibodies (table 1) were applied overnight at 4‘C in the
IHC buffer. The sections were washed 3 times for 5 minutes
each in the same buffer. The second antibody was
preadsorbed with 15-20% rat serum (v/v) for 30 minutes and
centrifuged to remove precipitate before use. Goat anti
mouse fluoroscein-conjugated antibody was used to visualise
macrophage or class II labelling. Goat anti-mouse rhodamine
conjugated antibody was used to visualise the BrdU label
and sheep anti-rabbit rhodamine conjugated antibody to
visualise SlOO. All antibodies were applied for 45 minutes
113
TABLE 1 : Antibodies used for immunohistochemistry.
ANTIBODY SOURCE ANTIGEN
EDI Di jkstra* Cytoplasmic/membrane
protein
ED2/ED3/ED7“ Di jkstra' Membrane protein
0X42 Seralab
Membrane protein,
complement receptor
(C3bi)
0X1 Seralab Leucocyte common
antigen (LCA)
0X4 Seralab Membrane, MHC
class II antigen
SlOO Sigma Cytoplasmic protein
BU20 Magaud et al 1988 Bromodeoxyuridine
(BrdU), Nuclear
* Antibodies were a gift from Christine Dijkstra.
** ED7 labels granulocytes
114at room temperature. The sections were washed a further 3
times as above and mounted in Citiflour (City University,
U.K.). Sections were viewed with a fluorescence microscope
(Zeiss Universal).
Histochemistry.To visualise monocytes which express endogenous
peroxidase (myeloperoxidase), frozen sections of rat or
mouse sciatic nerve removed 2 or 6 days post-crush were
exposed to 3,3’-diaminobenzidine tetrahydrochloride (DAB)
and 0.01% hydrogen peroxide (v/v) in either phosphate
buffer or Tris-HCl, for 5-6 minutes at room temperature.
The sections were then washed in PBS and mounted in
glycerol.
115
RESULTS.
Myelomonocytic cell distribution in rat and mouse sciatic
nerve after lesion.
A large infiltration of myelomonocytic cells into mouse
sciatic nerve, distal to a lesion site, was detected by the
DAB/HîOj reaction product catalysed by their endogenous
peroxidase (figure 3.1a). In contrast, very few cells
expressing endogenous peroxidase were seen in the
endoneurium in response to either a cut or crush injury to
rat sciatic nerve (figure 3.1b). There was, however, a
high density of peroxidase-positive cells in the sheath of
rat nerves which was comparable to that seen in mouse nerve
sheaths.
Sciatic nerve lesion and macrophage subtypes.
Control unlesioned nerve: A number of macrophages labelled
with either ED3, 0X42 or ED2 were observed in the length of
nerve endoneurium analysed (12mm) (figure 3.2a,b and 3.3a).
Cells expressing the other markers, namely EDI, LCA, and
Class II MHC were rarely seen in the endoneurium (figure
3.3b). The nerve sheath contained macrophages which can be
labelled with the above markers, in the following
approximate proportions: ED2=ED3> OX42>EDl>LCA>ClassII (see
also table 2). ED2 and ED3 positive (ED2' and ED3* ) cells
were the predominant macrophage populations in normal
nerves and their morphology was either ramified or
116
é I
& /«m
I / • • * . /
v ' : . .
.T : . *' '/ 9
1« •
9 •
Figure 3.1: Staining of myeloperoxidase-positive cells in
(a) mouse or (b) rat sciatic nerve, at least 3mm distal to the crush site. The nerves were analysed 6 days post-crush. The diaminobenzidine reaction product appears black. The
scale bar = 70um
117
Figure 3.2: Staining of (a) EDS' and (b) 0X42' macrophages
in the endoneurium of an unlesioned rat sciatic nerve. EDS mAh recognises a membrane antigen and 0X42 recognises the CSbi receptor on the cell surface. Scale bar = 45um
118
Figure 3.3; Staining of (a) ED2* and (b) EDI* macrophages in the endoneurium of an unlesioned rat sciatic nerve. ED2 mAh recognises a membrane antigen where as EDI mAb recognises an antigen located within the cytoplasm, but is also on the
cell surface. Scale bar = 45um
TABLE 2: Summary of macrophage staining and distribution within a 12mm length of sciatic nerve.
EDI ED3 ED2 LCA 0X42 ClassIIControl; Endoneurium occasional sparse sparse occasional sparse occasionalSheath V. sparse sparse sparse occasional sparse occasional
2hr post crush; EndoneuriumSheath
as above as above as abovemany at
crush sitedense
- -
3 day post-crush: Endoneurium proximal (3mm) crush site distal (6mm)
sparse dense(1.2mm) many
manymanysparse
same as ED3 & EDI
same as EDS & EDI
manymany
occasionalSheath (12mm) dense dense dense
6 day post-crush: Endoneurium proximal (3mm) crush site distal (6mm)
as above except, dense at crush site
for 2.4mm as above as above as above* as aboveSheath (12mm) as above*18 day post-crush: Endoneurium (12mm) dense dense* sparse sparse undetected sparseSheath (12mm) sparse many many sparse sparse many
o\
* - less intense
120
amoeboid-like, as was the morphology of EDI* cells. The
majority of stained cells found in the endoneurium,
however, were process-bearing and were close to blood
vessels (figure 3.4a and b).
2 hours post-crush: There was no change in the numbers of
macrophages at this time point compared with undamaged
nerves. There was, however, a large infiltration of
granulocytes around the crush site that was visualised with
ED 7 and also with OXl/LCA monoclonal antibody (mcAb)
(figure 3.5a,b and table 2). There were no ED7* cells in the
endoneurium, either proximal or distal to the lesion, but
there were a surprisingly large number of OXl/LCA* and ED7*
cells in the 12mm length of the nerve sheath, either
proximal and distal to the lesion (figure 3.6a and b).
3 day post-crush: Large numbers of bloated EDI* macrophages
were observed in a 1.2mm area at the crush site (figure
3.7a and table 2), with a gradual decrease in EDI* cells
extending 6-8mm distal ly from the crush site. The EDI, cells
further from the crush site were not swollen to the same
extent as those at the crush site (figure 3.7b). There were
also a number of EDI*, process-bearing cells, proximal to
the crush site (figure 3.8). The sheath contained a large
number of EDI* cells throughout the length of the nerve
examined (12mm). The staining with ED3 antibody showed a
similar cellular distribution compared to EDI (figure
3.9a). In contrast to EDI and ED3, there were fewer ED2*
121
ililii
Figure 3.4: Staining of (a) EDI' macrophages in a blood
vessel with (b) the corresponding phase photograph of the endoneurial blood vessel in a rat sciatic nerve.
Scale bar = SOum
122
'i
Figure 3.5: Staining of (a) ED7* and (b) OXl/LCA* leukocytes found in the endoneurium within 1.2mm of the crush site of a 2 hour post-crush rat sciatic nerve. ED7 mAh recognises a membrane antigen on granulocytes. Scale bar = 45um
123
Figure 3.6; Staining of (a) ED7* and (b) OXl/LCA* leukocytes found in the sheath of a 2 hour post-crush rat sciatic
nerve. Scale bar = 45um
124
r■•* > \. ‘ » V V'
• V * * tA A V . f
4' .. ’ -. V t;
. » t\ »Jv' ♦ i
V '■%
Figure 3.7: Staining of EDI' macrophages in the endoneurium
(a) within 1.2mm of the crush site and (b) 6mm distal to the crush site of a 3 day post-crush rat sciatic nerve. Scale bar = 45um
125
Figure 3.8: Staining of EDI* macrophages found in the
endoneurium, 3mm proximal to the crush site of a 3 daypost-crush sciatic nerve. Scale bar = 45um
126
-A •, f
Figure 3.9; Staining of (a) ED3* and (b) ED2* macrophages inthe endoneurium, within 1 .2mm of the crush site of a 3 daypost-crush rat sciatic nerve. Scale bar = 45um
127macrophages at the crush site or distal to the crush site
(figure 3.9b), although large numbers of the ED2* cells were
found in the sheath. A number of ED2* process-bearing cells
were found proximal to the lesion; these cells were more
intensely labelled than those at the crush site (figure
3.10). The 0X42 mcAb and OXl/LCA mcAb labelled macrophages
with a similar distribution to ED3 and EDI except that
there were very few LCA* cells distal to the crush (figure
3.11a and b) . The LCA* macrophages found in the sheath,
however, had a different morphology (rounded, lymphocyte
like) than with ED3* or 0X42* cells. Class II MHO antigens
labelled with 0X4 were found on a small number of cells
proximal to the lesion with increasing numbers towards the
site of lesion, but only a small number of cells expressed
class II around the site of crush and distally (figure
3.12); a large number of cells in the sheath expressed
class II antigens. Surprisingly, ED7 labelled only a few
cells around the crush site at this time.
6 day post-crush: The general distribution of EDI*
macrophages had not changed significantly from that of 3
days post-crush (table 2), except the mass accumulation of
cells around the crush site extended further distally
(2.4mm compared with 1.2mm at 3 days). The distribution of
ED3 was similar to that above, but the intensity of the
staining had reduced with time. A small number of ED2*
macrophages were found at the crush site and distally;
there were still a number of ED2*cells in the sheath, but
128
M ' * ' i '
\ \V '
Figure 3.10: Staining of ED2* macrophages in theendoneurium, 3mm proximal to the crush site of a 3 day
post-crush sciatic nerve. Scale bar = 45um
129
Figure 3.11: Staining of (a) 0X42* and (b) OXl/LCA*
macrophages within 1.2mm of the crush site in the endoneurium of a 3 day post-crush rat sciatic nerve.Scale bar = 45um
130
Figure 3.12: Staining of 0X4/MHC classll* macrophages in theendoneurium, within 1.2mm of the crush site of a 3 daypost-crush rat sciatic nerve. Scale bar = 45um
131less than the number of EDI* macrophages. 0X42 and OXl/LCA
labelled cells at the crush site, but like EDS, the
staining intensity had decreased. A few 0X42* cells were
found distal to the site of lesion. Finally, the number of
class II positive cells (0X4*) was similar to the earlier
time point and there were no ED7* cells found in the nerve
at this time.
18 day post-crush; A large number of EDI* and EDS*
macrophages extended throughout the length of the
endoneurium examined (12mm) (figure S.lSa,b and table 2).
This contrasts with a very low number of ED2* cells in the
endoneurium (figure S.14). The intensity of the EDS*
immunoreactivity had decreased further compared with 6 days
post-crush. There were, however, brightly labelled ED2* and
EDS* macrophages found in the nerve sheath (figure S.15a,b)
but a surprisingly low number of EDI* macrophages in the
sheath (figure S.16). There were a small number of small
round LCA* cells scattered in the endoneurium and in the
sheath, but the staining intensity was very low. The 0X42
labelling on the macrophages in the endoneurium was
virtually undetectable.
SlOO staining in unlesioned versus lesioned sciatic nerve.
Lesioning the sciatic nerve produced a very disorganised
SlOO staining pattern after S days compared to a normal
nerve; this loss of SlOO immunoreactivity was observed
around the lesion site extending for a short distance
132
Figure 3.13: Staining of (a) EDI* and (b ) ED3* macrophages in the endoneurium of an 18 day post-crush rat sciatic nerve. Scale bar = 45um
133
Figure 3.14; Staining of ED2* macrophages in the endoneurium
of an 18 day post-crush rat sciatic nerve. Scale bar = 45um
134
Figure 3.15: Staining of (A) ED3' and (B) ED2* macrophages in the sheath of an 18 day post-crush rat sciatic nerve.
Scale bar = 45um
135
t
Figure 3.16: Staining of EDI* macrophages in the sheath of
an 18 day post-crush rat sciatic nerve. Scale bar = 45um
136distally after 3 or 6 days post-crush. The lack of SlOO
staining corresponds with the mass accumulation of EDI* and
ED3* macrophages (figures 3.17a,b and 3.18a,b). SlOO at 18
days post-crush is virtually absent in the 12mm of nerve
analysed, with only discrete areas of staining observed
(figure 3.19a and b)
BrdU labelling in crushed sciatic nerve.
A large number of BrdU* cells were found around the crush
site (figure 3.20a). However, the majority of EDI*
macrophages found around the crush site were not BrdU*
(figure 3.20b). Only a very small number EDI* macrophages
found in the sciatic nerve after injury expressed BrdU.
Proliferating Schwann cells were the main cell type
expressing BrdU.
137
Figure 3.17: Staining of (a) EDI* macrophages with (b) the
corresponding SlOO staining in the endoneurium within 1.2mm of the crush site of a 6 day post-crush rat sciatic nerve. Scale bar = 45um
138
Figure 3.18: Staining of (a) EDI* macrophages with (b) the
corresponding SlOO staining in the endoneurium 6mm distal to the crush site of a 6 day post-crush rat sciatic nerve. Scale bar = 45um
139
■ V .. .Î-' ■
, -•: ,, * ’
Figure 3.19: Staining of (a) EDI* macrophages with (b) the
corresponding SlOO staining in the endoneurium of an 18 daypost-crush rat sciatic nerve. Scale bar = 45um
140
Figure 3.20: Staining of (a) BrdU* cells and (b) EDI* andBrdU* cells 2mm distal to the crush site of a 4 day postcrush rat sciatic nerve. Scale bar = 45um
141DISCUSSION»
Previous data has suggested that the classical
inflammatory response, that of early migration of
granulocytes into an inflammatory lesion, is muted in the
distal segment of a lesioned peripheral nerve injury
(reviewed by Perry and Brown, 1992). My data, however,
supports the presence of a classical inflammatory response
confined to the crush site. Although a large number of ED7*
granulocytes were recruited to the epineurial nerve tissue,
both proximal and distal to the crush, they failed to move
into the endoneurial tissue. The large number of
granulocytes in the epineurium could be caused by increased
blood supply to the nerve. Since these cells do not move
into the endoneurium, except for an area in and around the
crush site (which probably reflects damaged blood vessels)
they may not have a direct role in degeneration.
Conversely, large numbers of different macrophages
phenotypes/subpopulations were found in the epineurium and
endoneurium.
The distribution of ED3* , 0X42* and LCA* cells in the
lesioned sciatic nerve suggests the existence of one
population of macrophages expressing the three recognised
antigens on the cell surface. This could be further
analysed by double labelling. When the expression of the
C3bi receptor (0X42) and the leucocyte common antigen
(LCA/OXl) was monitored over a period of 18 days after
142
crush, there was a significant down regulation of both
antigens. The expression of the C3bi receptor is related to
the adhesive and phagocytic properties of macrophages as
well as their activation (Ding et al., 1987; Arnaout et
al., 1983; Brück and Friede, 1990). Therefore, it would
seem that the observed increase in expression of this
membrane protein following axotomy correlates with the
requirement to move into the endoneurium after injury and
the removal of myelin debris. Moreover, macrophage
recruitment is prevented in lesioned mouse sciatic nerve by
the antibody '5C6’ which, like 0X42, recognises the C3bi
receptor (Robinson et al., 1986). Down regulation and
virtual loss of C3bi receptor expression, demonstrated in
this study, may correspond with a decrease in phagocytosis
of myelin debris and a change in the activation state of
this macrophage subpopulation. Subsequently, the process of
regeneration could proceed as myelin phagocytosis is
reduced.
The response of macrophages to activation is dependant
upon the type of stimulus. For example, macrophages
activated by either 1ipopolysaccharide (LPS) or IFNy affect
gene expression differently (Yu et al., 1990), also
thyoglicolate-, peptone-, and con A-elicited macrophages
respond differently when IL-4 or IFNy is added to the cell
cultures (Molina and Huber, 1991). It seems that, although
the macrophages within an injured nerve are in an activated
state as they phagocytose myelin debris, the majority of
143
macrophages in the endoneurium fail to express class II
antigens. This is also observed in the mouse sciatic nerve
and the rat optic nerve (Beuche and Friede, 1986; Stoll et
al., 1989). In contrast, a large number of class II
expressing cells were present in the sheath of lesioned
sciatic nerves. The low level of class II expression in the
endoneurium may be due to either active inhibition of
expression, or alternatively activated macrophages occupied
with phagocytosing myelin do not trigger the necessary
machinery to cause class II expression. However, the fact
that a large number of class II expressing cells are found
in the perineurium with only very few in the endoneurium,
suggests that they may lose their ability to express class II antigens as they migrate into the endoneurium, or some
factor released from the lesioned axons is actively down-
regulating class II antigen expression. Such absence of
class II expression may be beneficial to the process of
nerve regeneration. If the macrophages that are
phagocytosing the myelin debris did express class II
antigens, then myelin could become processed within the
macrophage, with certain myelin proteins eventually
presented on the cell surface. This would lead to an immune
type inflammatory response and therefore prevent nerve
regeneration, since the nerve would be under constant
bombardment from the immune system. This may bear some
relation to some neurological diseases, such as multiple
sclerosis, which are thought to have an immunological
aspect to the pathology.
144
Although a normal sciatic nerve contains more ED3* than
EDI* macrophages, lesioning produced a similar accumulation
and distribution of both EDI* and ED3* macrophages in the
sheath and endoneurium of the sciatic nerve; the mass
accumulation of macrophages in endoneurium results from the
migration of EDI*,ED3‘,ED2‘ monocytes from the blood stream.
This is supported by the failure to observe significant
numbers of dividing macrophages, measured by BrdU
labelling. The distribution of EDI* and ED3* macrophages
within a lesioned sciatic nerve suggests that the
environment of the nerve is influencing macrophage
phenotype, causing the expression of ED3 by the cells as
they move into the tissue; this supports previous data
which suggests that environmental factors influence
macrophage phenotype (reviewed by Perry and Brown, 1992).
Moreover, since I have shown that the number of ED2*
macrophages increases to a lesser extent after lesioning,
it is possible that ED3* and EDI* macrophages are more
suited to the environment of a degenerating nerve, than is
the separate subpopulation of ED2* macrophages. In contrast,
no ED3* macrophages are found in the sciatic nerve during
the immune mediated model of experimental allergic neuritis
(Stevens et al., 1989). However, EDI* and ED2* macrophages
are present in increased numbers during experimental
allergic neuritis (Stevens et al., 1989). This data brings
into question the importance of the ED3* macrophage
subpopulation during peripheral nerve injury. It also
supports the hypothesis that the environment can influence
145
macrophage phenotype and that the expression of certain
antigens reflect a particular function.
The bloated morphology of all macrophage types around the
crush site, compared with a ramified/spindly appearance
proximally, probably reflects the phagocytosis of myelin.
This is supported by the observation of a reduction in
intensity of all ED proteins analysed, on or in the
macrophages around the crush site, compared with those
found proximally. This may reflect a constant renewal of
cell membrane during phagocytosis.
In addition to the ED macrophage phenotypic markers, the
presence of myeloperoxidase within monocytes
(myelomonocytic cells) is another marker routinely used to
detect the presence of macrophages in both mice and rat
tissue. However, the data presented in this chapter has
shown that the distribution of myelomonocytic cells found
in injured sciatic nerves of mice was significantly
different compared to rats. Although there was a low number
of myelomonocytic cells recruited into the peripheral
nervous system in the rat, compared with mouse sciatic
nerve, this does not appear to affect phagocytosis and
removal of myelin. This brings into question the
significance of the large number of myelomonocytic cells
found in the mouse sciatic nerve after injury. It is not
clear what importance, if any, the presence of
myeloperoxidase in these cells has for nerve degeneration
146
and regeneration.
The distribution of SlOO immunoreactivity observed in
this study correlates with the extent of nerve
degeneration. SlOO protein was originally isolated and
purified from bovine and rabbit brains and is used as a
marker for cultured Schwann cells (Moore, 1965; Spreca et
al., 1989). It is not clear whether the reduction in SlOO
protein levels observed after injury is related directly to
the loss of axonal contact of the Schwann cells or to the
action of macrophages. Loss of SlOO immunoreactivity
occurred within 3 days after nerve lesion and although the
loss was restricted to only a small area around the injury
site, it coincided with the presence of large numbers of
bloated macrophages. As the macrophage staining extended
along the nerve with time, so the SlOO was observed to
decline in the same direction, with only patches of SlOO
found 18 days after a crush. It is possible that
macrophages may cause the decrease in protein either
directly, by secretion of a cytokine, or indirectly via
disrupting the surrounding environment. Although down
regulation of SlOO mRNA has been observed after sciatic
nerve lesion (De Leon et al., 1991), there is conflicting
data concerning the regulation of SlOO expression after
injury. Spreca et al. (1989) found that crushing did not
affect the intensity or localisation of SlOO but a decrease
was seen after cutting the nerve. The reason for the
discrepancy between the data presented in this chapter as
147
well as that of Kate and Saroh (1983), with that of Spreca
et al. (1989) is not entirely clear. One possibility is
that the extent to which the nerve is damaged may be
important.
This study has provided evidence for the existence of
different macrophage phenotypes/subpopulations within the
peripheral nervous system. It has also described how crush
injury to the peripheral nervous system alters the
distribution of the macrophage phenotypes. Although similar
macrophage phenotypes, defined by the ED markers, have also
been observed in the central nervous system (Sminia et al.,
1987), it still remains unclear as to whether this is a
reflection of different macrophage activation states or
whether separate macrophage populations perform different
functions. Furthermore, the expression of ED3 by recruited
macrophages may be important during peripheral nerve injury
but appears not be required during immune mediated
experimental allergic neuritis (Stevens et al., 1989).
Further studies are required to determine the function of
the ED3* macrophages following peripheral nerve injury.
148
CONCLUSIONS
149
Communication between the nervous system and the immune
system has been hypothesised for a number of years. The
presence of receptors for neuropeptides, along with the
identification of nerve fibres within primary and secondary
lymphoid tissue support such a hypothesis. This thesis has
provided further evidence of communication between elements
of the nervous system and the immune system. It has shown
how a component of the inflammatory response, produced as
a consequence of a peripheral nerve lesion, may be
regulated by one of a number of neuropeptides expressed at
elevated levels after injury.
Lesioning of a peripheral nerve leads to a inflammatory
response that can be split into two major events: 1) the
classical inflammatory response at the site of injury with
the granulocytes found at the site within hours of
lesioning the nerve but with no granulocytes recruited into
the endoneurium, distal to the crush; 2) a recruitment of
distinct macrophage phenotypes/subpopulations throughout
the distal segment of the lesioned nerve, which occurs at
a later time point of 3-4 days after injury. This thesis
has provided evidence that the neuropeptide, CGRP, observed
within a subset of axons in the sciatic nerve, is an
important regulatory factor of the macrophage response to
peripheral nerve injury. Furthermore, the time course of
expression of the lymphokine, IFNy, provided by this
thesis, along with documented evidence that IFNy influences
macrophage recruitment (Mebius et al., 1990; Sethna and
150
Lampson, 1991), suggests a potential role for IFNy in
macrophage recruitment.
Gamma interferon was thought be expressed only within the
immune system. During an immune response it is produced and
released by the CD4* subset of T cells. Its production by
certain neurons in the central and peripheral nervous
system (Ljungdahl et al., 1989; Olsson et al, 1989; Kiefer
and Kreutzberg, 1990) and its transportation along the
axons was intriguing. Subsequently it was suggested that
IFNy, may be considered as a new member of the neuropeptide
family. Moreover, the pattern of IFNy-like immunoreactive
fibres observed within the spleen was found to resemble
tyrosine hydroxylase-positive fibres which were previously
proposed to be important in neuronal control of immune cell
activity. Activation of macrophages by gamma interferon has
been known for a considerable period. Increase in both
macrophage HzOj production and MHC class II expression are
two of the changes which take place as a consequence of the
addition of IFNy to macrophages. In addition to these
direct effects upon macrophages, strong evidence exists to
support the above finding that recruitment of both
lymphocytes and monocytes/macrophages into injury sites is
influenced by IFNy* Injection of IFNy into rat popliteal
lymph nodes, which has the afferent lymphatic severed,
mediated an accumulation of EDI* macrophages into the
operated node (Mebius et al., 1990). A similar study has
also shown that intracerebral injection of IFN-y caused the
151
recruitment of 0X42' macrophages and CD4' T cells into the
brain parenchyma (Sethna and Lampson, 1991). The ability of
IFNy to influence lymphocyte traffic (Issekutz et al.,
1988) has been shown to involve the regulation of MECA-325,
an antigen specific for endothelial cells involved in the
above trafficking (Duijvestijn et al., 1986).
Although there is no documented data supporting IFNy as
a chemotactic factor for macrophages, there is data
suggesting that IFNy regulates the expression of adhesion
proteins found on macrophage and endothelial cells (Dustin
et al., 1986; Blanchard and Djeu, 1991). One such adhesion
molecule is intercellular adhesion molecule (I-CAM). During
an inflammatory response I-CAM expression on endothelial
cells increases. Subsequently it was found that a number of
inflammatory cytokines, including IL-1 and IFNy caused an
increase in I-CAM expression on endothelial cells (Dustin
et al., 1986). One of the ligands for I-CAM is leukocyte
function-associated antigen-1 (LFA-1), also referred to as
CDlla/CD18. This antigen appears to be essential for the
migration of inflammatory cells into sites of inflammation.
The expression of this antigen on macrophages is increased
by IFNy (Blanchard and Djeu, 1991). Therefore, it is
possible that IFNy in the axons is secreted from the
damaged axons after nerve injury, thus causing an increase
in expression of the above adhesion molecules. This would,
consequently, allow the macrophages to migrate into the
endoneurium of the sciatic nerve. Since IFNy-like
152
immunoreactivity is not restricted to lymphoid organs it is
possible that it regulates inflammatory processes in
peripheral tissue.
The finding that CGRP can prevent macrophage activation
(Nong et al., 1989) supports the hypothesis that it
regulates the macrophage response after peripheral nerve
injury. Although the simultaneous addition of CGRP and IFNy
to macrophage cultures fails to inhibit macrophage H Oz
production, a marker for activation, preincubation with
CGRP inhibited the ability of IFNy to activate the cells
(Nong et al., 1989). Since CGRP remained expressed at a
high level in the distal stump for a significantly longer
period than IFNy, constant release of CGRP around the
macrophages, as the axons regenerate, may lead to the
eventual down regulation of the macrophage activation
state. Potentially, CGRP could regulate macrophages
recruited into the injured nerve via increasing macrophage
intracellular cAMP levels. The inhibition of both
macrophage phagocytosis and class II MHC expression has
been linked with increasing intracellular cAMP (Eppell et
al., 1989; Figueiredo et al., 1990; Newman et al., 1991).
Moreover, the small number of class II expressing cells in
the endoneurium, compared to a large number of such cells
in the sheath following trauma, may be related to CGRP
expression following peripheral nerve injury. Since IFNy
increases class II MHC expression on macrophages and it was
present at elevated levels for a short while after nerve
153
injury, then it is possible that an inhibitory signal is
present in the nerve to prevent over expression of such
class II in the endoneurium. This inhibitory signal, like
CGRP and VIP, probably acts via an ability to elevate cAMP.
If nerve regeneration is going to be successful then
regulation of the inflammatory and immune response could be
essential. Regeneration of the peripheral nervous system
appears to be one particular situation where regulation of
the most influential cell type of the immune system, the
macrophage, may be essential for reformation of a
peripheral nerve. Furthermore, since macrophages are
fundamental to the immune response, then regulation of
macrophage function in the nervous system following trauma
is probably essential if autoimmunity within the peripheral
nervous system is to be prevented. This thesis has provided
data supporting the ability of CGRP, present in a lesioned
peripheral nerve, to prevent such autoimmunity within the
PNS by its capacity to regulate the response of recruited
macrophages via elevation of cAMP.
However, CGRP is not only important for regulation of the
macrophage response. It also seems to have a role in the
Schwann cell proliferative response following injury.
Furthermore, with its vasodilatory properties and its
ability to regulate the expression of AChR on muscle, CGRP
can be classed as a pleiotropic factor. The ability of
other neuropeptides, like VIP, to interact with different
154
cell types including immune cells, expands the regulatory
role of neuropeptide secretion from the nervous system.
Neuropeptides can no longer be thought of as simple
chemical transmitters.
155
REFERENCES
Abercrombie, M. and Johnson, M.L. (1946) Quantitative
histology of Wallerian degeneration I. Nuclear population
in rabbit sciatic nerve. Journal of Anatomy 80:37-50.
Ackerman, K.D., Felten, S.Y., Dijkstra, C.D., Livnat, S.
and Felten, D.L. (1989) Parallel development of
noradrenergic innervation and cellular compartmentation in
the rat spleen. Experimental Neurology 103:239-255.
Aebischer, P., Guénard, V., Winn, S.R., Valentini, R.F.
and Galletti, P.M. (1988) Blind-ended semipermeable
guidance channels support peripheral nerve regeneration in
the absence of a distal nerve stump. Brain Research
454:179-187.
Aebischer, P., Salessoitis, A.N. and Winn, S.R. (1989)
Basic fibroblast growth factor released from synthetic
guidance channels facilitates peripheral nerve regeneration
across long nerve gaps. Journal of Neuroscience Research
23:282-289.
Alberts, B., Bray, D., Lewis, J ., Raff, M.C., Roberts,
K. and Watson, J.D. (1989) Molecular biology of the cell,
New York:Garland publishing Inc., Ed. 2nd pp. 1062-1063.
156
Alitalo, K., Hovi, T. and Vaheri, A. (1980) Fibronectin is
produced by human macrophages. Journal of Experimental
Medicine 151:602-613.
Amara, S.G., Arriza, J.L., Leff, S.E., Swanson, L.W.,
Evans, R.M. and Rosenfeld, M.G. (1985) Expression in brain
of a messenger RNA encoding a novel neuropeptide homologous
to calcitonin gene-related peptide. Science 229:1094-1097.
Amara, S.G., Jonas, V., Rosenfeld, M.G., Ong, E.S. and
Evans, R.M. (1982) Alternative RNA processing in calcitonin
gene expression generates mRNAs encoding different
polypeptide products. Nature 298:240-244.
Anderson, P.N. and Turmaine, M. (1986) Peripheral nerve
regeneration through grafts of living and freeze-dried CNS
tissue. Neuropathology and Appl led Neurobiology 12:38 9-399.
Arnaout, M.A., Todd III, R.F., Dana, N. , Melamed, J.,
Schlossman, S.F. and Colten, H.R. (1983) Inhibition of
phagocytosis of complement C3- or immunoglobulin G-coated
particles and of C3bi binding by monoclonal antibodies to
a monocyte-granulocyte membrane glcoprotein (Mo-1). Journal
of Clinical Investigation 72:171-179.
Arvidsson, J., Ygge, J. and Grant, G . (1986) Cell loss
in lumbar dorsal root ganglia and transganglionic
157degeneration after sciatic nerve resection in the rat.
Brain Research 373:15-21.
Arvidsson, U. , Johnson, H. , Piehl, F. , Cullheim, S.,
Hokfelt, T., Risling, M. , Terenius, L. and Ulfhake, B.
(1990) Peripheral nerve section induces increased levels of
Calcitonin Gene-related Peptide (CGRP)-like
immunoreactivi ty in axotomized motoneurons. Experimental
Brain Research 79:212-216.
Assouline, J.G., Bosch, P., Lim, R., Kim, I.S., Jensen,
R. and Pantazis, N.J. (1987) Rat astrocytes and Schwann
cells in culture synthesize nerve growth factor-like neurite-promoting factors. Developmental Brain Research
31 ; 103-118 .
Baichwal, R.R., Bigbee, J.W. and DeVries, G.H. (1988)
Macrophage-mediated myelin-related mitogenic factor for
cultured Schwann cells. Proceedings of the National Academy
of Sciences USA 85:1701-1705.
Bellinger, D.L., Lorton, D. , Romano, T.D., Olschowka,
J.A., Felten, S.Y. and Felten, D.L. (1990) Neuropeptide
innervation of lymphoid organs. Annals of the New York
Academy of Science 594:17-33.
158
Benfey, M. and Aguayo, A.J. (1982) Extensive elongation
of axons from brain into peripheral nerve grafts. Nature
296:150-152.
Berry, M., Rees, L., Hall, S., Yui, P. and Sievers, J.
(1988) Optic Axons Regenerate Into Sciatic Nerve Isografts
Only in the Presence of Schwann Cells. Brain Research
Bulletin 20:223-231.
Beuche, W. and Friede, L. (1986) Myelin phagocytosis in
Wallerian degeneration of peripheral nerves depends on
silica-sensitive, bg/bg-negative and Fc-positive monocytes.
Brain Research 378:97-106.
Beuche, W. and Friede, R.L. (1984) The role of
non-resident cells in Wallerian degeneration. Journal of
Neurocytology 13:767-796.
Bigbee, J.W., Yoshino, J.E and DeVries, G.H. (1987)
Morphological and proliferative responses of cultured
Schwann cells following rapid phagocytosis of a
myelin-enriched fraction. Journal of Neurocytology
16:487-496.
Bisby, M.A. and Pollock, B. (1983) Increased
regeneration rate in peripheral nerve axons following
double lesions: Enhancement of the conditioning lesion
phenomenon. Journal of Neurobiology 14(6):467-472.
159
Bixby, J.Lw Lilien, J. and Reichardt, L.F. (1988)
Identification of the major proteins that promote neuronal
process outgrowth on Schwann cells in vitro. The Journal of
Cell Biology 107:353-361.
Blanchard, D.K and Djeu, J.Y. (1991) Differential
modulation of surface antigens on human macrophages by
IFN-gamma and GM-CSF: Effect on susceptibility to LAK
lysis. Journal of Leukocyte Biology 50:28-34.
Bonnekoh, P.G.H., Scheidt, P. and Friede, R.L. (1989)
Myelin phagocytosis by peritoneal macrophages in organ
cultures of mouse peripheral nerve. A new model for
studying myelin phagocytosis in vitro. Journal of
Neuropathology and Experimental Neurology 48(2):140-153.
Boudard, F. and Bastide, M. (1991) Inhibition of mouse
T-cell proliferation by CGRP and VIP: effects of these
neuropeptides on IL-2 production and cAMP synthesis.
Journal of Neuroscience Research 29:29-41.
Brown, M.G., Perry, H.V., Lunn, E.R., Gordon, S. and
Heumann, R. (1991) Macrophage Dependence of Peripheral
Sensory Nerve Regeneration: Possible Involvement of Nerve
Growth Factor. Neuron 6:359-370.
160
Brück, W. and Friede, R.L. (1990) Anti-macrophage CR3
antibody blocks myelin phagocytosis by macrophages in
vitro. Acta Neuropathologica 80:415-418.
Bunge, R.P. (1987) Tissue culture observations relevant
to the study of axon-Schwann cell interactions during
peripheral nerve development and repair. Journal of
Experimental Biology 132:21-34.
Chiu, S.Y. and Wilson, G.F. (1989) The role of potassium
channels in Schwann cell proliferation in Wallerian
degeneration of explant rabbit sciatic nerves. Journal of
Physiology 408:199-222.
Clemence, A., Mirsky, R. and Jessen, K.R. (1989)
Non-myelin-forming Schwann cells proliferate rapidly during
Wallerian degeneration in the rat sciatic nerve. Journal of
Neurocytology 18:185-192.
Danielsen, N., Pettmann, B., Vahlsing, H.L., Manthorpe,
M. and Varon, S. (1988) Fibroblast growth factor effects on
peripheral nerve regeneration in a silicone chamber model.
Journal of Neuroscience Research 20:320-330.
Daniloff, J.K., Levi, G . , Grumet, M. , Rieger, F . and
Edelman, G.M. (1986) Altered expression of neuronal cell
adhesion molecules induced by nerve injury and repair. The
Journal of Cell Biology 103:929-945.
161
David, S., Bouchard, C ., Tsatas, 0. and Giftochristos,
N. (1990) Macrophages can modify the nonpermissive nature
of the adult mammalian central nervous system. Neuron
5: 463-469.
Davis, J.B. and Stroobant, P. (1990) Platelet-derived
growth factors and fibroblast growth factors are mitogens
for rat Schwann cells. Journal of Cell Biology
110:1353-1360.
De Leon, M. , van Eldik, L.J. and Shooter, E.M. (1991)
Differential regulation of SIOOB and mRNAs coding for
SlOO-like proteins (42A and 420 during development and
after lesion of rat sciatic nerve. Journal of Neuroscience
Research 29:155-162.
Diamond, J., Holmes, M. and Coughlin, M. (1992)
Endogenous NGF and Nerve impulses regulate the collateral
sprouting of sensory axons in the skin of the adult rat.
The Journal of Neuroscience 12(4):1454-1466.
Diamond, M.S., Staunton, D.E., de Fougerolles, A.R.,
Stacker, S.A., Garcia-Aguilar, J. , Hibbs, M.L. and
Springer, T.A. (1990) ICAM-1 (CD54): A counter receptor for
Mac-1 (GDIlb/CD18 ) . The Journal of Cell Biology
111:3129-3139.
162
Dijkstra, C.D., Dopp, E.A., Joling, P. and Kraal, G.
(1985) The heterogeneity of mononuclear phagocytes in the
lymphoid organs; distinct macrophage subpopulations in the
rat recognized by monoclonal antibodies EDI, ED2 and ED3.
Immunology 54:589-599.
Dimaline, R. and Dockray,G.J. (1982) Molecular forms of
VIP in normal tissue. In "Vasoactive intestinal peptide
S.I. Said, ed, pp 23-33. Raven Press, New York.
Ding, A., Wright, S.D. and Nathan, C. (1987) Activation
of mouse peritoneal macrophages by monoclonal antibodies to
Mac-1 (complement receptor type 3). Journal of Experimental
Medicine 165:733-749.
DiStefano, P.S. and Johnson, E.M.,Jr. (1988 ) Nerve
growth factor receptors on cultured rat Schwann cells.
Journal of Neuroscience 8(1):231-241.
Duijvestijn, A.M, Schreiber, A.B. and Butcher, E.G.
(1986) Interferon- regulates an antigen specific for
endothelial cells involved in lymphocyte traffic.
Proceedings of the National Academy of Science USA
83:9114-9118.
Dumoulin, F.L., Raivich, G., Streit, W.J. and
Kreutzberg, G.W. (1991) Differential regulation of
163
Calcitonin Gene-related Peptide (CGRP) in regenerating rat
facial nucleus and dorsal root ganglion. European Journal
of Neuroscience 3:338-342.
Dustin, M.L., Rothlein, R. , Bhan, A.K., Dinarello, A.
and Springer, T.A. (1986) Induction by IL-1 and interferon-
: Tissue distribution, biochemistry, and function of a
natural adherence molecule (ICAM-1). The Journal of
Immunology 137(1):245-254.
Eppell, B.A., Newell, A.M. and Brown, E.J (1989)
Adenosine receptors are expressed during differentiation of
monocytes to macrophages in vitro. Journal of Immunology
143:4141-4145.
Farel, P.B. (1989) Naturally occurring cell death and
differentiation of developing spinal motoneurons following
axotomy. The Journal of Neuroscience 9:2103-2113.
Felten, D.L., Felten, S.Y., Carlson, S.L., Olschowka,
J.A. and Livnat, S. (1985) Noradrenergic and peptidergic
innervation of lymphoid tissue. The Journal of Immunology
135 :755s-765s.
Felten, D.L., Ackerman, K.D., Wiegand, S.J. and Felten,
S.Y. (1987) Noradrenergic sympathetic innervation of the
spleen: I. Nerve fibers associated with lymphocytes and
164
macrophages in specific compartments of the splenic white
pulp. Journal of Neuroscience Research 18:28-36.
Felten, D.L. and Olschowka, J. (1987) Noradrenergic
sympathetic innervation of the spleen: II. Tyrosine
hydroxylase (TH)-positive nerve terminals form synaptic-
like contacts on lymphocytes in the splenic white pulp.
Journal of Neuroscience Research 18:37-48.
Felten, S.Y. and Felten, D.L. Psychoneuroimmune 1ogy,
London : Academic Press, Inc., 1990. Ed. 2nd pp. 27-61.
Figueiredo, F., Uhing, R.J., Okonogi, K., Gettys, T.W.,
Johnson, S.P., Adams, D.O. and Prpic, V. (1990) Activation
of the cAMP cascade inhibits an early event involved in
murine macrophage la expression. Journal of Biological
Chemistry 265:12317-12323.
Fitzgerald, M., Wall, P.D., Goedert, M. and Emson, P.C.
(1985) Nerve growth factor counteracts the
neurophysiological and neurochemical effects of chronic
sciatic nerve section. Brain Research 332:131-141.
Fontaine, B., Klarsfeld, A., Hokfelt, T. and Changeux,
J-P. (1986) Calcitonin gene-related peptide present in
spinal cord motoneurons, increases the number of
acetylcholine receptors in primary cultures of chick embryo
myotubes. Neuroscience Letters 71:59-65.
165
Foreman, J. and Jordan, C. (1983) Histamine release and
vascular changes induced by neuropeptides. Agents and
Actions 13:105-116.
Foreman, J.C. and Jordan, C.C. (1984) Neurogenic
inflammation. Trends in Pharmacology 5:116-119.
Freid, G . , Franck, J. , Brodin, E. , Born, W. , Fischer,
J.A., Hiort, W. and Hokfelt, T. (1989) Evidence for
differential storage of calcitonin gene-related peptide,
substance P and serotonin in synaptosomal vesicles of rat
spinal cord. Brain Research 499:315-324.
Fuji, K . , Senba, E. , Ueda, Y. and Tohyama, M. ( 1983)
Vasoactive intestinal polypeptide (VIP)-containing neurons
in the spinal cord of the rat and their projections.
Neuroscience Letters 37:51-55.
Fuxe, K . , Hokfelt, T., Said, S.I. and Mutt, V. ( 1977)
Vasoactive intestinal polypeptide and the nervous system:
immunohistochemical evidence for localisation in central
and peripheral neurons, particularly intracortical neurons
of the cerebral cortex. Neuroscience Letters 5:241-246.
Gamse, R. , Posch, M. , Saria, A. and Jansco, G. ( 1987 )
Several mediators appear to interact in neurogenic
inflammation. Acta Physiologica Hungary 69:343-354.
166Giachetti/ A. and Said, S.I. (1979) Axonal transport of
vasoactive intestinal peptide in sciatic nerve. Nature
281:574-575.
Gibson,S.J., Polak, J.M., Bloom, S.R., Sabate, I.M.,
Mulderry, P.M., Ghatei, R.A., McGregor, G.P., Morrison,
J.F.B., Kelly, J.S., Evans, R.M. and Rosenfeld, M.G. (1984)
Calcitonin gene-related peptide immunoreactivity in the
spinal cord of man and eight other species. The Journal of
Neuroscience 4: 3101-3111.
Gibson, S.J., Polak, J.M., Giaid, A., Hamid, Q.A., Kar,
S., Jones, P.M., Denny, P., Legon, S., Amara, S.G., Craig,
R.K., Bloom, S.R., Penketh, R.J.A., Rodek, C., Ibrahim,
N.B.N. and Dawson, A. (1988) Calcitonin gene-related
peptide messenger RNA is expressed in sensory neurones of
the dorsal root ganglia and also in spinal motoneurones in
man and rat. Neuroscience Letters 91:283-288.
Gorin, P.D. and Johnson, E.M. (1979) Experimental
autoimmune model of nerve growth factor deprivation:
effects on developing peripheral sympathetic and sensory
neurons. Proceedings of the National Academy of Science USA
79 ( 10): 5382-5386.
167
Graeber, M.B., Raivich, G. and Kreutzberg, G.W. (1989)
Increase of transferrin receptors and iron uptake in
regenerating motor neurons. Journal of Neuroscience
Research 23:342-345.
Gulbenkian, S., Merighi, A., Wharton, J., Varndell, I.M.
and Polak, J.M. (1986) U1trastructural evidence for the
coexistence of calcitonin gene-related peptide and
substance P in secretory vesicles of peripheral nerves in
the guinea pig. Journal of Neurocytology 15:535-542.
Hægerstrand, A., Dalsgaard, C.-J., Jonzon, B., Larsson,
0. and Nilsson, J. (1990) Calcitonin Gene-related Peptide
stimulates proliferation of human endothelial cells.
Proceedings of the National Academy of Science USA
87:3299-3303.
Hall, S. ( 1986) The effect of inhibiting Schwann cell
mitosis on the re-innervât ion of acellular autografts in
the peripheral nervous system of the mouse. Neuropathology
and Applied Neurobiology 12:401-414.
Hamburger, V., Brunso-Bechtold, J.K. and Yip, J.W.
(1981) Neuronal death in the spinal ganglia of the chick
embryo and its reduction by nerve growth factor. Journal of
Neuroscience 1(1):60-71.
168
Helme, R.D. and Andrews, P.V. (1985) The effect of nerve
lesions on the inflammatory response to injury. Journal of
Neuroscience Research 13:453-459.
Hendry, I.A. The response of adrenergic neurones to
axotomy and nerve growth factor. (1975) Brain Research
94:87-97.
Hendry, I.A., Stockel, K., Thoenen, H. and Iversen, L.L.
(1974) The retrograde axonal transport of nerve growth
factor. Brain Research 68:103-121.
Henry, J.L. (1976) Effects of substance P on
functionally identified units in cat spinal cord. Brain
Research 114:439-451.
Heumann, R., Korsching, S., Bandtlow, C. and Thoenen, H.
(1987a) Changes of Nerve Growth Factor synthesis in
nonneuronal cells in response to sciatic nerve transection.
The Journal of Cell Biology 104:1623-1631.
Heumann, R. , Lindholm, D. , Bandtlow, C ., Meyer, M. ,
Radeke, M.J., Misko, T.P., Shooter, E. and Thoenen, H.
(1987b) Differential regulation of mRNA encoding nerve
growth factor and its receptor in rat sciatic nerve during
169development, degeneration, and regeneration: Role ofmacrophages. Proceedings of the National Academy of Science
USA 84:8735-8739.
Hokfelt, T., Elde, R. , Johansson, 0., Luft, R. , Nilsson,
G. and Arimura, A. (1976) Immunohistochemical evidence for
separate populations of somatostatin-containing and
substance P-containing primary afferent neurons in the rat.
Neuroscience 1:131-136.
Hokfelt, T., Johansson, 0., Ljungdahl, A., Lundberg,
J.M., Schultzberg, M. (1980) Peptidergic neurons. Nature
284:515-521.
Hokfelt, T. (1991) Neuropeptides in perspective: The
last ten years. Neuron 7:867-879.
Ide, C . , Tohyama, K . , Yokota, R. , Nitatori, T. and
Onodera, S. (1983) Schwann Cell Basal Lamina and Nerve
Regeneration. Brain Research 288:61-75.
Issekutz, T.B., Jeanette, M. , Stoltz, P. and Van der
Meide, P. (1988) The recruitment of lymphocytes into the
skin by T cell lymphokines: the role of interferon.
Clinical and Experimental Immunology 73:70-75.
170
Jacob, J.M. and McQuarrie, I.G. (1991) Axotomy
accelerates slow component b of axonal transport. Journal
of Neurobiology 22(6):570-582.
Jenq, C.B. and Coggeshall, R.E. (1986) Regeneration of
Transected Rat Sciatic Nerves after Using Isolated Nerve
Fragments as Distal Inserts in Silicone Tubes. Experimental
Neurology 91:154-162.
Johnson, E.M.,Jr., Gorin, P.D., Brandeis, L.D. and
Pearson, J. (1980) Dorsal root ganglion neurons are
destroyed by exposure in utero to maternal antibody to
nerve growth factor. Science 210:916-918.
Johnson, E.M.,Jr., Osborne, P.A., Rydel, R.E., Schmidt,
R.E. and Pearson, J. (1983) Characterization of the effects
of autoimmune nerve growth factor deprivation in the
developing guinea-pig. Neuroscience 8(3):631-642.
Johnson, E.M.,Jr., Taniuchi, M., Clark, H.B., Springer,
J.E., Koh, S., Tayrien, M.W. and Loy, R. ( 1987)
Demonstration of the retrograde transport of nerve growth
factor receptor in the peripheral and central nervous
system. Journal of Neuroscience 7(3):923-929.
Joseph, J. (1950) Further studies in changes of nuclear
population in degenerating non-myelinated and finely
myelinated nerves. Acta Anatomy 9:279-288.
171
Kashihara, Y., Sakaguchi, M. and Kuno, M. (1989) Axonal
transport and distribution of endogenous Calcitonin
Gene-related Peptide in rat peripheral nerve. Journal of
Neuroscience 9(11):3796-3802.
Kato, K. and Satoh, T. ( 1983) Changes in the
concentration of enolase isozymes and S-100 protein in
degenerating and regenerating rat sciatic nerve. Journal of
Neurochemistry 40:1076-1081.
Keucherer-Ehret, A., Graeber, M.B., Edgar, D., Thoenen,
H. and Kreutzberg, G.W. (1990) Immunoelectron microscopic
localization of laminin in normal and regenerating mouse
sciatic nerve. Journal of Neurocytology 19:101-109.
Kiefer, R. and Kreutzberg, G.W. (1990) Gamma
interferon-like immunoreactivity in the rat nervous system.
Neuroscience 37:725-734.
Kleitman, N. , Wood, P., Johnson, M.I. and Bunge, R.P.
(1988) Schwann cell surfaces but not extracellular matrix
organised by Schwann cells support neurite outgrowth from
embryonic rat retina. The Journal of Neuroscience
8(2): 653-663.
Laufer, R. and Changeux, J-P. (1989) Calcitonin
gene-related peptide and cyclic AMP stimulate
172
phosphoinositide turnover in skeletal muscle cells. Journal
of Biological Chemistry 264 (5): 2683-2689.
Laufer, R. and Changeux, J.-P. (1987) Calcitonin gene
related peptide elevates cyclic AMP levels in chick
skeletal muscle: possible neurotrophic role for a
coexisting neuronal messenger. EMBO 6(4): 901-906.
Lazar, P., Reddington, M., Streit, W., Raivich, G. and
Kreutzberg, G.W. (1991) The action of calcitonin
gene-related peptide on astrocyte morphology and cyclic AMP
accumulation in astrocyte cultures from neonatal rat brain.
Neuroscience Letters 130:99-102.
Leiberman, A.R., (1971) The axon reaction: a review of
the principal features of perikaryal responses to axon
injury. International Reviews of Neurobiology 14:49-124.
Levi-Montalcini, R. and Angeletti, P.U. (1963) Essential
role of the nerve growth factor in the survival and
maintenance of dissociated sensory and sympathetic
embryonic nerve cells in vitro. Developmental Biology
7:653-659.
Levi-Montalcini, R. and Hamburger, V. (1951) Selective
growth stimulating effects of mouse sarcoma on the sensory
and sympathetic nervous system of the chick embryo. Journal
of Experimental Zoology 116:321-362.
173
Levi-Montalcini, R. , Meyer, H. and Hamburger, V. ( 1954)
In vitro experiments on the effects of mouse sarcomas 180
and 37 on the spinal and sympathetic ganglia of the chick
embryo. Cancer Research 14:49-57.
Lindholm, D. , Heumann, R. , Meyer, M. and Thoenen, H.
(1987) Interleukin-1 regulates synthesis of nerve growth
factor in non-neuronal cells of rat sciatic nerve. Nature
330:658-659.
Lisak, R.P. and Bealmear, B. (1991) Antibodies to
interleukin-1 inhibit cytokine-induced proliferation of
neonatal Schwann cells in vitro. Journal of Neuroimmunology
31:123-132.
Lisak, R.P., Sobue, G . , Kuchmy, D. , Burns, J.B. and
Pleasure, D.E. (1985) Products of activated lymphocytes
stimulate Schwann cell mitosis in vitro. Neuroscience
Letters 57:105-111.
Ljungdahl, A., Olsson, T., Van der Meide, P.H.,
Holmdahl, R. , Klareskog, L. and Hojeberg, B . ( 1989)
Interferon-gamma-like immunoreactivi ty in certain neurons
of the central and peripheral nervous system. Journal of
Neuroscience Research 24:451-456.
Longo, P.M., Hayman, E.G., Davis, G.E., Ruoslahti, E.,
Engvall, E. , Manthorpe, M. and Varon, S. (1984 )
174
Neurite-Promoting Factors and Extracellular Matrix
Components Accumulating In Vivo Within Nerve Regeneration
Chambers. Brain Research 309:105-117.
Lorton, D. , Bellinger, D.L., Felten, S.Y. and Felten,
D.L. (1990) Substance P innervation of the rat thymus.
Peptides 11:1269-1275.
Lotz, M., Vaughan, J.H. and Carson, D.A. (1988) Effect
of neuropeptides on production of inflammatory cytokines by
human monocytes. Science 241:1218-1221.
Lundberg, J.M., Fahrenkrug, J. and Brimijoin, S. (1981)
Characteristics of the axonal transport of vasoactive
intestinal polypeptide (VIP) in nerves of the cat. Acta
Physiologica Scandanavia 112:427-436.
Lundborg, G. (1988) Nerve injury and repair, New
York :Churchi11 Livingstone.
Lunn, E.R., Brown, M.C. and Perry, V.H. ( 1990) The
pattern of axonal degeneration in the peripheral nervous
system varies with different types of lesion. Neuroscience
35:157-165.
Lunn, E.R., Perry, V.H., Brown, M.C., Rosen, H. and
Gordon, S. (1989) Absence of Wallerian degeneration does
175
not hinder regeneration in peripheral nerve. European
Journal of Neuroscience 1:27-33.
Magaud, J.P., Sargent,!., and Mason, D.Y. (1988)
Detection of human white cell proliferative responses by
immunoenzymatic measurement of bromodeoxyuridine uptake.
Journal of Immunological Methods 106:95-100.
Martin, D.P., Schmidt, R.E., DiStefano, P.S., Lowry,
O.H. , Carter, J.C. and Johnson.E.M., Jr. ( 1988) Inhibitors
of protein synthesis and RNA synthesis prevent neuronal
death caused by nerve growth factor deprivation. The
Journal of Cell Biology 106:82 9-844.
Martini, R. and Schachner, M. (1988) Immunoelectron
microscopic localisation of neural cell adhesion molecules
(LI, N-CAM and myelin-associated glycoprotein) in
regenerating adult mouse sciatic nerve. The Journal of Cell
Biology 106:1735-1746.
Matsuoka, I., Meyer, M. and Thoenen, H. (1991)
Cell-type-specific regulation of nerve growth factor (NGF)
synthesis in non-neuronal cells: comparison of Schwann
cells with other cell types. Journal of Neuroscience
11 ( 10 ): 3165-3177.
Mebius, R.E., Hendriks, H.R., Breve, J. and Kraal, G.
(1990) Macrophages and the activity of endothelial venules.
176
The effect of interferon-Y• European Journal of Immunology 20:1615-1618.
McQuarrie, I.G. and Grafstein, B. (1973) Axon outgrowth
enhanced by previous nerve injury. Archives of Neurology
29:53-55.
McQuarrie, I.G. and Jacob, J.M. (1991) Conditioning
nerve crush accelerates cytoskeletal protein transport in
sprouts that form after subsequent crush. Journal of
Comparative Neurology 305:139-147.
Mizobe, F. , Kozousek, V., Dean, D.M. and Livett, E.G.
(1979) Pharmacological characterization of adrenal
paraneurons: substance P and somatostatin as inhibitory
modulators of the nicotinic response. Brain Research
178:555-566.
Molina, I.J. and Huber, B.T. (1991) Regulation of
macrophage activation markers by IL-4 and IFN-y is
subpopulation-specific. Cellular Immunology 134:241-248.
Moore, B.W. (1965) A soluble protein characteristic of
the nervous system. Biochemical and Biophysical Research
19:739-744.
Mulderry, P.K., Ghatei, M.A., Spokes, R.A., Jones, P.M.,
Pierson, A.M., Hamid, Q.A., Kanse, S., Amara, S.G., Burrin,
177
J.M., Legon, S., Polak, J.M. and Bloom, S.R. (1988)
Differential expression of a-CGRP and &-CGRP by primary
sensory neurons and enteric autonomic neurons of the rat.
Neuroscience 25(1):195-205.
Nathan, C. (1987) Secretory products of macrophages.
Journal of Clinical Investigation 79:319-326.
New, H.V. and Mudge, A.W. (1986) Calcitonin gene-related
peptide regulates muscle acetylcholine receptor synthesis.
Nature 323:80 9-811.
Newman, S.L, Mikus, L.K. and Tucci, M.A. (1991)
Differential requirements for cellular cytoskeleton in
human macrophage complement receptor- and Fc
receptor-mediated phagocytosis. The Journal of Immunology
146:967-974.
Nielsch, U. and Keen, P. (1989) Reciprocal regulation of
tachykinin and vasoactive interstinal peptide-gene
expression in rat sensory neurones following cut and crush
injury. Brain Research 481:25-30.
Noguchi, K ., Senba, E., Morita, Y. , Sato, M. and
Tohyama, M. (1990) a-CGRP and 8-CGRP mRNAs are
differentially regulated in the rat spinal cord and dorsal
root ganglion. Molecular Brain Research 7:299-304.
178
Nong, Y.H., Titus, R.G., Ribeiro, J.M.C. and Remold,
H.G. ( 1989) Peptides encoded by the calcitonin gene inhibit
macrophage function. The Journal of Iimunology 143:45-49.
Ohmori, Y., Strassman, G . and Hamilton, T.A. (1990) cAMP
differentially regulates expression of mRNA encoding IL-1
and IL-1 in murine peritoneal macrophages. The Journal of
Inununology 145:333 3-3339.
Olsson, T., Kristensson, K., Ljungdahl, A., Maehlen, J.,
Holmdahl, R. and Klareskog, L. (1989) Gamma-interferon-like
immunoreactivity in axotomized rat motor neurons. The
Journal of Neuroscience 9:3870-3875.
Ottaway, C.A. and Greenberg, G.R. (1984) Interaction of
vasoactive intestinal peptide with mouse lymphocytes:
specific binding and the modulation of mitogen responses.
The Journal of Immunology 132:417-423.
Pellegrino, R.G., Politis, M.J., Ritchie, J.M. and
Spencer, P.S. (1986) Events in degenerating cat peripheral
nerve: induction of Schwann cell S phase and its relation
to nerve fibre degeneration. Journal of Neurocytology
15:17-28.
Perry, V.H., Brown, M.C. and Gordon, S. (1987) The
macrophage response to central and peripheral nerve injury.
Journal of Experimental Medicine 165:1218-1223.
179
Perry, V.H., Brown, M.C., Lunn, E.R., Tree, P. and
Gordon, S. (1990a) Evidence that very slow Wallerian
degeneration in C57Bl/01a mice is an intrinsic property of
the peripheral nerve. European Journal of Neuroscience
2:802-808.
Perry, V.H., Lunn, E.R., Brown, M.C., Cahusac, S. and
Gordon, S. ( 1990b) Evidence that the rate of Wallerian
degeneration is controlled by a single autosomal dominant
gene. European Journal of Neuroscience 2:408-413.
Perry, V.H., Brown, M.C. and Lunn, E.R. (1991a) Very
slow retrograde and Wallerian degeneration in the CNS of
C57Bl/01a mice. European Journal of Neuroscience 3:102-105.
Perry, V.H. and Gordon, S. (1991b) Macrophages and the
nervous system. Internat ionl Reviews in Cytology
125:203-244.
Perry, V.H. and Brown, M.C. (1992) Role of macrophages
in peripheral nerve degeneration and repair. BioEssays
14 ( 6): 401-406.
Politis, M.J., Ederle, K. and Spencer, P.S. ( 1982)
Tropism in Nerve Regeneration In Vivo. Attraction of
Regenerating Axons by Diffusible Factors Derived from Cells
in Distal Nerve Stumps of Transected Peripheral Nerves.
Brain Research 253:1-12.
180
Raivich, G., Hellweg, R. and Kreutzberg, G.W. (1991) NGF
receptor-mediated reduction in axonal NGF uptake and
retrograde transport following sciatic nerve injury and
during regeneration. Neuron 7:151-164.
Raivich, G. and Kreutzberg, G.W. (1987) Expression of
growth factor receptors in injured nervous tissue. I.
Axotomy leads to a shift in the cellular distribution of
B-nerve growth factor binding in the injured and
regenerating PNS. Journal of Neurocytology 16:689-700.
Ramon yCajal, S.R. (1928) Dégénérât ion and regeneration
of the peripheral nervous system, New York:Hafner.
Rappolee, D.A. and Werb, Z. (1988) Secretory products of
phagocytes. Current Opinion in Immunology 1:47-55.
Reiger, F., Nicolet, M. , Pincon-Raymond, M. , Murawsky,
M. , Levi, G . and Edelmann, G.M. ( 1988 ) Distribution and
Role in Regeneration of N-CAM in the Basal Laminae of
Muscle and Schwann Cells. The Journal of Cell Biology
107:707-719.
Remsen, L.G., Strain, G.M., Newman, M.J., Satterlee, N.
and Daniloff, J.K. (1990) Antibodies to neural cell
adhesion molecule disrupt functional recovery in injured
nerves. Experimental Neurobiology 110:268-273.
181
Rich, K.M., Luszczynski, J.R., Osborne, P.A. and Johnson
Jr, E.M. (1987) Nerve growth factor protects adult sensory
neurons from cell death and atrophy caused by nerve injury.
Journal of Neurocytology 16:261-268.
Rich, K.M., Yip, H.K., Osborne, P.A., Schmidt, R.E. and
Johnson, E.M.,Jr. (1984) Role of nerve growth factor in the
adult dorsal root ganglia neurons and its response to
injury. Journal of Comparative Neurology 230:110-118.
Ridley, A.J., Davis, J.B., Stroobant, P. and Land, H.
(1989) Transforming growth-factor-Bl and B2 are mitogens
for rat Schwann cells. Journal of Cellular Biology
109:3419-3424.
Roa, M. and Changeux, J.-P. (1991) Characterisation and
developmental evolution of a high-affinity binding site for
Calcitonin Gene-related Peptide on chick skeletal muscle
membrane. Neuroscience 41:563-570.
Robinson, A.P., White, T.M. and Mason, D.W. (1986)
Macrophage heterogeneity in the rat as delineated by two
monoclonal antibodies MRC 0X41 and MRC 0X42, the latter
recognising complement receptor type 3. Immunology 57:239.
Rosen, H. and Gordon, S. (1987) Monoclonal antibody to
the murine type 3 complement receptor inhibits adhesion of
182
myelomonocytic cells in vitro and inflammatory cell
recruitment in vivo. Journal of Experimental Medicine
166:1685-1701.
Said, S.I. and Mutt, V. ( 1970) Polypeptide with broad
biological activity: isolation from small intestine.
Science 169:1217-1218.
Saika, T., Senba, E. , Noguchi, K., Sato, M. , Yoshida,
S., Kubo, T., Matsunaga, T. and Tohyama, M. (1991) Effects
of nerve crush and transection on mRNA levels for nerve
growth factor receptor in the rat facial motoneurons.
Molecular Brain Research 9:157-160.
Salzer, J.L. and Bunge, R.P. (1980) Studies of Schwann
cell proliferation I. An analysis in tissue culture of
proliferation during development, Wallerian degeneration
and direct injury. The Journal of Cell Biology 84:739-752.
Scaravilli, F. (1984) The influence of distal
environment on peripheral nerve regeneration across a gap.
Journal of Neurocytology 13:1027-1041.
Scheldt, P., Waehneldt, T.V., Beuche, W. and Friede,
R.L. (1986) Changes of myelin proteins during Wallerian
degeneration in situ and in millipore diffussion chambers
preventing active phagocytosis. Brain Research 379:380-384.
183
Sethna, M.P. and Lampson, L.A. (1991) Immune modulation
within the brain: recruitment of inflammatory cells and
increased major histocompatibility antigen expression
following intracerebral injection of interferon-y• Journal
of Neuroimmunology 34:121-132.
Shehab, S.A.S. and Atkinson, M.E. (1986) Vasoactive
intestinal polypeptide increases in areas of the dorsal
horn of the spinal cord from which other neuropeptides are
depleted following peripheral axotomy. Experimental Brain
Research 62:422-430.
Sketelji, J., Bresjanac, M. and Popovic, M. (1989) Rapid
growth of regenerating axons across the segment of sciatic
Nerve devoid of Schwann cells. Journal of Neuroscience
24:153-162.
Skofitsch, G. and Jacobowitz, D.M. (1985) Calcitonin
gene-related peptide: detailed immunohistochemical
distribution in the central nervous system. Peptides
6:721-745.
Sminia, T., De Groot, C.J.A., Dijkstra, C.D., Koetsier,
J.C. and Polman, C.H. ( 1987 ) Macrophages in the central
nervous system of the rat. Immunobiology 174:43-50.
Spreca, A., Rambotti, M.G., Rende, M. , Saccardi, C .,
Aisa, M.C., Giambanco, I. and Donato, R. (1989)
184
Immunocytochemical localisation of S-100J3 protein in
degenerating and regenerating rat sciatic nerves. Journal
of Histochemistry and Cytochemistry 37(4):441-446.
Steenbergh, P.H., Hoppener, J.W.M., Zandberg, J., Lips,
C.J.M. and Jansz, H.S. (1985) A second human
calcitonin/CGRP gene. FEBS 183(2):403-407.
Stevens, A., Schabet, M. , Schott, K. and Wietholter, H.
(1989) Role of endoneurial cells in experimental allergic
neuritis and characterisation of a resident phagocytic
cell. Acta Neuropathologica 77:412-419.
Stoeckel, K., Schwab, M. and Thoenen, H. (1975)
Specificity of retrograde transport of nerve growth factor
(NGF) in sensory neurons: a biochemical and morphological
study. Brain Research 89:1-14.
Stoeckel, K ., Guroff, G. , Schwab, M. and Thoenen, H.
(1976) The significance of retrograde axonal transport for
the accumulation of systemically administered nerve growth
factor (NGF) in the rat superior cervical ganglion. Brain
Research 109:271-284.
Stoll, G . , Griffin, J.W. , Li, C.Y. and Trapp, B.D.
(1989) Wallerian degeneration in the peripheral nervous
system: participation of both Schwann cells an macrophages
in myelin degradation. Journal of Neurocytology 18:671-683.
185
Stoll, G. and Muller, H.W. (1986) Macrophages in the
peripheral nervous system and astroglia in the central
nervous system of rat commonly express apolipoprotein E
during development but differ in response to injury.
Neuroscience Letters 72:233-238.
Stoll, G., Trapp, B.D. and Griffin, J.W. (1989)
Macrophage function during Wallerian degeneration of rat
optic nerve: clearance of degenerating myelin and la
expression. The Journal of Neuroscience 9:2327-2335.
Streit, W.J., Dumoulin, F.L., Raivich, G . and
Kreutzberg, G.W. (1989) Calcitonin Gene-related Peptide
increases in rat facial motoneurons after peripheral nerve
transection. Neuroscience Letters 101:143-148.
Tacke, R. and Martini, R. (1990) Changes in expression
of mRNA specific for cell adhesion molecules (LI and NCAM)
in the transected peripheral nerve of the adult rat.
Neuroscience Letters 120:22 7-230.
Takami, K., Kawai, Y., Uchida, S., Tohyama, M. ,
Shiotani, Y., Yoshida, H., Emson, P.C., Girgis, S.,
Hillyard, C.J. and MacIntyre, I. (1985) Effect of
calcitonin gene-related peptide on contraction of striated
muscle in the mouse. Neuroscience Letters 60:227-230.
186
Taniuchi, M., Clark, H.B., Schweitzer, J.B. and Johnson
Jr, E.M. (1988) Expression of Nerve Growth Factor Receptors
by Schwann Cells of Axotomised Peripheral Nerves;
U1trastructural Location, Suppression by Axonal Contact,
and Binding Properties. The Journal of Neuroscience
8:664-681.
Terzis, J.K. and Smith, K.L. ( 1990) The peripheral
nerve: Structure, function and reconstruction, New
York:Raven Press.
Tessler, A., Himes, B.T., Kreiger, N.R., Murray, M. and
Goldberger, M.E. (1985) Sciatic nerve transection produces
death of dorsal root ganglion cells and reversible loss of
substance P in spinal cord. Brain Research 332:209-218.
Tetzlaff, W. , Bisby, M.A. and Kreutzberg, G.W. ( 1988 )
Changes in cytoskeletal proteins in the rat facial nucleus
following axotomy. The Journal of Neuroscience 8:3181-3189.
Tohyama, K ., Ide, C. and Osawa, T. (1990) Nerve
regeneration through the cryoinjured allogeneic nerve graft
in the rabbit. Acta Neuropathologica 80:138-144.
Troy, C.M., Muma, N.A., Greene, L.A., Price, D.L. and
Shelanski, M.L. (1990) Regulation of perpherin and
neurofilament expression in regenerating rat motor neurons.
Brain Research 529:232-238.
187
Tsujimoto, T. and Kuno, M. (1988) Calcitonin
Gene-related Peptide prevents disuse-induced sprouting of
rat motor nerve terminals. Journal of Neuroscience
8 ( 10 ): 3951-3957.
Uchida, S., Yamamoto, H ., lio. S., Matsumoto, N., Wang,
X-B. , Yonehara, N. , Imai, Y. , Inoki, R. and Yoshida, H.
(1990) Release of calcitonin gene-related peptide-like
immunoreactive substance from neuromuscular junction by
nerve excitation and its action on striated muscle. Journal
of Neurochemistry 54:1000-1003.
Umeda, Y. and Arisawa, M. (1989) Characterization of the
calcitonin gene-related peptide receptor in mouse T
lymphocytes. Neuropeptides 14:237-242.
Vigney, A., Wang, F. and Ganz, M.E. (1991) Macrophages
express functional receptors for calcitonin gene-related
peptide. Journal of Cellular Physiology 149:301-306.
Wakisaka, S., Kajander, K.C. and Bennett, G.J. (1991)
Increased neuropeptide Y (NPY)-like immunoreact ivi ty in rat
sensory neurons following peripheral axotomy. Neuroscience
Letters 124:200-203.
Wawryk, S.O., Novotny, J.R., Wicks, I.P., Wilkinson, D.,
Maher, D., Salvaris, E., Welch, K., Fecondo, J. and Boyd,
A.W. ( 1989) The role of the LFA-l/ICAM-1 interaction in
188
human leukocyte homing and adhesion. Immunological Eeviews
108:135-161.
Wehrle, B. and Chiquet, M. (1990) Tenascin is
accumulated along developing peripheral nerves and allows
neurite outgrowth in vitro. Development 110:401-415.
Weihe, E., Muller, S., Fink, T. and Zentel, H.J. (1989)
Tachykinins, calcitonin gene-related and neuropeptide Y in
nerves of the mammalian thymus: interaction with mast cells
in autonomic and sensory neuroimmunomodulation?
Neuroscience Letters 100:77-82.
Weimaster, G. and Lemke, G. (1990) Cell-specific cyclic
AMP-mediated induction of the PDGF receptor. EMBO
9( 3 ) : 915-920.
Welgus, H.G., Campbell, E.J., Bar-Shavit, Z ., Senior,
R.M. and Teitelbaum, S.L. (1985) Human alveolar macrophages
produce a fibroblast-like collagenase and collagenase
inhibitor. Journal of Clinical Investigation 76:219-224.
Wiik, P. (1989) Vasoactive intestinal peptide inhibits
the respiratory burst in human monocytes by a cyclic
AMP-mediated mechanism. Regulatory Peptides 25:187-197.
Williams, L.R., Powell, H.C., Lundborg, G . and Varon, S.
(1984) Competence of Nerve Tissue as Distal Insert
189
Promoting Nerve Regeneration in a Silicone Chamber. Brain
Research 293:201-211.
Wong, J. and Oblinger, M.M. ( 1990a) A comparison of
peripheral and central axotomy effects on neurofilament and
tubulin gene expression in rat dorsal root ganglion
neurons. The Journal of Neuroscience 10:2215-2222.
Wong, J. and Oblinger, M.M. (1990b) Differential
Regulation of peripherin and neurofilament gene expression
in regenerating rat DRG neurons. Journal of Neuroscience
Research 27:332-341.
Wood, S.J., Pritchard, J. and Sofroniew, M.V. (1990)
Re-expression of nerve growth factor receptor after axonal
injury recapitulates a developmental event in motor
neurons: differential regulation when regeneration is
allowed or prevented. European Journal of Neuroscience
2:650-657.
Woolf, C.J., Chong, M.S. and Ainsworth, A. (1984)
Axotomy increases glycogen phosphorylase activity in
motoneurones. Neuroscience 12:1261-1269.
Yamamoto, A.I. and Tohyama, M. ( 1989) CGRP in the
peripheral nervous system. Progress in Neurohiology
33:353-386.
190
Yip, H.K. and Johnson, E.M.,Jr. (1984) Developing dorsal
root ganglion neurons require trophic support from their
central processes: Evidence for a role of retrogradely
transported nerve growth factor from the central nervous
system to the periphery. Proceedings of the National
Academy of Science USA 81:6245-624 9.
Yip, H.K., Rich, K.M., Lampe, P.A. and Johnson, E.M.,Jr.
(1984) The effects of nerve growth factor and its antiserum
on the postnatal development and survival after injury of
sensory neurons in the rat dorsal root ganglia. Journal of
Neuroscience 4:2986-2992.
Yu, S.F., Koerner, T.J. and Adams, D.O. ( 1990) Gene
regulation in macrophage activation: Differential
regulation of genes encoding for tumor necrosis factor,
interleukin-1, JE and KC by interferon-y and
1ipopolysaccharide. Journal of Leukocyte Biology
48:412-419.
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