INFORMAnON TO USERS

This manuscript has been reproduced from the microfilm master. UMI films the text directJy from the original or copy submitted. Thus, sorne thesis a-xi dissertation copies are in typewriter face, while others may be trom any type of computer printer.

The quality of this reproduction la dependent upon the quality of the copy submitted. Broken or indistinct print. coIored or pool" quality iNustralions and photographs, print bleedthrough, substandard margins, and improper alignment can adverselyaffect reproduction.

ln the unJikely event that the author did not send UMI a compfete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indïcate the deletion.

Oversize materials (e.g., maps, drawings, char1s) are reproduced by sectioning the original, begiming at the upper Ieft-hand camer and continuing trom left ta right in equal sections with small avertaps.

Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6- x 9" black and white photographie prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contad UMI direclly to arder.

Bell & Howell Information and Leaming 300 North Zeeb Raad, Ann Arbor, MI 48106-1346 USA 800-521-0600

•

IMPLICATION OF THE -RELATED AND ITS RECEPTOR BINDING SITES IN ANIMAL MODELS OF CHRONIe PAIN

Julie Rémillard •

Department ofPhannacology and Therapeutics McGill University, Montreal, Canada FebrualJ' 1999

A thesis submitted to the Faculty ofGraduate Studies and Research in partial fulfillment of the requirements for the degree ofMaster's in Science

• ©Julie Rémillard, 1999. National Ubrary Bibliothèque nationale 1+1 of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395. rue Wellington Ottawa ON K1A 0N4 Oftawa ON K1 A 0N4 canada Canada

The author has granted a non­ L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la N ationaI Library ofCanada to Bibliothèque nationale du Canada de reproduce, loan, distribute or seO reproduire, prêter, distribuer ou copies ofthis thesis in microfo~ vendre des copies de cette thèse sous paper or electronic formats. la fonne de microfiche/film, de reproduction sur papier ou sur fonnat électronique.

The author retains ownership ofthe L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts from it Ni la thèse ni des extraits substantiels may he printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son penmSSlOD. autorisation.

0-612-50866-8

Canadi fi • Abstract Calcitonin gene-related peptide (CGRP) is a 37 peptide arising from

the ofthe calcitonin gene. The "ide distribution ofCGRP in

the central and peripheral nervous systems suggests a raie for CGRP in sensory

processing. The primary foeus of this thesis was to study the involvement of

CGRP and its binding sites in rat models of chronic inflarnrnatory and

neuropathic pain. The expression and binding sites of and

were aIso examined to assess the specificity ofthe observed changes.

No changes were observed in CGRP, galanin and substance P immunostaining

in the dorsal horn ofthe lumbar regions 4 and 5 (L4-L5) ofthe spinal cord al 24

hours, 4, 7 and 14 days following the injection of Freund's complete adjuvant • or sciatic nerve constriction. However, binding sites at the L4-L5 levels were found to be modulated during the development ofchronic pain. CGRP binding

sites were found to be increased and decreased in models of inflammatory and

neuropathic pain, respectively. Substance P binding sites were increased in

both models \vhile galanin binding sites were decreased only in this neuropathy

pain mode!.

In conclusion, the changes in CGRP binding levels observed following the

injection ofFreund's complete adjuvant or constriction ofthe seiatic nerve were

not mirrored by similar changes in substance P and galanin binding sites. This

finding indicates differential involvement of these and their • receptors in chronic pain mechanisms. ID • Résumé Le peptide relié au gène de la calcitonine (CGRP) est un peptide composé de 37

acides aminés résultant de l'épissage alternatif du gène de la calcitonine. La

distribution du CGRP et de ses sites de liaison dans les systèmes nerveux

central et périphérique suggère un rôle pour le CGRP dans les processus

sensoriels. Le but principal de ce mémoire était d ~ étudier les modifications du

CGRP et de ses sites de liaison dans des modèles de douleur chronique de type

inflammatoire et neuropathique chez le rat. Les galanine et substance

P furent aussi examinés pour comparaison.

Aucun changement n'a été observé au niveau du marquage

immunohistochimique du CGRP, de la galanine et de la substance P dans la

come dorsale des régions lombaires L4 et L5 de la moelle épinière à 24 heures, • 4, 7 et 14 jours suivant l'injection de l'adjuvant de Freund ou la constriction du nerf sciatique. Par contre, des changements ont été observés pour les sites de

liaison localisés au niveau L4-L5 de la moelle épinière. Les sites de liaison du CGRP ont augmenté dans le modèle de douleur inflammatoire et diminué dans

le modèle de douleur neuropathique. Les sites de liaison de la substance Pont augmenté dans les deux modèles tandis que les sites de liaison de la galanine

ont diminué dans le modèle de neuropathie.

En conclusion, les changements observés pour les sites de liaison du CGRP suivant lïnjection de l'adjuvant de Freund ou la constriction du nerf sciatique sont spécifiques. Ces résultats suggèrent que les neuropeptides CGRP, galanine

et substance P et leurs sites de liaison ont des rôle différents dans le • développement de la douleur chronique. IV • Acknowledgements

This thesis could not have been completed without the facilities and equipment

provided by the Astra Research Centre Montreal. In addition, the guidance and

advice provided by Dr. Daniel. Ménard and Dr. Andy Dray were greatly

appreciated.

Special thanks to Dr. Rémi Quirion from the Douglas Hospital Research Centre

for rus supervision throughout the duration ofthis thesis. •

• v • Table of Contents ABSTRACT n

RÉSliMÉ ID

ACKNO\VLEDGEMENTS IV

TABLE OF CONTENTS v

ARBREVIATIONS .4....l\Tf> GLOSSARY VII

CH.<\.PTER 1 REVIE\V OF THE LITERATURE 1

• CALCITO~IN GE~E-REL-\TED PEPTIDE 2 CALCITONlN GENE MRNA PROCESSING ,:}.. CGRP RECEPTORS 6 DISTRIBUTION OF CGRP A..'ID ITS BINDING SITES 13 CGRP Fœ-.1CTIONS 18 SUBSTAl'lCE P 26 NEUROKININ RECEPTORS 27 DISTRIBUTION OF SUBSTANCE P ~'.:D ITS BINDING SITES IN THE SPINAL CORO 28 SUBSTANCE P FUNCTIONS 28 GALAl'"IN 29 DISTRIBUTION OF GALANlN AND ITS BINDING SITES 30 .. ., GALANIN FUNCTIONS ,:}- CHRO~IC PAIN 34 AI~I OF THE THESIS 37

CHAPTER II MATERIALS Al\n METHODS 38

A) BEHAVIOUR 39 1) CHRO!':IC PAIN MODELS 39 2) BEHAVIOURAL TESTING 40 3) STATISTICS 41 B) IMMUNOHISTOCHEMISTRY 42 • 1) PERFUSIONS 42 VI

2) IMMUNOHISTOCHEMISTRY 45 C) RADIOAUTOGRAPHY 51 1) [12Sl]HCGRP BINDING 51 • 2) [12S[]BH_SUBSTANCE P 53 3) [12S[]PORCINE GALANIN 54 4) QUAl'ollTATIVE IMAGE ANALYSIS 56 5) STATIsncs 56

CHAPTER nI RESULTS 57

A) BEHAVIOURAL EXPERIMENTS 58 1) INFLAM?-.1ATORY PAIN MaDEL 58 2) NEUROPATHIC PAIN MaDEL 61 B) IMMUSOHISTOCHEMICAL STAINING 64 1) CALCITO~IN GENE-RELATED PEPTIDE 64 2) GALA.'!lN 64 3) SUBSTA.;'-:CE P 64 C) BI1"DI!'iG SITES 69 1) C.-\.LCITO~IN GENE-RELATED PEPTIDE BINDING SITES 69 2) G:\.LANIN BINDr.-:G SITES 72 3) SUBSTA."\CE P BrNDING SITES 76

CHAPTER IV DISCUSSION 80

A) CHROSIC PAI!'i MODELS 82 • 1) I~FLA.\-IMATORY PAIN MaDEL 82 2) NECROPATHIC PAIN MaDEL 85 B) PLASTICITY Of SPINAL CORn NEUROPEPTIDES 87 l )1~f~flJ~OHISTOCHEMICAL STAINn\G 87 2)BfNDI~G SITES 92 C) COSCLt:SION 95

REFERENCES 96

• VII • Abbreviations and Glossary a.a.: amino acid allodynia: pain resulting from innocuous stimuli CFTR assay: cystic fibrosis transmembrane conductance regulator CGRP: calcitonin gene-related peptide CGRP-RCP: calcitonin gene-related peptide receptor component protein CNS: central nervous system CSF: cerebrospinal fluid CT: calcitonin DRG: dorsal root ganglia dysesthesia: spontaneous pain

EC50: concentration required ta produce 50% ofthe maximal response FCA: Freund's complete adjuvant Gs: stimulatory G-protein a-subunit hCGRP: human calcitonin gene-related peptide

• IC 50: concentrations ofcompetitors needed to compete for 500/0 of specifie radioactive ligand binding i.c.v.: intracerebroventricular IR: immunoreactive

Kd : dissociation constant LM: like-material NK-l: neurokinin 1 NK..A.: NKB: NP-y: y (gamma) NPK: NPY: organ ofCorti: mammalian hearing organ • PK.A: protein kinase A vm

PPT-A: preprotachykinin-A • PPT-B: preprotachykinin-B SP: substance P VIP: vasoactive intestinal peptide

•

• • Chapter 1 Review of the Literature

•

• Chapter 1 Review ofthe Literature 2

Calcitonin Gene-Related Peptide

• Calcitonin gene-related peptide (CGRP) is a 37 amino acid peptide arising from the alternative splicing of the RNA transcript of the calcitonin gene (Rosenfeld

et al.! 1983; MacIntyre et al. 1992; Feurstein et al. 1995; Wimalawans~ 1996;

Wimalawansa, 1997; for review see van Rossum et al., 1997). CGRP is widely

distributed in the central and peripheral nervous systems where it plays a role in

a number of biologicaJ actions including cardioexcitatory effects ( Brain et al.,

1985b; Brain and Cambridge, 1996), inhibition of gastric acid secretion

(Hughes et a/., 1984) and food intake ( Krahn et al., 1984; Morley et al., 1996).

CGRP shares considerable homology with a number of other peptides of the

calcitonin family. Calcitonin (CT) was the first member within this family to be • isolated and sequenced. Following the molecular cloning of the calcitonin gene, Rosenfeld et al. discovered CGRP by using a recombinant DNA (Amara

et al.! 1982; Rosenfeld et al., 1983). Subsequently, the related peptides amylin

and adrenomedullin haye been isolated and found to share considerable

homology with CGRP, 46% and 240/0. respectively. These single-chain

peptides ail have an amidated C-terminal and two N-tenninal cysteines forming

a disulfide bridge (Figure 1).

Two forms of the calcitonin gene-related peptide have been identified, CGRPa

and CGRP~ (Bennett and Am~ 1992). Both of the encoding the two

CGRP isofonns are located on chromosome Il and are thought to have arisen • from gene duplication. The physiological significance of the two isofonns is Chapter 1 Review ofthe Literature 3

unknown and it still remains unclear which peptide is the evolutionary • precursor. CGRPa and CGRPJ3 are highly homologous differing by only 1 amino acid in rats and 3 amine acids in humans (Figure 1). Both isoforms are

present in the rat nervous system and exhibit nearly identical pharmacological

profiles.

Calcitonin gene mRNA processing

The calcitonin gene is comprised of six exons and encodes two different

mR.NAs that share an identical 5' sequence but have unique 3 ~ sequences.

Splicing of the first four exons generates calcitonin mRNA, which represents

over 98% of the mature transcripts in th}Toid C ceUs (Rosenfeld et al., 1992).

The calcitonin rnRNA encodes a 17,500 molecular weight caIcitonin precursor • protein, which is proteolytically processed to yield the calcium-regulating honnone calcitonin. Alternative processing ofthe ealcitonin gene results in the

production ofa mature transcript in neural tissue distinct from the predominant

rnRNA in thyroid C eeUs (Figure 2). In the central and peripheral nervous

systems, splicing ofthe fifth and sixth exons generates the mRNA encoding the

16.000Dalton precursor ofaCGRP (Rosenfeld et al. ~ 1992). It is suggested that

the calcitonin and CGRP exons share a common primordial genomic origin.

The complex caIcitoninlCGRP gene arose either by duplication and sequence

divergence of the primordial calcitonin-like exon itself or as a consequence of • gene duplication and rearrangement (Rosenfeld et al., 1992). • • •

1 1 human CGRPa. AC0TATCVTHRLAGLl SRSGGVV K N N f V P T N V G S K A f-NHZ

, 1

human CGRPp "C[!JT A TCV TH R LA GLL SR SG G[â]v K(i]N f V PT NVG Sie" f-NH2

1 1

rat CGRPa. [Ue[UT A TC V TH R L" GLL SR SGGVV KlUN FV PT N VG sliJ" f-NHZ

1 - --1

ratCGRPp (§Je [i]T AT CV THR LA GLL SR SGGVV K [ilN FV PT NVGS 1( A f-NHl ,--, rat amyUn lLl C [!] T " TC I!J T li] RL " lLJ(fJ L (ïJ liLJ S lU [UrHJ(fJ1iJlA](jJ(jJli]lU TNVG s lLlliJliJ NH 2 J -- 1

salmon calcitonin l'JGJ[I][]GJ TCV liJ(iJlLJ L GJ[;][fJ L (j!JlillU~[i] ***** liJ[fJliJ , N li] GS (U[j][f] NH 2 J--- 1

human calcitonin ~lillUl1J[!] T c lilliJlU[i][!][i][g] lPJ[fJliJ[!]lLIlilllJ * *** * F (fJ lU T ~][iJ G liJliJ" [f] NH 2

J , human adrenomedullin C l!J(fJ[ij] TC ~[UliJliJl!JliJ[2] ~(fJ[i][L][Llliù [j] **** * F [fJ [Q] T ~[l] G GJli] " ILl N H 2

lïJlBJ[gJGJ~[lJIJJ[fJ[g]~(JlBJlU[fJ

Figure 1: Amino a~id sequen~es for CG RI) and related peptides Spaccs, indicatcd by ., wcrc insertcd to allow for sequence comparison. Amino acids diffcring from human CGRPa arc frmncd. • • •

Prlmary Transcrlpt 5' 'A n" c ]-l·Ju' H.C~lci~_~::I.~iCltl:·H::ççi~i:.:·i:~--~~ 3' Calcltanln ----1 H H Gene 1 ItNA IJltOCESSING

TI IYROII> "C" CELLS NmWOlJS SYSTEM

Malure mRNA 3' 5'~-~~--3'

mRNA Trnnsllliion ~ Primary l l ~,···· ~ Translation Product "',I··,,·F...... Ik'&.... l IIIIII NIII ....l--...... m011

Prolculylic Itroccssing Malure Peptides ~ CAl.nmNIN nau'

anll ('l'"

allli N' IClllllllalllcpll\lc and N' &. C' lerminal pepliilcs

Figure 2: Altermltil'c Sillicing of the cnlcitonin/C(;IU· gcnc The cnld'onin gene is nllcrnativcly splicctl gcncraling calcilonin und CGR.. in il lissue-specifie manner. Chapter 1 Review ofthe Literature 6

CGRP recebtors • A wide spectrum ofbiological functions have been described for CGRP. These effeets are mediated through specifie reeeptor sites. By evaluating the

phannaeological properties of numerous CGRP fragments and analogues in

severa! peripheral tissue preparations, the existence of multiple CGRP receptor

sites has been proposed, namely CGRPI based on the antagonistic properties of

the CGRP antagonist, CGRPS-37, and CGRP2, based on the agonistic properties

of the linear analogue of CGRP, [CYS(ACM)2.7]hCGRP ( Dennis et al., 1989;

Dennis el al., 1990; Quirion et al., 1992; Quirion et Dumont, 1998).

Pharmacological classification

The C-tenninal fragment of hCGRP, hCGRP8-37, eompetes for [I25I]hCGRPa.

• binding sites \vith very high affinity (1C 0= 0.5-1 nM) bath in the CNS and 5

peripheral membrane preparations (Dennis et al., 1990). Its iodinated

counterp~ [I25I]hCGRPg_37 , binds with high affinity (Ko=0.075-0.215nM) to

CGRP receptors in brain, atrium and vas deferens membrane preparations (van

Rossum et al., 1994). CGRPg.37did not induee any biologieal activity in neither

guinea pig atrial and iIeai preparations nor a variety of behavioural assays.

However, CGRPg-37 was able to inhibit sorne ofthe effects of hCGRPa in these

preparations and is thus considered a relatively potent, competitive CGRP

receptor antagonist (Dennis et al., 1989; Dennis et al., 1990). The antagonistic

effects of CGRPg-37 were shown to be much weaker in the rat vas deferens • (Dennis el al., 1989; Dennis et al., 1990) (Table 1). Table 1: Effect of various concentrations of hCGRP8-J7 on the sensitivity of ,rarious tissues to native bCGRPa Values taken from Dennis el a/., 1990. The EC50 is the concentration needed to produce 50% ofthe maximal effect.

The differential antagonistic potencies of hCGRPg.37 in various bioassays • suggest the existence of multiple CGRP receptor subtypes (Table 2). It was therefore proposed by Dennis el al. (1990) that the hCGRPs_37-sensitive sites be

classified as CGRPI receptor and CGRPS-37 rather resistant sites be classified as

the CGRP2 subtype. The linear analogue of hCGRP, [Cys(ACM)2.7]hCGRP,

binds to rat whole brain membranes with an affinity (3.0 ± l.4Ili\1) similar to

the native peptide (2.4 ± O.6nM). This synthetic linear peptide does not indùce

the cardioexcitatory activity characteristic of hCGRP in atrial preparations.

However, [Cys(ACM)2,7]hCGRP does retain sorne ofthe agonistic properties of

hCGRP in the rat vas deferens (Dennis et al., 1989). These results suggest that

[Cys(ACM)2,7]hCGRP acts as a fairly selective agonist for the CGRP2 receptor • subtype located in tissues resistant to the antagonistic properties of hCGRPS-37. Chapter 1 Review ofthe Literature 8

• CGRP1 CGRP2 hCGRPs_37 Potent antagonist Weak antagonist pA2 up to 7.8 (PA2 6.0) or inactive

[Cys(ACM)2.7]a- Inactive Agonist hCGRP Prototypic Assay Atria Vas deferens (rat & guinea pig) (rat & guinea pig)

Table 2: Profile of proposed CGRP receptor subtypes Values taken from Quirion el aL, 1992. The pAl values represents the negative logarithm of the molar concentration of antagonist required to produce a twofold increase in ECso (sec Table 1) of hCGRPa..

G-Protein Coupling and Second Messenger Systems

G-Protein Coupling Evidence from biochemical and pharmaeological studies suggests that CGRP • receptors belong to the family of G protein eoupled receptors. Various reports have described the effeet of GTP or its analogues on et 25I]CGRP binding

affinity in a variety 0 f tissue preparations (Roa and Changeux, 1991; Chanerjee

and Fischer, 1991; Chatterjee et al., 1991; van Rossum et al.. 1993; Chatterjee

et al., 1993). van Rossum et al. (1993) studied the effeets of the non-

hydrolizable GTP nucleotide analogue Gpp(NH)p in the brain, cerebel1um, atria

and vas deferens and showed that Gpp(NH)p induced a shift to a lower affinity

receptor state, as expected for G-protein coupled receptors.

Second Messenger Systems Various reports have shown that CGRP's actions may he mediated through an • increase in cAMP. Various tissue preparations such as muscle (Edwards et al., Chapter 1 Review ofthe Literature 9

1991; Sun and Benishin, 1995; Yousufzai and .~bdel-Latif, 1998; Wellman et • al., 1998), blood vessels (Jansen-Olesen et al., 1996; Wellman et al., 1998), Langerhans cells (Torii et al., 1997), isolated rat thymocytes (Kun et al., 1995),

rat cardiac myoC}1eS (Chatterjee et al., 1991), primary cultures of neonatal rat

spinal cord (Parsons and Seybold, 1997), heart and spleen (Sigrist et al., 1986)

show an increase in cAMP in the presence of CGRP. CGRP has also been

shown to modulate K+ channels via cAMP-dependent protein kinase, protein

kinase A, in vascular smooth muscles (Miyoshi and Nakaya, 1995) and guinea

pig ureter (Santicioli et al., 1995; Maggi et al., 1995). CGRP-dependent

increases in cAMP have been shown to be selectively antagonized by CGRPs-37

(Jansen-Olesen et aL, 1996; Parsons and Seybold, 1997; Yousufzai and Abdel- • Latif, 1998). CGRP Receptors

CGRP Receptor Clones Over the last few years, putative CGRP receptor clones have been isolated

(Kapas el al.. 1995; Luebke et aL, 1996; Aiyar et al., 1996). However, doubt

remains as to the real identity ofthese receptor clones since they share very low

homology (0 each other and do not seem to be expressed extensi\"ely in the

central nervous system or on blood vessels where CGRP binding sites have

been docwnented (Tschopp et al., 1985; Wimalawansa and EI-Kholy, 1993; van

Rossum et al., 1994). • Chapter 1 Review ofthe Literature 10

In 1995, Kapas et af. (1995) identified a canine orphan receptor, RDC-I, as a • CGRPI receptor. This cDNA cloned from the dog thyroid gland shares 30% homology (47% in conserved region) with the cloned adrenomedullin receptor.

The RDC-l gene encodes a seven transmembrane domain protein that confers

sensitive and specifie responsiveness to CGRP in transfected COS-7 cells. This

receptor showed a dose-dependent increase in cAMP in response to CGRP

(ECso= 3x10-9 M) and to adrenomedullin (ECso=lxIO-7 M), an effect blocked by

the CGRP antagonist, CORPS-37, (ICso=5xIO- 10 M). No cAMP response was

recorded in response to amylin or the linear analogue of CGRP,

[Cys(ACM)2.7]aCGRP. Ligand binding studies confirmed the high affinity of

this receptor for rCGRP (Ko=9.2xIO·9M), CGRPs-37 (Ko=13.4xl0-9M) and

adrenomedullin (Ko=1.9XI0·7M). Northern blot analysis identified the heart • and kidneys as the major sites of expression of the RDC-l receptor (Libert et al., 1989). Weaker signaIs were also revealed in the brain and spleen (Libel1 et

al., 1989). However, these data are difficult to replicate as indicated by the

authors (personal communication). Moreover, Tong et al. (1998) shewed that

the RDC-l receptor mRNA was observed in brain regions centaining only low

to very lo\\' levels of CGRP binding sites. Therefore, it seems unlikely that the

RDC-l is a true CGRP receptor and hence remains an "orphan" recepter.

Aiyar el al. (1996) aIse isolated a second clone from a human cDNA library.

This 461 amino acid protein shares homology with the prototypical G-protein­

coupled receptor and 55.5% homology with the human calcitonin receptor. It is • therefere referred to as the "calcitonin receptor like receptor". Northem blot Chapter 1 Review ofthe Literature Il

analysis revealed the expression of this putative CGRP1 clone in cardiac • myocytes and a1veolar cells of the lung. HEK 292 cells expressing the clone show high affinity 1251-CGRP-binding (Ko=19x1O·12M) as weIl as a 60-fold

increase in cAMP in the response to CGRP. This functional response to CGRP

is competitively antagonised by CGRP8-37 (PA2= 7.57). The linear analogue of

CGRP, [Cys(ACM)2.7]aCGRP, failed to induce an increase in cAMP in these

cells. However, it bas been difficult to replicate these findings.

Accessory Proteins Recentiy, McLatchie et al. (1998) cloned a series of receptor-activity modifying

proteins (RAMPs). The RAMP-I was isolated from Xenopus oocytes and

encoded a 148 amino acid protein. RAL\I1.P-l is not~ by itself, a CGRP receptor • as the expression of RAMP-I in mammalian cells did not induce cAMP responses to CGRP or specifie binding to 125I-Iabelled CGRP. The expression

of both G-protein-coupled receptors, ROC1 and CRL~ in Xenopus oocytes did

not alter the endogenous response to CGRP. The interaction of CRLR and

RDCl with RAMP-I was studied in HEK293T cells which are known not to

express any endogenous CGRP or calcitonin receptors. Neither RAMP-l nor

CRLR induced significant responses to CGRP when transfected alone, but

expression of both produced cells that responded to CGRP by increasing

intracellular cAMP levels and binding specifically to 125I-Iabelled CGRP.

ROC 1 did not induce binding or responses to CGRP in HEK293T cells, with or

without the expression of RAMP1. The requirement for CRLR and RA.MP 1 to • reconstitute a CGRP receptor explains why it has been difficult to use Chapter 1 Review ofthe Literature 12

expression cloning for CGRP receptors. Furthermore, the prerequisite • coexpression of CRLR and RAMP1 for CGRP receptor function explains the failure of CRLR alone to function in oocytes and the observation that CRLR

can only function as a CGRP receptor in certain cell lines which presumably

express an endogenous RAMP1. It was shawn by fluorescence-activated cell

sorting CFACS) that RAMP1 increased the cell surface expression ofCRLR. It

is also believed that RAMPI is necessary for the terminal glycosylation of

CRLR.

RAMP1 seems to be part of a family of receptor-activity modifying proteins.

RAMP2 and RAMP3 were isolated from SK-N-MC cell cDNA library and

human spleen mRNA, respectively. In contrast to RAMPI, RAMP2 and

RA.MP3 were unable to potentiate the oocyte response to CGRP. In HEK293T • ceUs, neither RAMP2 nor RAMP3 enabled the CRLR to function as a CGRP receptor. However, the coexpression of RAMP2 and the CRLR resulted in a

large response to adrenomedullin. This exciting discovery implies that the same

G-protein coupled receptor can demonstrate different pharmacological profiles

contingent on its coexpression with either RAMPI or RAMP2.

In 1996, Luebke et al. (1996) isolated a CGRP responsive protein from the

guinea pig organ of Corti. This short hydrophilic protein (146 a.a.) does not

belong to the typical seven transmembrane G-protein coupled receptors and it

has no homology to any known receptor. It is not known whether this protein is

a full functional CGRP receptor or part of a CGRP receptor complex. It is • therefore referred to as the CGRP receptor component protein (CGRP-RCP). It Chapter l Review ofthe Literature 13

was first believed that the CGRP-RCP demonstrated functional activity upon • co-transfection into Xenopus laevis OOC)1eS with the cystic fibrosis transmembrane conductance regulator (CFTR). However, it was later

demonstrated that there is a small endogenous response to CGRP in Xenopus

OOC}1eS (McLatchie et al., 1998). It is still unclear as ta the real raIe of the

CGRP-RCP. It is possible that (i): the CGRP-RCP could be an atypical

receptor resembling the mannose-6-phosphate receptor which is G-protein

coupled despite its lack of the prototypical seven transmembrane helices

(Luebke et al., 1996), or (ii): the CGRP-RCP could be part of a complex of

proteins fonning the CGRP receptor (Luebke et al., 1996). The interleukin 6

and the ciliary neurotrophic factor receptors are examples of such complexes

where a small extracellular membrane-associated protein binds the ligand and • interacts \Vith a membrane-spanning protein for signal transduction (Taga and Kishimoto, 1997; Heinrich et al. 1998).

Distribution of CGRP and its Binding Sites

Calcitonin gene-related peptide distribution

Calcitonin gene-related peptide is widely distributed throughout the rat nervous

system (Skofitsch and Jacobwi~ 1985; Lee et al., 1985; Kawai et al., 1985; for

review see Hôkfelt et al., 1992). In the periphery, CGRP is highly expressed in

the bladder (Wimalawansa et al., 1987a; Wimalawansa, 1992), pancreas

(Wimalawansa, 1992; Ding et al., 1998), penis (Wimalawansa et al., 1987a; • Wimalawansa, 1992), smooth muscle layers of blood vessels (Rosenfeld et al. Chapter 1 Review ofthe Literature 14

1983; Wimalawansa et al., 1987a; Wimalawansa, 1992; Bell and McDennott, • 1996) and the thyroid gland (Wimalawansa et al., 1987a; Wimalawansa, 1992). CGRP was aIso observed in low amounts in the lung (Rosenfeld et al., 1983;

Wimalawansa et al., 1987a; Hôkfelt et al., 1992), gastrointestinai tract (GD

(Rosenfeld et al., 1983; Hôkfelt et al., 1992), adrenai glands (Rosenfeld et al.,

1983; Hokfelt et al., 1992) and in the heart (Rosenfeld et al., 1983;

Wimalawansa et al., 1987a; Wimalawansa and MacIntyre, 1988; Bell and

McDerrnott, 1996) where 4-fold higher levels 'Nere observed in the atria

compared to the ventricles (Franco-Cereceda et al., 1987b).

Systemic capsaicin pretreatment in adult guinea pigs (Franco-Cereceda et al.,

1987b) and newborn rats (Wimalawansa, 1993) decreases CGRP­

immunoreactivity in the circulation, cardiovascular tissues, lungs, GI tract,

• genitourinary tract and nervous tissues. This suggests that CGRP-

immunoreactivity in the peripheral organs is associated \Vith sensory neurones

(Franco-Cereceda el al.. 1987b).

a- and ~-CGRP are detected in the circulation in both human plasma and

cerebrospinal fluid (CSf) (Wimalawansa and MacIntyre, 1987b; Wimalawansa

el al., 1989). It is believed that the high circulating levels of CGRP originate

from the th)Toid and perivascular nerves suggesting that CGRP may be an

important regulator ofvascular tone (Girgis et ol., 1985; Zaidi et al.., 1986). • Chapter 1 Review ofthe Literature 15

Central Nervous System In the central nervous system, CGRP is expressed in discrete brain regions and

• Jacobwi~ in the spinal cord (Skofitsch and 1985; Kawai et al., 1985; Hokfelt

et al., 1992). In the brain, CGRP-immunoreactivity (CGRP-IR) can be

observed in various areas ofthe hypothalamus, hippocampus, dentate gyms and

ail cranial motor nuclei (Rosenfeld et al., 1983; Skofitsch and Jacobwitz, 1985;

H6kfelt et al., 1992). In humans, the pituitary shows high levels ofCGRP with

10wer amounts in cerebral and cerebellar cortices (Tschopp et al., 1985). In the

spinal cord, CGRP-IR fibres fonn a dense network in lamina IIII of the spinal

cord (Wiesenfeld-Hallin et al., 1984; Skofitsch and Jacobwitz, 1985;

Wimalawansa et al., 1987a). In humans, the highest levels of immunoreactive

CGRP are detected in the dorsal horn of the spinal cord while 10wer amounts • are observed in the ventral horn ofthe spinal cord (Tschopp et al., 1985). Sensory System The high levels of CGRP-immunoreactivity in the dorsal horn suggests a role

for CGRP in sensory processes. CGRP is the most abundant peptide in

neurones of the dorsal root ganglia (DRG) with approximately 50% ofthe cells

showing immunoreactivity (Rosenfeld et al., 1983; Skofitsch and Jacob\\'itz,

1985; \Vimalawansa et al., 1987a). In spinal ganglia, aU substance P-

immunoreactive (SP-IR) cell bodies aIso contain CGRP-immunoreactivity

while not alI CGRP-immunoreactive ceIls contain SP-irnmunoreactivity

(\Viesenfeld-Hallin et al., 1984; Lee et al., 1985). CGRP and substance P are

also co-Iocalized in axonaI boutons of the superficial dorsal horn (Ribeiro-da- • Silva~ 1995). CGRP-immunoreactive neurones have their tenninals in various Chapter l Review ofthe Literature 16

areas of the spinal cord (laminae l, n, v and X) (Gibson et al., 1984) and • brainstem (Rosenfeld et al., 1983) and are believed ta relay somatic cutaneous pain and temperature information. CGRP-rich nerve fibres fonn part of the

primary afferent nervous system, comprising capsaicin-sensitive A(eS) and C

fibre afferent nerves (Franco-Cereceda et al., 1987b). Sensory neurones are

enriched in CGRPa, containing three to six limes more CGRPa than CGRP~

(Gibson et al., 1984; Mulderry et al., 1988). Both CGRPa and CGRP~

rnRJ.'JAs are located in the dorsal root ganglia (Mulderry et al., 1988).

Calcitonin gene-related peptide binding sites distribution

CGRP binding sites have been studied in man and rat and are widely distributed

throughollt the nervous system (Skofitsch and Jacobwitz, 1985; Tschopp et al., • 1985: Dennis et al., 1991). CGRP receptors are also present in abundance in the cardiovascular system (Sigrist et al., 1986; Wimalawansa et al., 1987a;

\\ïrnalawansa and MacIntyre, 1988; Wimalawansa, 1992; Wirnalawansa and

EI-Kholy. 1993), blood vesse1s (Sigrist et al., 1986; \Vima1awansa et al., 1987a;

\'"irnalawansa and MacInt)Te, 1988; Wimala~\.'ansa, 1992; Wimalawansa and

EI-Kholy. 1993), spleen (Sigrist et al., 1986; Wimalawansa et al., 1987a;

\\ïrnalawansa, 1992; Wimalawansa and EI-Kholy, 1993), penis (Wimalawansa

el al.. 1987a; \Vimalawansa, 1992; Wimalawansa and EI-Kholy, 1993), vas

deferens (Dennis et al., 1990), lungs (\Vimalawansa, 1992) and adrenal gland

(\Virnalawansa et al., 1987a; Wimalawans~ 1992; Wimalawansa and E1-Kholy, • 1993). Moderate levels of binding are also observed in the pancreas Chapter 1 Review ofthe Literature 17

(Wimalawansa, 1992) and bladder (Wimalawansa et al., 1987a; Wimalawansa, • 1992). Negligible amounts are detected in kidneys (Wimalawansa, 1992), muscle and liver (Wimalawansa et al., 1987a).

Nervous System In the central and peripheral nervous systems, high densities of CGRP binding

sites are observed in the cerebellum, dorsal spinal cord, nucleus accumbens,

amygdaloid complex, mammilary body, superior colliculus, inferior olive and

temporal and frontal cortices (Dennis et al., 1991; Wimalawansa, 1992). The

substantia nigra, rnedulIa, pons, striatum, hypothalamus, hippocampus, medial

geniculate nucleus and inferior colliculus show intennediate levels of CGRP

binding ( Dennis et al., 1991; Wirnalawansa, 1992). In humans, binding sites

for 125I_CGRP are detected in high quantities in the cerebellar cortex, spinal • cord and nucleus dentatus; intermediate levels in the inferior colliculus and substantia nigra while oruy low amounts in the hippocampus, amygdala,

superior coUiculus, thalamus, hypothalamus and globus pallidus (Tsehopp et

al., 1985; Wimalawansa and EI-Kholy, 1993).

The highest density of specifie 12sI-hCGRPa. binding in the rat spinal cord was

observed around the central canal while the deeper dorsal horn and \"entrai horn

shov;ed moderate labelling ( Yashpal et a/., 1992, Kar and Quirion, 1995). The

superficial dorsal horn and white matter showed relatively low densities of

specifie labelling. The widespread distribution of 125I_hCGRP binding sites in

the developing spinal cord suggests the possible involvement ofthis peptide and • its receptor in the growth, development and normal maturation ofthe cord (Kar Chapter 1 Review ofthe Literature 18

and Quirion, 1995). No binding was observed in the dorsal root ganglia of • mature rats (Tschopp et al., 1985; Wimalawan~ 1992; Wirnalawansa and El­ Kholy, 1993).

CGRP Functions

Although the exact biological functions of CGRP have not been fully

characterised, its anatomical distribution suggests raIes ln autonomie,

somatosensory, integrative and motor functions (Rosenfeld et aL, 1983;

Rosenfeld et al., 1992; Wimalawansa, 1996; WimaIav~'ansa, 1997; for review

see van Rossum et al., 1997).

Ingestive Behaviour

CGRP is located in brain areas involved in the control of ingestive behaviour

• (Rosenfeld et al.~ 1983). Intracerebroventricular (lCV) and peripheral

administrations of CGRP suppress food intake in both rats (Krahn et al., 1984)

and mice(~10rley et al., 1996). leV eGRP injection aise inhibits gastric acid

secretion(Hughes et al., 1984; Rossowski et al., 1997).

Cardiovascular Functions

CGRP is probably the most potent vasodilator currently kno\\n (for review see

Bell and McDermon, 1996; Brain and Cambridge, 1996). Intradermai injection

of eGRP in fentomole doses induces microvasculature vasodilatation

(arterioles) resulting in increased blood flow 10-100 times greater that that of

the synthetic ~-adrenergic stimulant, isoprenaline (Brain et al., 1985b). • Intravenous injection in humans has been shown to cause and Chapter 1 Review ofthe Literature 19

hypotension (Franco-Cereceda el al., 1987a). CGRP produces inotropic and • chronotropic effects in humans (Franco-Cereceda et al., 1987a) and in isolated heart preparations (Franco-Cereceda and Lundberg, 1985; Sigrist et al., 1986;

Giuliani et al., 1992). These effects are blocked by CGRPs-37 (Giuliani et al.,

1992). CGRP has aIso been shown to relax arterioles (Edwards et al., 1991),

small and large coronary arteries in vitro ( Foulkes et al., 1991; Jansen-Olesen

et al., 1996; Wellman et al., 1998). Finally, there is a significant reduction of

CGRP immunoreactive fibres in the skin of patients suffering from Raynaud's

disease (Bunker et al., 1990). Raynaud's disease is characterized by intense

spasm of local small arterles and arterioles (Kumar et al., 1992). Although the

cause is unknown, it appears to be based on an exaggeration of nonnal central

and local vasomotor responses to coId or to emotion.

• Nociception The presence of CGRP in areas known to be involved in sensory processes and

its co-localisation \\ith the neuropeptide substance P suggest raIes for CGRP in

nociception.

Intrathecal injections of CGRP do not produce aversive reactions (\Viesenfeld­

Hallin et al., 1984) nor analgesic responses (Jolicoeur et al., 1992). However,

when injected along with substance P, it potentiates the scratchinglbiting

behaviour observed following the administration of SP alone (\Viesenfeld­

Hallin et al., 1984), possibly by inhibiting the metabolic degradation of • substance P (Le Greves et al., 1989; Mao et al., 1992). Chapter 1 Review ofthe Literature 20

Intracerebroventrieular (Le.v.) administration of CGRP produces • antinociceptive responses in aeute pain assays such as the tail·fliek and hot· plate tests, an effect blocked by CGRPS.37 (Bates et al., 1984; Pecile et al.,

1987; Jolicoeur et al., 1992). Hence, CGRP plays a role in the transmission of

nociceptive information.

1nflammation

The vasodilatory effects of CGRP have been shown to increase

and neutrophil accumulation as weil as potentiate the vascular leakage mediated

by SP (Brain and Williams, 1985a; Brain et al., 1992).

The implication of CGRP in inflammation has been studied in severa! animal

models. Several groups have shown that CGRP markers are modulated during • the development ofarthritis. CGRP rnRNA (Donaldson et al., 1992; Galeazza et al., 1995) and CGRP·content (Smith et al., 1992) 'was increased in ipsilateral

ORO ofarthritic rats. CGRP was also sho\\'n to be decreased in the superficial

dorsal horn at 1 and 2 days following injection of Freund's complete adjuvant

(FeA) and then increased to levels greater than controls at 8 days (Seybold et

al., 1995; Galeazza et al., 1995).

I.25 I.hCGRP binding was decreased in laminae 1111 4 days following the injection

of fCA (Galeazza el al., 1992) but not at 2 and 8 days (Abbadie el a!., 1996).

Release of immunoreactive CGRP from spinal cord dorsal horn is also

enhanced (Garry and Hargreaves, 1992; Collin et al., 1993) or unchanged

(Galeazza el al. 'J 1995; Malcangio and Bowery, 1996) following the induction • ofarthritis. Chapter 1 Review ofthe Literature 21

Intrathecal administration of antiserum against CGRP produced analgesic • responses in thermal and mechanical pain assays follov.ing the injection of carrageenan (Kawamura et al., 1989) and fCA (Kuraishi et al., 1988).

CGRPs-37 increased the withdrawal latencies to both thennal and mechanical

stimulation in normal rats (Yu et al., 1994) as well as rats with unilateral

carrageenan-induced inflammation (Yu et al., 1996).

Neuropathic Pain

Modulation of CGRP and CGRP binding sites have aIso been shown following

neuropathic injury. CGRP was shown to be decreased in the spinal cord

following chronic constriction injury (Bennett et al., 1989; Kajander and Xu,

1995; Carlton and Coggeshall, 1996; Xu et al., 1996). Garry et al. (1991) found • no change in 125I_human CGRP binding in the dorsal spinal cord of rats at 2, 5, 10 and 20 days folIo\\ing chronic constriction injury. However, the same group

showed a large increase in 125I_hCGRP binding ipsilateraI to the lesion at 4 and

8 days fol1owing dorsal rhizotomy (Garry el al., 1991).

As for inflammation, CGRPS-37 increased the withdrawal Iatencies to both

thennal and mechanical stimulation in rats with unilateral mononeuropathy (Yu

et al., 1996).

CGRP in the development of morphine tolerance

Considerable efforts have been made in order to understand the modifications • occurring in the central nervous system CCNS) during the long tenn Chapter 1 Review ofthe Literature 22

administration of opioids, particularly during the development of tolerance to • their antinociceptive effeets. Tolerance is deseribed as a diminution of effeets after exposure to a drug or the need for a higher dose of a drug to maintain a

given response (Portenoy, 1994). Exposure to a drug is thought to be the

'"driving force" for the development oftolerance and the need for a higher dose

due to progressing pathology should not he eonsidered as toleranee (portenoy,

1994). It is welI known that chronie morphine administration produees

tolerance to the analgesie, thermal, respiratory depressant, euphorie, locomotor

depressant and stimulant effeets ofthis drug (Bhargav~ 1994).

Opioids produee their pharmaeologieal effeets tbrough three types of receptors,

Jl, 8, and le opioid reeeptors loeated in several regions of the brain and spinal

cord (Fowler and Fraser, 1994; Bhargav~ 1994). Investigators first

• hypothesized that toleranee oceurred primarily through critical alterations in

opiate receptors as weIl as endogenous opiate systems. However, the nature of

these critical alterations is not yet resolved as some authors show no change

(Hallt el al., 1975; Gouardères et al., 1993), an upregulation ( Pert and Snyder,

1976; Brady el al., 1989; Rothman el al., 1991) or a downregulation (Werling et

al., 1989; Bhargava and Gulati, 1990) of the opioid receptors. Consequently,

other mechanisms that could possibly be involved in the modulation of

tolerance have been studied. Substance P (SP) is known to he located in smail

primary afferent neurons involved in pain transmission. It is aIso thought that

opiates act partly by inhibiting the release of SP in vivo (Yaksh el al., 1980). • This suggests that SP and neurokinin-VSP reeeptor sites could be modified Chapter 1 Review ofthe Literature 23

during the development oftolerance. However, it was sho\W that SP receptors • were not significantly altered during morphine tolerance (Gouardères et al., 1993).

CGRP has been shown to be involved in nociception and to be co-Iocalised with

substance P (SP) in dorsal root ganglia (Lee et al., 1985) and superficial dorsal

horn (Plenderleith et al., 1990; Ribeiro-da-Silv~ 1995). Thus, it seemed

plausible that CGRP may have a raIe in the development ofmorphine tolerance.

Ménard et al. (1995a) studied the effects of CGRP on the development of

tolerance in animals administered a chronic intrathecal infusion of morphine

(7.5Jlg/hr) (Ménard et al., 1995b). Initially, the tail-immersion latencies, in

response to morphine, increased ta reach a maximum on day 3. On day 5, the

latencies were not significantly different from controis indicating the beginning

• of tolerance to the effects of morphine. Tolerance ta morphine analgesia was

maintained up to the 14th and last day of infusion. A marked increase ofCGRP

immunostaining was observed in the superficial laminae (1 & II) of the spinal

cord after 5 days of treatment. Substance P immunostaining was slightly

increased after 5 days only while no change \\'as observed in galanin

immunostaining. On the Sth day of infusion, (25I]hCGRPa binding was

reduced by SO% in the superficiai Iaminae (II & III) ofthe dorsal homo Similar

changes were observed following the chronic infusion of the delta agonist

OPOPE, [D-Pen2, D-Pen 5], but not of the kappa agonist, U-S0488H (Ménard

125 125 125 et al., 1995a). [ I]BH-SP, [ I]galanin, [125I] and [ I]NPY • receptor binding sites in the dorsal horn of morphine-treated animaIs were not Chapter 1 Review ofthe Literature 24

significantly different from saline-treated animaIs on the 5th day of infusion. • Ménard et al. (l995~ b) postulated that chronie morphine administration causes the tolerance to the inhibitory effect of morphine on the release of CGRP.

Consequently, there is an increase in CGRP aIong with a concomitant

downregulation of i15 receptors. Moreover, Ménard et al. (1996) has shown

that the potent CGRP antagonis1, hCGRPS-37, could prevent the development of

morphine tolerance in acute pain models such as the paw pressure and tail

immersion tests as weIl as the changes in hCGRPa immunostaining and binding

sites (fvlénard el aL, 1996).

Morphine is frequently used for the treatment of moderate to severe pain

associated with carcinoma, biliary or renaI colic and surgery (Bhargava, 1994). • In chronic pain treatment, toleranee to its analgesic properties leads to dose escalation and increases in side-effects including respiratory depression. It is

therefore critical to study the interactions ofCGRP and opiates and to test ifthe

antagonist hCGRPs-37 can black the development of morphine tolerance in

chronic pain models.

It is hypothesised that the potent CGRP antagonist hCGRPs-3i will block the

development of morphine tolerance in both acute and chronic pain models. As

a first step towards this goal, the aim of the present thesis is to study the

modulation of CGRP and its binding sites in models of chronic pain. The

outcome of these experiments are intended to complement the study of

morphine tolerance in models ofchronic pain as weIl as to provide insights into • the efficacy of the CGRP antagonist, hCGRPs-37, in preventing the development Chapter 1 Review ofthe Literature 25

of morphine tolerance in chronic pain. In order to detennine whether the • possible changes observed in CGRP binding sites or immunostaining in response to chronic pain are specifie for CGRP, substance P and galanin

binding sites and immunostaining were aIso monitored since both neuropeptides

are believed to he involved in chronic pain (Henry et al. 1976, \Viesenfeld­

Hallin et al. 1992). The following sections present a brief summary on

substance P and galanin as weIl as their implications in pain mechanisms.

•

• Chapter 1 Review ofthe Literature 26

Substance P

• Substance P ~ an Il amino acid neuropeptide, was discovered in 1931 by von Euler and Gaddum (1981) but it was not until 1971 that it was purified and its

structure isolated by Leeman and colleagues (Chang and Leeman, 1970). It is a

member ofthe tachykinin family along \\'Îth neurokinin A (NKA)~ neurokinin B

(NKB), neuropeptide K (NPK) and neuropeptide gamma (NP-y) (Tatemoto et

al., 1985). AU of the mammalian so far isolated have a

common COOH-tenninal sequence, Phe-X-Gly-Leu-Met-NH2 where the "X"

residue is either Phe or Val. The gene organization for the preprotachykinin-A

and preprotaehykinin-B precursors are very similar (Kotani et al., 1986). This

suggests that the mammalian tachykinin system has acquired diversity through • various cellular mechanisms including gene duplication, differential expression ofduplieated genes, and alternative RNA splicing (Kotani et al.~ 1986).

The rat preprotachykinin-I (PPT-O gene rnRNA is altematively spliced in a

tissue-specifie manner to yield four different rnRNA, alpha-~ beta-~ delta- and

gamma-preprotachykinin (Nav..·a et al., 1984; MacDonald et al. ~ 1989; Lai et

al. 1998). Alpha-PPT is processed ta the mature undecapeptide substance P.

Beta-PPT is proeessed into various products including substance P, neurokinin

A~ neurokinin (3-10) and neuropeptide K while gamma-PPT (Kawaguchi et al.,

1986) gives rise ta substance P, neurokinin A, neurokinin A (3-10) and

neuropeptide y. A delta isoform of preprotachykinin rnRNA has also been

identified in the rat intestine (Khan et al. 1994), dorsal root ganglia (Hannar et • al., 1990) and human mononuclear phagoc~1es and lymphocytes (Lai et al., Chapter 1 Review ofthe Literature 27

1998). The sequence analysis of the ô-PPT isoform predicts the existence of a • novel tachykinin precursor polypeptide containing substance P. However, it is still to be determined ifthis new precursor is translatable (Lai et ai., 1998). The

second mammalian tachykinin precursor, preprotachykinin-II (pPT-II) is

processed to yield neurokinin B (Kotani et al., 1986).

Neurokinin Receptors

Three neurokinin receptors have been isolated: neurokinin-l ~l(-I),

neurokinin-2 (NK-2) and neurokinin-3 (l\llC-3) (Nakanishi, 1991; Fong, 1996).

Neurokinin receptors share significant homology with receptors of the G­

protein coupled receptor family (Routh and Helke 1995). Like ail G-protein­

coupled receptors, these receptors have a-helical transmembrane domains, three

• extracellular loops, three cytoplasmic loops and a cytoplasmic C-tenninal

region. AlI three receptors are encoded in five exons that are very similar

between the three genes. Thus, the neurokinin receptors share 54-66%

homology in their transmembrane and cytoplasmic regions (Routh and Helke,

1995). This considerable homology between the receptor subtypes is consistent

\\ith the fact that all three tachykinins are able to bind to aIl three receptors.

Substance P binds preferentially to the NK-l receptor while neurokinin-A and

neurokinin-B bind with higher affinity to the NK-2 and NK-3 receptors

respectively (Routh and Helke, 1995). The activation of all three receptors

resuit in hydrolysis of phosphoinositols and increased levels of cA.~P • (Nakajima et al. 1991, Krause el al. 1993). Chapter 1 Review ofthe Literature 28

Distribution of Substance P and ils bindina sites in the • spinal cord

Substance P is highly concentrated in the superficiaI layers (I-III) of the dorsal

horn where most primary afferent fibres terminate. Unilateral rhizotomy results

in a dramatic decrease in substance P content suggesting its presence in primary

afferent terminaIs (Takahashi and Otsuk~ 1975; Hokfelt el al., 1975; Kajander

and Xu, 1995)

Substance P Binding Sites in the Spinal Cord

The highest density ofsubstance P binding sites was observed in the superficial

dorsal horn and in the region surrounding the central canal (Rossler el al., 1993; • Kar and Quirion, 1995). Moderate to low densities were detected in deeper laminae and in the ventral homo

Substance P Functions

In addition to their raie in sensory transmission, tachykinins have been

implicated in a variety ofCNS functions such as the control ofmotor activities,

autonomie and endocrine functions and memory processing. This section \vill

concentrate on the raie of substance P in sensory processing (for review see

CueHo, 1993).

The involvement of substance P in the development of chronic pain has been

studied in sorne details. It has been shown that substance P and its binding sites • are altered in various models ofchronic pain. In models ofinflammation, it has Chapter 1 Review ofthe Literature 29

been dernonstrated that substance P immunoreactivity (Smith el al., 1992) and • preprotachykinin mRNA (Donaldson et al., 1992) were iDcreased in dorsal root ganglia neurones. An increase in the release of substance P( Ok'U el al., 1987;

Garry and Hargreaves, 1992) and the expression of its binding sites has also

been demonstrated (Abbadie et al., 1996).

Differentiai effects have been observed in models of neuropathic pain.

Substance P immunoreactivity was significantly decreased in the dorsal horn

(Takahashi and Otsuk~ 1975; Hokfelt et al., 1975) following neuropathy while

substance P receptor (NK-l) immunoreactivity \\·as increased in nerve injury

models of chronic pain (Abbadie et al., 1996). Hence, substance P has been • proposed as a pain transmitter (Henry, J.L. 1976; De Koninck et al., 1992). Galanin

Galanin is a 29/30 amino acid peptide isolated from the porcine upper small

intestine (Tatemoto et al., 1983). Its name is derived from its N-tenninal

and C-terminal alanine. It shows no homology to any other known

peptides and therefore forms its O\Nn family. Galanin is a phylogenetically oid

peptide and was weIl conserved throughout evolution; human, porcine and rat

galanin showing 90% homology. AIl galanin sequences determined so far

consist of29 arnino acids except the human galanin \\'hich has 30 amino acids.

The tirst 15 N-tenninal residues are fully conserved while the C-tenninal • portion shows a higher degree of variability. This ubiquitous neuropeptide Chapter 1 Review ofthe Literature 30

controls numerous functions such as endocrine secretions (Bauer el al., 1986) • and ingestive behaviour (McDonald el al., 1985; Crawley el al., 1990).

Distribution of Galanin and ils Binding Sites

Galanin is abundant in both the central and peripheral nervous systems. In the

central nervous system, galanin -immunoreactive (Gal-IR) structures were

observed in high levels in the superficial layers and intemeurons of the spinal

cord (Melander el al., 1986). In the peripheral nervous system, sensory dorsal

root ganglion cells (DRG) show moderate levels ofgalanin-irnmunoreactivity.

Galanin Binding Sites in the Spinal cord

Galanin binding sites show a widespread distribution in the nervous system • (Melander et al., 1986) and in neurons innervating the GI tract (King et al., 1989). Their distribution seems to be weIl conserved among different species

(Ma and Bisby, 1997). Autoradiographie mapping shows that the expression of

galanin binding sites is in good correlation with the distribution of galanin-like

immunoreactivity. In the dorsal spinal cord, the highest density oflabelling was

observed in superficial layers of the dorsal hom \vhile moderate labelling was

detected around the central canal and laminae IV-V( Melander et al., 1986; Kar

and Quirion, 1995). • Chapter 1 Review ofthe Literature 31

Galanin Receptors • Two galanin receptors have recently been cloned, GalR-l and GalR-2. GALR-1

A cDNA coding for a human galanin receptor \.\ras isolated from a Bowes

melanoma cellline (Habert-Ortoli et al., 1994). It is a 349 amino acid protein

\vith 7 putative hydrophobie transmembrane domains and it shows significant

homology to the members of the guanine nuc1eotide binding protein-coupled

receptor family. The cloned receptor expressed in COS-cells specifically binds

human, porcine and rat galanin with high affinity (Ko=O.8±O.2nM). The

primary sequence shows homology (30%) to the human 4 and

human delta opioid receptors.

• GALR-2 The GaIR-2 receptor cDNA was recently isolated from the rat hypothalamus

('Nang el al., 1997). The receptor is 372 amine acids in length and shares 40%

homology with the rat GalR-1 receptor. It contains seven putative

transmembrane domains. Northem blot analysis revealed a more -widespread

distribution for GaIR-2 suggesting a broader functional range than for GaiR-I.

125I_hurnan galanin binds with high affinity to the GalR-2 receptor expressed in

COS-} cells (KD=0.59nM). The pharmacological profiles ofGalR-2 and GaIR­

1 differ in their affinities for the galanin fragment, galanin2-30 (Wang el al.,

1997). Activation of the cloned GALR-2 receptor by galanin led to the • inhibition ofcAMP production. Chapter 1 Review ofthe Literature 32

Galanin Functions • Although galanin is involved in ingestive behaviour (McDonald et al., 1985; Crawley et al., 1990) and the neuroendocrine system (Bauer et al., 1986), this

section will focus on the involvement of galanin within the sensoI)' system (for

review see Wiesenfeld-Hallin et al. 1991).

Galanin has been shown to be inhibitory to excitatory peptides in the spinal cord

and to produce a tonic inhibition of spinal cord neurone excitability (XU et al.,

1989). For example, galanin antagonized the effect of substance P on the

nociceptive flexor reflex in the rat (XU et al., 1989).

Galanin has been shown to have weak analgesic effects in acute pain assays

such as mechanical and thermal tests when administered intrathecally in mice • (Post et al., 1988) and rats (Wiesenfeld-Hallin et al., 1993). A low dose of galanin, not antinociceptive by itself, was aIso shown to potentiate the

antinociceptive effects of morphine in the hot plate test (Wiesenfeld-Hallin et

al., 1990).

Galanin has aiso been sho\\TI to be modulated in models of chronic pain.

Galanin immunoreactivity significantly increased in the superficiai laminae of

the dorsal hom following chronic constriction injuI)', partial injury and

complete transection of the sciatic nerve (Wiesenfeld-Hallin et al., 1992;

Carlton and Coggeshall, 1996; Ma and Bisby, 1997). A marked increase in

ipsilateral galanin immunoreactive ganglion cell bodies was observed in rats • with unilateral transection of the sciatic nerve and in rats with peripheral Chapter 1 Review ofthe Literature 33

axotomy (Hôkfelt et al., 1987). However, no change was observed in galanin • binding sites in the dorsal horn following peripheral axotomy (Zhang et al., 1995).

Although galanin seems to he involved in neuropathic pain, its involvement in

inflammation is still uncertaÏn. The induction of inflammation did not produce

any change in galanin-immunoreactivity in laminae 1and II ofthe lumbar spinal

cord 3 days after the injection ofcarrageenan (Ji et al., 1995). However, a 63%

increase in galanin mRNA in the superficial dorsal horn and significant

decreases (47%) in immunoreactivity and mRNA levels (39%) were observed

in the dorsal root ganglia. No galanin binding "pas observed in dorsal root • ganglia ofcontrol and treated animais (Ji et al., 1995).

• Chapter 1 Review ofthe Literature 34

Chronic Pain

• Pain serves as a physiological waming for potentially tissue-damaging situations. It is normaUy evoked by a transient stimulus that is not associated

with significant tissue damage. Most persistent pain associated with

hyperalgesia, tendemess and generally inflammation can also he considered a

normal protective response ta mild injury. In this case, the pain would resolve

rapidly once the injury bas healed (Dray and Urban, 1996).

A number ofchronic pain conditions occur in which the stimulus and pain are

unrelated and pain can no longer be regarded as a pbysiologically protective

symptom. Sucb cases include chronic pathological lesion, degenerative

process, arthritis, low-back pain, cancer, neuropathic pain, migraine, pelvic and • abdominal pain (Dray and Urban, 1996). During normal physiological conditions, nociceptive signals are generated by

intense thennal, mechanical or chemical stimuli. In general, the adequate

stimuli for nociceptors are stronger than those needed to activate

mechanoreceptors or specific thennoreceptors (Dickenson, 1995). These

stimuli activate specialised nerve fibres, fine myelinated Aô and unmyelinated

C fibres. A8 fibres are thought to be responsible for sharp pricking pain

sensations (Kelly, 1985). The polymodal C-fibres respond to high threshold

mechanical, chemical and thermal stimulations which mediate long lasting • buming pain (Kelly, 1985). Dorsal root ganglion ceUs giving rise to nociceptors Chapter 1 Review ofthe Literature 35

belong to the small cell c1ass and generally contain one or more peptides such • as substance P, somatostatin and CGRP (Dickenson, 1995). Signais are conducted through these nociceptors ta the spinal cord where they

will be modified by local mechanisms or from higher centres and then will be

transmitted to the thalamus and cerebral cortex where further processing occurs

resulting in pain awareness (Berkley and Hubscher, 1995). Disease,

inflammation or injury to the peripheral and/or central nervous system induces

remarkable changes in the nociceptive pathways: heightened excitability,

alterations in gene regulation and the expression of new molecules including

, enzymes and receptors. Chemical mediators are produced in

the peripheral nervous system in association ""ith tissue damage and

inflammation. These products arise from the damaged tissue themselves, the

• vasculature, sensory and sympathetic neurones and immune ceUs (Dray and

Urban~ 1996).

Recently, experimental models of pathological pain have been developed that

permit the study of mechanisms on inflammatory and neuropathic pain states.

An understanding of the pathophysiology in these experimental models may

lead to improvements in our understanding of comparable human states and

hopefully improved therapy. In this present thesis, two models of chronic pain

have been investigated: the Freund's complete adjuvant inflammatory pain

model and the sciatic nerve constriction. • Chapter 1 Review ofthe Literature 36

Freund's Complete Adjuvant

• Freund's adjuvant polyarthritis in rats bas been used extensively to study pain processes of long duration. However, the severe systemic changes provoked

ethical concems and also affected behaviour, physiology and biochemistry.

Severa! groups presented limited arthritic models for the study of chronic pain

in rats as an alternative to the polyarthritic rat (Butler et al., 1992; Donaldson et

al., 1993). The complete adjuvant (Mycobacterium butyricum) injected in the

tibio-tarsal joint produces a stable predictable model of monoarthritis (Butler et

al., 1992). Animais gain weight and remain active indicating the absence of

systemic disturbances as opposed to the polyarthritic rat (Butler et al., 1992).

Monoarthritic models have yielded much useful data on mechanisms specific to

the inflammatory process and its spread. For these reasons, the monoarthritic

• inflanunatory model was chosen and used in the present thesis.

Chronic Constriction lnjury

This peripheral mononeuropathy is produced in adult rats by placing 4 loose

chromic gut sutures around the sciatic nerve (Bennett and Xie, 1988). The

animais show post-operative hyperalgesi~ allodynia and possibly spontaneous

pain. Several groups have successfully used the chronic constriction injury

(CCI) model to induce relatively consistent nocifensive behaviours but

variability has been reported in the alteration of fibre spectrum in the portion of

the ne.rve distal to the constriction. Mosconi and Kruger (1996) hypothesized • that the extent ofvariation reported using the ligation model is sufficiently large Chapter 1 Review ofthe Literature 37

to render quantitative analyses unreliable, especially analyses of CNS changes. • Most likely, the variability is due to the difficulty in controlling the tightness of the constriction. Mosconi and Kruger (1996) aIso proposed a new model of

neuropathic pain. This model aims at standardizing the degree of injury to the

sciatic nerve in an attempt to decrease inter-animal variability. Fixed-diameter

polyethylene tubing cuffs are applied to the rat sciatic nerve and produced

nociceptive behaviours similar to that produced by the CCI model of Bennett

and Xie (1988) with the important advantage that cuffneuropathy appears more

consistent in the magnitude ofthe nerve injury. Application ofcuffs is easy and

standardised cuff sizes produce a controlled, reproducible nerve injury that

should render quantitative analyses of central alterations more reliable and

easily interpretable. In addition, the cuff model induces nociceptive responses • suggestive of pain similar to human neuropathic pain. This model aIso has the advantage oflimiting the self-mutilatory behaviours so that this behaviour is the

exception rather that the mie as in most human neuropathies.

Aim of the Thesis

The aim of this thesis is to study possible alterations in CGRP markers in

neuropathic and inflammatory models ofchronic pain. Galanin and substance P

markers were also studied to assess the specificity ofthe observed changes. • • Chapter Il Materials and Methods

•

• Cbapter fi Materials and Methods 39

A) Behaviour

• 1) Chronic Pain Models

Adult maie Sprague-Dawley rats (l75-225g) were obtained from Charles River

(St-Constant, Quebec, Canada). Animais were rnaintained according to the

guidelines of the Canadian Council on Animal Care and given free access to

food and water. The protocols were reviewed and accepted by the internai

committees of the institutions where the experiments were conducted: Institut

Armand-Frappier (Laval, Quebec) and the Astra Research Centre Montreal (St­

Laurent~ Quebec).

a) Monoarthritic model

Materials • Isoflurane was purchased from CDMV mc. (St-Hyacinthe, Quebec, Canada). Freund"s complete adjuvant (FCA) (i.e. mycobacterium bUt)TÎcum suspended in

mineraI oil, [lmg/ml]) and mineraI oil were obtained from Sigma Aldrich

Canada Ltd. (Oakville, Ontario. Canada).

Methods A solution of four parts of FCA and one part of minerai oil were mixed

thoroughly. Under brief isoflurane anaesthesi~ 50JlI of the FCA solution was

injected into the articulation of the left hindpaw using a 26 gauge 5/8 needle

(Butler el al., 1992). An equal volume of minerai oil was injected into the paw

ofcontrol rats. Paw inflammation was estimated by plunging the hindpaw into a • beaker filled with water and placed on a balance. Volume displacement was Chapter il Materials and Methods 40

measured in grams and reported in millilitres according to the fol1owing rule: • one millilitre ofH20 weighs one gram. b) Neuropathic model

Materials Ketamine/xylazine was purchased from Research Biochemicals International

(MA~ USA). Polyethylene tubing {lntramedic, Clay Adams (ID 0.03"» was

obtained from VWR Canlab (Mississaug~ Ontario, Canada). Suture, silk 4.0,

was obtained from CDMV me. (St-Hyacinthe, Quebec, Canada).

Methods Surgeries were perfonned under ketamine/xylazine (O.Icc/lOOg i.p.)

anaesthesia. A skin incision of 1cm was made at the mid-thigh level, the

muscle layers were gently separated and the sciatic nerve was isolated. A 2mm­ • euff of PE-60 polyethylene tubing was eut longitudinally and used to encapsulate the left sciatic nerve (Mosconi and Kruger~ 1996). Sciatic nerves of

sham animals were exposed but not eneapsulated. The skin was sutured in

three areas using 4.0 silk suture.

2) Behavioural Testing

a) Thermal hyperalgesia

Paw-fliek latencies (Ugo Basile, Stoelting Co.~ IL., USA) were recorded over 14

days. • Chapter II Materials and Methods 41

b) Mechanical allodynia • Rats were placed on a wire mesh platform covered by a plastic cage that allowed full access to the paws. Rats were acclimatised to their new

environment 20-30 minutes prior to the experiment. A modified version ofthe

up-do\\n method was used to assess mechanical allodynia. The mid-plantar left

hindpaw region was touched with 1 of a series of 7 von Frey filaments with

logarithmically incremental stiffness (0.45, 0.98, 2.35, 4.64, 7.37, 12.50,

20.90g) (Semmes-Weinstein monofilaments, Stoelting Co., IL., USA) as

routinely used in our laboratory. The von Frey hair was presented perpendicular

to the plantar surface with sufficient force to cause a slight buckling against the

paw, and held in place for approxirnately 5-6 seconds. A positive response was

noted if the paw was withdrawn, the animal flinched upon removal of the hair • or prevented the application of the stimulus by lifting its paw. Walking was considered an ambiguous response and the stimulus was repeated. The eut-off

of20.9 grams \Vas chosen since stiffer hair raised the entire limbe

3) Statistics

StatisticaI analyses were performed in SigmaStat (landeI Scientific Software,

San Rafael, CA, USA) or GraphPad Prism (San Diego, CA, USA). Statistics on

mechanical allodynia were calculated using the Friedman test (non-parametric

repeated measures ANOVA) followed by post-hoc Student-Newman-Keuls test.

Thennal hyperalgesia and inflammation were calculated using a repeated • Chapter II Materials and Methods 42

measures two-way ANOVA followed by a Bonferroni post-hoc analysis. • Statistical significance was set at p

B) Immunohistochemistry

1) Perfusions

Rats (n=4-6 per group) were perfused 24 hours, 4 days, 7 and 14 days following

neuropathic surgery or FCA injection.

Materials Sodium pentobarbitaI (SomnotoI, 65mg/ml) was obtained from CDMV Ine. (St-

Hyacinthe, Quebee, Canada). Picric acid was purchased from Fisher Scientific

(Montreal, Quebec, Canada). Poly-L-lysine, poly-prep sIides, sodium nitrite, • sodium carbonate and disodium phosphate were obtained from Sigma-Aldrich Canada Ltd. (Oakville, Ontario, Canada). AIl other chemicals were of

analytical grade and obtained from VWR Canlab (Mississauga, Ontario,

Canada).

Methods Rats \Vere deeply anaesthetised with sodium pentobarbital (1 OOmg/kg i.p.). The

animaIs were restrained 10 a board and a transverse incision of the skin and

muscle wall was made beneath the sternum and on both sides of the thoracic

cage. The flap forrned by the ventral wall of the thoracic cage was lifted and

pinned to the board using 20 gauge needles. The heart was released from the • pencardium and a blunt stainless steel needle (20g I~~I/2) was insened through Chapter II Materials and Methods 43

the left ventricle and then upward into the aona. The cannula was held in place • using a hemostatic clamp. An incision was made through the right atria. The animaIs were then perfused intracardially at a rate of SOmllminute using

approximately lOOml of phosphate buffer followed by 300ml of Bouin's

solution or 4% paraformaIdehyde solution.

Preparation ofsolutions Phosphate buffered saline (PBS)

Phosphate buffered saline (PHS) (lM) was prepared using:

NaCI 87.9g Na2HP04 23.9g KH2P04 2.7g

The ingredients \Vere dissolved in a sufficient quantity of H20. Water was

added to a fmal volume of one litre and the pH was adjusted to 7.4. This • solution was kept at room temperature. PBS 0.1 M was prepared prior to the

experiment by mixing 100mi ofPBS lM and 900ml ofH20.

Bouin's solution

Saturated picric acid 750ml 40% formaldehyde 250ml Glacial acetic acid IOml

The saturated picric acid was prepared by adding H10 to the picric acid boule

and mixing by inverting the bonle. The solution was left to saturate ovemight.

The saturated solution ofpicric acid was decanted and vacuum-filtered. Bouin's

solution was prepared by adding 2S0ml of 400/0 formaldehyde to 750ml of • saturated picric acid. Glacial acetic acid (lOmI) was added just before use. Chapter II Materials and Methods 44

Sodium Sorenson's phosphate buffer (PB) (O.2M)

• Stock solution A: NaH2P04 (O.2M) Stock soultion B: Na2HP04 (O.2M) Stock solution A was added to stock solution B until the pH reached 7.4

(approximately 19m1 ofsolution A to 81 ml ofsolution B). The O.2M phosphate

buffer solution was kept at 4°C for a few weeks.

Perfusion Buffer

The perfusion buffer was prepared by mixing the following:

PBO.2M 50rnlll NaCI 8g1l KCI 0.25g!l NaHCO] 0.50gll To induce vasodilation, 1% sodium nitrite was added to the perfusion buffer.

• 4% Paraformaldehvde in 0.1 M Phosohate Suffer (4%PFA)

Under a fumehood, a solution of80g/1 ofPFA in H20 was heated to 60°C while

stirring constantly. Drops of lM NaOH was added until the solution became

clear. It was then cooled on ice and filtered under vacuum. This 8% solution

was kept at 4oC for a few weeks. Prior to use, equaI parts of 8%

parafonnaldehyde and 0.2M PB were mixed.

Preparation ofslides A solution of poly-L-Iysine was prepared folIo\\ing the manufacturer's

instructions. Microscope siides were dipped into the poly-L-Iysine solution for • Chapter fi Materials and Methods 4S

5 minutes and then air-dried. Poly-prep sIides coated with poly-L-lysine • obtained from Sigma-Aldrich were also used. Perfusions using Bouin's solution AnimaIs were perfused intracardially with IOOmi of phosphate buffered saline

(O.IM PBS, pH 7.4) followed by 200-300mI of Bouin's solution. Spinal cords

were dissected and post-fixed in the same fixative for at least 2 hours. Samples

were then washed and stored in PBS containing 15% sucrose and 0.01% sodium

azide for at Ieast 24 hours before further processing. Transverse sections

(20J.lm) were eut and mouoted onto poly-L-Iysine-eoated slides.

Perfusions using 4% paraformaldehyde AnimaIs were perfused intracardially with 100mi ofperfusion buffer containing

1% sodium nitrite followed by 200-300ml of 4% parafonnaldehyde solution. • Spinal cards were dissected and post-fixed in the same fixative for 2-3 hours. Samples were then stored in PB containing 10% sucrase for at least 24 hours

before further processing. Prior ta the experiment, transverse sections (40~)

\vere eut and placed in PBS.

2) Immunohistochemistrv

Materials Rabbit anti-CGRP polyclonai antiserum was kindly provided by Dr. S. Kar

(Douglas Hopsital Research Centre~ ~fontréal~ Québec). Goat anti-rabbit IgG,

peroxidase anti-peroxidase (PAP) immunogiobulin, 3~3' -diaminobenzidine

(DAB), glucose oxidase and nonnal goat serum \Vere obtained from Sigma- • Aldrich Canada Ltd. (OakviIle, Ontario, Canada). Rabbit anti-rat galanin (IHC- Chapter II Materials and Methods 46

7141, lot # 0301-9541), anti-substance P (RGG·7451, lot # 970653) and anti­ • CGRP (IHC-6006, lot # 971497) antisera were purchased from PeninsuJa Laboratories (Belmont, CA, USA). Nickel ammonium sulphate and Permount

were obtained from Fisher Scientific Canada (Montreal, Quebec, Canada). AlI

other chemicals were of analytical grade and obtained from VWR Canlab

(Mississauga, Ontario, Canada).

a) CGRP Immunostaining

CGRP immunostaining performed on spinal cords of animals killed 24h, 4 and

7 days following surgery or injection of FCA was conducted according to the

protocol described below. Spinal cords of animaIs killed 14 days following

treatment were immunostained for CGRP following the procedures described in • section b) for galanin and substance P immunostaining. Methods Preparation ofsolutions Phosphate Buffered Saline (PBS. 0.1 M)

The phosphate buffered saline was prepared as described pre'-iously in the

methods section ofperfusions using Bouin's solution.

Antisera Solutions

The rabbit anti-rat CGRPCL antiserum was diluted 1:2000 in PBS. Secondary

antibodies, goat anti-rabbit IgG and peroxidase anti-peroxidase complex were

diluted in PBS containing 0.2% Triton X-100 1:25 and 1:50 respectively. • Chapter II Materials and Methods 47

3.3' Diaminohenzidine Solution (PAB) • The DAB solution was prepared in the following manner: 3~3' diaminohenzidine 25mg PBS SOml

H202 (30%) 50JlI

This solution was prepared immediately before use. Before adding the

hydrogen peroxide, the 3~3'DAB was diluted in PBS and the solution filtered.

Immunostaining Hvdration: The sections (20J.LlIl), mounted onto poly-L-Iysine-coated slides,

were obtained from animaIs perfused with Bouin's solution. Sections were

cleared in xylene twice for S minutes each then hydrated in a graded aIcohol

gradient. Slides were dipped for 5 minutes in each of the following ethanol • solutions: 100%, 100% , 90%, 90% 70%. Slides were then dipped in H20 for 5 minutes, rinsed and washed in PBS containing 0.2% Triton X-IOO twice for 5

minutes each.

Preincubation: The slides were incubated in methanol containing 1% hydrogen

peroxide (30%) for 30 minutes to remove anyendogenous peroxidase activity.

Incubation with primarv antisera: Sections were then incubated at 4°C for 48h

with polyclonal antisera to rat aCGRP diluted 1:2000 in PBS. Sections were

then rinsed and washed in PBS twice for S minutes each.

Incubation with secondarv antibodies: Siides were incubated for 60 minutes • with goat anti-rabbit IgG. After rinsing and washing in PBS twice for S minutes Chapter n Materials and Methods 48

each, slides were incubated with the PAP complex for an hour and then • developed in DAB solution for 10 minutes. Sections were rinsed and washed twice in PBS, dipped in H20 for 5 minutes,

dehydrated in graded alcohol, cleared in xylene and mounted in Permount. To

monitor specificity, conventional controls such as omission of one step in the

PAP method were employed. The characteristic and specificity of CGRP

antibody in relation to the spinal cord were described in detail previously

(Gibson et al., 1984).

b) CGRP, Substance P and Galanin Immunostaining

Preparation ofsolutions Phosphate buffered saline (PHS, O.IM)

Phosphate buffer (PB) SOrnl1l • NaCI 8.8g11 KCI O.2g1l

The phosphate buffered saline was freshly prepared for each experiment as

described previously in the methods' section of perfusions using 4%

parafonnaldehyde.

Phosphate buffered saline! Triton solution (PBS+T)

A solution of 0.2% Triton X-IOOIPBS solution \\'as freshly prepared for each

experiment. 200J.11 ofTriton X- i 00 was added to 100mi of PBS. • Chapter il Materials and Methods 49

Antisera solutions • AlI the antibodies were diluted in PBS+T containing 1% normal goat serum. The rabbit anti-rat galanin antisera, rabbit anti-rat substance P and rabbit

anti-CGRP were diluted 1:4000. The secondary antibodies, goat anti-rabbit IgG

and peroxidase anti-peroxidase complex were diluted 1:25 and 1:50

respectively.

Acetate Buffer

A stock solution of 0.2M acetate buffer was prepared by mixing 13.61g of

sodium acetate in SOOml of H20. The pH \Vas adjusted to 6.0 using a 10%

solution of acetic acid. The 0.1 M acetate buffer was diluted just before the

experiment in an equal part ofH20. • Glucose Oxidase-3.3' Diaminobenzidine-Nickel Solution Nickel ammonium sulphate (NAS) solution was prepared by adding 2.Sg/50ml

ofnickel ammonium sulphate to O.2M sodium acetate buffer. The 0.12% DAB

solution was prepared by mixing 60mg of DAB into SOml of H20. The DAB

solution was poured into the NAS solution and the resulting solution was

filtered. 200mg of D-glucose, 40mg of ammonium chloride and 1.5mg of

glucose oxidase were added to the DABINAS solution. This solution was used

immediately.

Immunostaining Sections (40Jlm) from 4% PFA-perfused animais were placed in 24 weil-plates • containing PBS. The sections were washed m"Ïce in PBS for 15 minutes. Chapter II Materials and Methods so

Preincubation: To remove any endogenous peroxidase activity, the sections • were preincubated in 0.3% H202IPBS for 20 minutes at room temperature and then rinsed twice for 15 minutes each. The sections were then incubated in

10% nonnal goat serumlPBS+T for 30 minutes at room temperature to reduce

non-specifie staining.

Incubation with primarv antibody: Sections were incubated in 250J,l1 of primary

antibody solution for 48 hours at 4oC.

Incubation with secondarv antibodies: The following steps were aIl carried out

at room temperature. The sections were washed twice for 15 minutes each

using PBS+T. The secondary antibody, goat anti-rabbit IgG (l:25) was added

and sections were incubated for one hour. Sections were washed in PBS+T • twice for 15 minutes each. The peroxidase anti-peroxidase complex (1 :50) was added and sections were incubated for one hour. Slides were washed in O.IM

acetate buffer twice for 10 minutes.

Deyelopment: Sections were developed in the DABINAS solution. Sections

were washed twice in 0.1 t\.1 acetate buffer for 10 minutes. Sections were then

mounted onto poly-L-lysine-coated sIides and dried ovemight at room

temperature. The slides were then dehydrated in a water/ethanol/xylene

gradient as described previously for CGRP immunostaining. Slides were finally

mounted with Permount. • Chapter il Materials and Methods 51

C) Radioautography • A minimum of 5 rats per group (naïve, sham, cuff, oil, fCA) was used for each time point (4, 7 and 14 days) studied. Rats were decapitated and their spinal

cords dissected and snap-frozen in isopentane. The tissues were then serially

eut (16).Ull) using a microtome cryostat and mounted onto gelatine-coated

slides. Slides were kept at -SO°C until the day ofthe experiment.

Preparation ofGelatine-coated siides Deionized water was heated to 50°C. Then, gelatine powder (5g11itre of

deionized water) was slowly added while stirring. The solution was cooled to

30°C and chromium potassium sulphate (O.5g/litre of deionised water) was

added. The gelatine solution was filtered before use. Clean slides were dipped • in gelatine and dried at room temperature or in an aven at 37°C ovemight. 1) ~hCGRP bindinq

Materials

[115I]hCGRPa (-2000Cilmmol)~ microscales and 3H-Hyperfilms were obtained

from Amersham Canada (Oakville, Ontario, Canada). Bovine serum albumin

(SSA), bacitracin, leupeptin, chymostatin, MgCh and HEPES were purchased

from Sigma Aldrich Canada Ltd. (Oakville, Ontario, Canada). hCGRPa was

bought from Baehem Califomia Inc.(CA, USA). CaCh and KCI were obtained

from Anachemia Canada Ine. (Ville St-Pierre, Quebec, Canada). AIl other

reagents were ofanalytical grade and obtained from VWR Canlab (Mississauga, • Ontario, Canada). Chapter II Materials and Methods 52

Methods • Preparation ofsolutions HEPES Buffer (lOmM)

10mM HEPES 9.50g 150mM NaCI 35.06g 5mM KCf 1.49g ImM MgCb 0.81g 2mM CaCl2 1.18g

The ingredients were dissolved in a sufficient quantity of dH20 and dH20 was

added to a fmal volume of one litre. The pH was adjusted to 7.4. The 1OmM

HEPES buffer was prepared the day before the experiment.

Incubation Buffer

The incubation buffer was composed ofthe fol1owing: • HEPES buffer Bovine serum albumin 0.1 % Bacitracin 4Jlg/m1 Leupeptin 4J.lglm1 Chymostatin 2J.lg/ml C25]hCGRP SOpM

The incubation buffer was freshly prepared the day of the experiment. The

radioactive CGRP was diluted (lOJ.lCi/l OOmI) in the incubation buffer. The

incubation buffer had an activity ofapproximately 20 000 CPMlI00J.ll.

Experiment Preincubation: The sections were pre-incubated at room temperature in HEPES

buffer for 15min. • Chapter il Materials and Methods 53

Incubation: Sections were then incubated at room temperature for 90 minutes in • the incubation buffer. Non specifie binding was defined in the presence of IJ,LM ofunlabelled hCGRPa.

Washes: Following incubation, slides were washed t\\ice for 4 minutes each in

ice-cold HEPES buffer, dipped in cold deionized water, dried ovemight and

then exposed against 3H-Hyperfilms for 6 days.

2) ~BH-substance P

Materials

[1:!5I]-Bolton-Hunter Substance P (-2000Cilmmol), microscales and 3H_

Hyperfilms were obtained from Amersham Canada (Oakville, Ontario, Canada).

Bovine serum albumin (BSA), bacitracin, leupeptin, chymostatin, Tris and • substance P were purchased from Sigma Aldrich Canada Ltd. (Oakville, Ontario. Canada). AIl other reagents were ofanalytical grade and obtained from

V\VR Canlab (Mississauga, Ontario, Canada).

Methods Preparation ofsolutions Tris buffer

A solution of sûmM Tris was prepared. The pH was adjusted to 7.4 using

hydrochloric acid. This buffer was prepared the day prior to the experiment. • Chapter II Materials and Methods 54

Incubation Buffer • Bovine serum albumin 0.02% Bacitracin 4011g/ml Leupeptin 411g!ml Chymostatin 2fJg!ml MnCh 3mM [125I]BH-substance P SOpM

The ingredients were mixed in Tris buffer the day ofthe experiment.

Experiment Pre-incubation: Slide-mounted sections were pre-incubated at room

temperature in Tris buffer for 15minutes.

Incubation: The slides were then incubated in the incubation buffer containing

SOpM [125I]BH-substance P for 90 minutes at room temperature. Non-specific

binding was determined in the presence of 1~M substance P.

• \Vashes: At the end ofthe incubation, slides were washed four times in periods

of 1 minute in ice-cold Tris buffer, rinsed in coId water, dried ovemight and

then exposed against 3H-Hyperfilrns for 4 days.

3) Ôporcine galanin

Materials

[125I]porcine galanin (-2000Ci/rnmol), microscales and 3H-Hyperfilms were

obtained from Amersham Canada (Oakville, Ontario, Canada). Bovine serum

albumin (BSA), bacitracin, leupeptin, pepstatin A and Tris were purchased from

Sigma Aldrich Canada Ltd. (Oakville, Ontario, Canada). Human galanin was • obtained from Bachem Califomia Inc. (CA. USA). Ail other chemicals were of Chapter II Materials and Methods 55

analytical grade and obtained from VWR Canlab (Mississauga, Ontario, • Canada).

Methods Preparation ofsolutions Tris buffer CSOmJill

The pH was adjusted to 7.4. This buffer was prepared the day prior to the

experiment.

Preincubation buffer

MgCh 5mM EGTA 2mM

The ingredients were added to Tris buffer the day ofthe experiment. • Incubation buffer Bovine serum albumin 1% Leupeptin O.OsolO Pepstatin 0.001% 125 [ I]porcine galanin SOpM

The BSA and peptidase inhibitors were added to the preincubation buffer prior

to the experiment.

Experiment Preincubation: Slide-mounted sections were pre-incubated at 4°C for 30

minutes in Tris-preincubation buffer. • Chapter fi Materials and Methods S6

Incubation: The slides were then incubated for 60 minutes with SOpM of • rt 25I]porcine galanin at room temperature in Tris incubation buffer. Non­ specific binding 'vas determined in the presence of 1J.lM human galanin.

Washes: Slides were then washed 4 times for 1 minute each in ice·cold Tris­

wash buffer, rinsed in cold deinoized water, air-dried ovemight and exposed

against 3H·Hyperfllms for 3 days.

4) Quantitative Image Analysis

Specifie binding of CGRP, substance P and galanin in lamina I-V and X was

quantified by using commercially available [IlS!] standards (Amersham Canad~

Oakville, Ontario, Canada) and computerized image analysis system (MCID

System, lmaging Research Inc., St-Catharines, Ontario, Canada) (Ménard et al.,

• 1996). A minimum of 3 sections was analysed for each animal. Each lamina was identified carefully and a density reading recorded.

5) Statistics

Statistical analysis were performed in GraphPad Prism (San Diego, CA, USA)

using a two-way ANDVA followed by a Bonferroni post-hoc analysis.

Statistical significance was set at p

•

• Chapter III Results 58

A) Behavioural Experiments • 1) Inflammatorv Pain Model a) Inflammation

AnimaIs developed a significant inflammation 8 hours following the injection

of Freund~s complete adjuvant (p

peaked 24 hours after the injection (Figure 3). It was long-lasting and present

for the 4 weeks of testing. The animaIs showed no significant differences in

their mean weight gain over the course ofthe experiment (p>O.05) (Figure 4).

Inflammation 1.5 * ~Saline ~ u- --"*-- Oil CO 1.0 • f»1ï ~FCA ~ c ~~ .- ~ * *:p 0.0

-0.5 1 i i i 1 0 2 4 6 8 10 15 20 25 30 Days post-injection

Figure 3: Inflammatory Pain Model- Paw Inflammation The inflammation was assessed by the difference in volume between the ipsilateral and contralateral paws. Values are expressed as means ± S.E.M. of 10 animais per group. Data demonstrate the induction of inflammation by the injection of 40J.LI of fCA (~) compared to saline injection (.). A slight inflammation is noticeable fol1owing the injection of mineraI oil (vehicle) (.). • Chapter ID Results 59

• Mean Weight Gain 50

o 40 ~ Il C 30 ~ w fi) 20 +Q ::::===::::::::: 10 ~ O...L-_~-_--L._.....&.-::::::_-=-~=~:::-~: -~ Saline Qil Treatment

Figure 4: Inflammatory Pain Model - Mean Weight Gain The mean weight gain was calculated following 28 days oftreatment. Values are expressed as means ± S.E.M. of 10 animais per group. Data demonstrate no statistical differences between saline-, oil- and FCA­ treated animaIs. Control animais received 50JlI of saline or 50JlI of ail. Treated animais • received ..tOJlI ofFCA and 1O~1 ofoil.

b) Mechanical Allodynia

i\1echanical allodynia was noticeable tWQ days following the injection of FCA

(Figure 5). The allodynia was significant for the first 10 days. Although not

significan~ the allodynia seemed to be present until the third week and reached

baseline Ievel by week 6 post-injection. • Chapter III Results 60

• Mechanical Allodynia

20

o- 32"o c:: ­ ..cn~ 10 .cenfw ~+f en - ~Control ---6- FCA *: p< 0.05 O~~-r--r--r--r-"""T'"....,...-n----"'j---,jr-----,..j--""jr---"""'j o 3 6 9 12 20 30 40 50 60 Days post-injection

Figure 5: InOammatory Pain Model - Mecbanical Allodynia The threshold was measured using von Frey filaments. Values are presented as means ± • S.E.M. for 10 animais per group. Data show significant mechanical allod)nia (p

c) Thermal Hyperalgesia

Thermal hyperalgesia was present 2 hours following the injection of FCA

(Figure 6). This hyperalgesia was, however, of short duration and was

significant for the first 24 hours ooly. Latencies returned to baseline values by

day 5 post-injection. • Chapter m Results 61

• Thermal Hyperalgesia

(1) 1 .S! _ ~ u~ cC"') ..QI co1 nJ Il a ~ C C-~ 1 .-(l)W uc/) -1 c + (1) 0 .. Q) I\ ---Control :!~ * .,,,.., -2 /f .-.-- FCA ê \j t,/' *: p< 0.05 1..

-3 1 i i 1 i 0 1 2 4 6 8 10 12 14 Days post-injection

Figure 6: Inflammatory Pain Model - Thermal Hyperalgesia Thermal hyperalgesia was assessed by measuring withdrawal latencies to a radiant heat beam. • Results are presented as the difference in latency between the ipsiJateral and contralateral paws and expressed as means ± S.E.M. for at least 8 animais per group. Data show a significant decrease in latencies (p

2) Neuropathic Pain Model

The neuropathic rats mean weight gains were not significantly àifferent from

control (p>O.OS) (Figure 7). Autotomy, i.e. self-mutilation~ was not observed

for any ofthe animaIs. • Chapter mResults 62

• Mean Weight Gain

300-

...... 1 ...... o ...... Il 200­ ...... c ...... :2 ...... w ...... ri) ...... ~ ...... 100- ......

Treatment

Figure 7: Inflammatory Pain Model - Mean Weight Gain The mean weight gain was calculated following S6 days of treabnent. Values are expressed as means ± S.E.M. of 10 • animaIs per group. Data demonstrate no statistical differences between sham and cuffanimaIs. a) Mechanical Allodynia

AnimaIs showed significant mechanical allodynia 2 days following the

encapsulation of the sciatic nerve (see Figure 8 on the following page). The

allodynia peaked 14 days post-surgery and lasted for the 8-week period of

testing.

b) Thermal Hyperalgesia

The animaIs showed hyperalgesia to thermal stimuli 4 days after surgery. This

hyperalgesia lasted for the 8 week period of testing (see Figure 9 on the • following page). Chapter ID Results 63

Mechanical Allodynia • 20 0- "C Il (5 -c: ~ en ~ 4» 10 ~ u.i -.-...- Sham ~ cJi --r- Cuff t- +1 0) *: p< 0.05 - *

o-+-""'-'"'T'""""T"""....,...... -....,...... ,....-t i i i i • 1 a 3 6 9 12 20 30 40 50 60 Days post-surgery

Figure 8: Neuropathic Pain Model-Mechanical Allodynia Threshold was was measured using von Frey filaments. Values are presented as means ± S.E.M. for 10 animais per group. Data show significant mechanical allodynia (p

20

* 10 ~Sham * -'-Cuff *: p< 0.05

a+-.....-....,....""T"""-r-.,.-..,.-.-r-~ i i i 1 1 o 3 6 9 12 20 30 40 50 60 Days post-surgery

Figure 9: Neuropathic Pain Model-Thermal Hyperalgesia Thermal hyperalgesia was assessed by measuring withdrawal latencies to a radiant heat beam. Values are expressed as means ± S.E.M. for 10 animais per group. Data show significant thermal hyperalgesia (p

B) Immunohistochemical Staining

• 1) Calcitonin Gene-Related Peptide

Immunoreactive fibres formed a dense network in the superficial dorsal horn of

the spinal cord (Figure 10). Low to moderate numbers of fibres were observed

in the deeper layers of the dorsal horn. No significant differences were

observed in CGRP·like immunostaining at 24h, 4, 7 and 14 days following

injection ofFreund's complete adjuvant (Figure 10a) or following sciatic nerve

constriction (Figure 1Ob).

2) Galanin

Galanin-like immunostaining was observed in the superficial dorsal hom ofthe • spinal cord (Figure 11). No significant differences were observed in galanin· like immunostaining at 24h, 4, 7 and 14 days following injection of Freund"s

complete adjuvant (Figure lia) or sciatic nerve constriction (Figure II b).

3) Substance P

Substance P-like immunostaining was observed in the superficial dorsal horn of

the spinal cord (Figure 12). No significant differences were observed in

substance P-like immunostaining at 24h, 4, 7 and 14 days following injection of

Freund"s complete adjuvant (Figure 12a) or sciatic nerve constriction (Figure

12b). • Chapter III Results 65

• On the following pages: Figure 10a: CGRP-like Immunostaining - InOammatory Pain Model CGRP-like immunostaining in the superficial dorsal horn at L4-L5 following injection of40".11 of fCA (1 mg/ml) or vehicle. No differences were observed al 24 hours, 4, 7 and 14 days following rreatrnent.

Figure lOb: CGRP-like Immunostaining ... Neuropathic Pain Model CGRP­ like immunostaining in the superficiaJ dorsal horn at L4-L5 foUowing constriction ofthe sciatic nerve or sham surgery. No differences were observed at 24 hours, 4, 7 and 14 days following surgery.

Figure lIa: Galanin-like Immunostaining - Inflammatory Pain Model Galanin-like immunostaining in the superficial dorsal hom at L4-L5 following injection of 40~.d of fCA (1 mg/ml) or vehicle. No differences were observed al 24 hours, 4, 7 and 14 days following treatrnent.

Figure Ilb: Galanin-like Immunostaining ... Neuropatbic Pain Model Galanin-like immunostaining in the superficial dorsal horn at L4-L5 following constriction of the sciatic nerve or sham surgery. No differences were observed at 24 hours. 4, 7 and 14 days • following surgery.

Figure 12a: Substance P-like Immunostaining ... InOammato!1' Pain Model Substance P-like immunostaining in the superficial dorsal hom at L4-L5 following injection of 40~1 offCA (1 mg/ml) or vehicle. No differences were observed at 24 hours. 4, 7 and 14 days following rreatrnent.

Figure 12b: Substance P-like Immunostaining ... Neuropathic Pain Model Substance P-like Immunostaining in the superficial dorsal horn al L4-L5 following constriction of the sciatic nerve or sham surgery. No differences were observed at 24 hours, 4, 7 and 14 days following surgery. • FIGURE 10 CGRP IMMUNOREACTMTY IN THE DORSAL HORN AT L4-L5 SPINAL CORD LEVELS • Ipsllateral Contralateral

•

• FIGURE 11 GALANIN IMMUNOREACTIVITY IN THE DORSAL HORN AT L4-L5 SPINAL CORD LEVELS • Ipsilateral Connlateral

•

• FIGURE 12 SUBSTANCE P IMMUNOREAcnvrrv IN THE DORSAL HORN AT L4-L5 SPINAL CORD LEVELS • Contralatenl'

•

• Cbapter ID Results 69

C) Binding Sites • 1) Calcitonin Gene-Related Peptide Binding Sites

The highest level of 125I_CGRPa binding was particularly observed in the

region surrounding the central canal, lamina X. Low leveIs of binding were

observed in the superficiaI dorsal homo Moderate levels of binding were

observed in deeper laminae of the dorsal born and in the ventral horn (Figure

13).

•

Following page:

Figure 13a: CGRP binding sites in the U-L5 spinal cord - Inflammatof)' Pain Model CGRP binding sites in the spinal eord (L4-L5) following injection of 40~.d of fCA (1 mg/ml) or minerai oil (vehicle). Sections were incubated in the presence of 50 pM 1~;

Figure 13b: CGRP binding sites in the L4-L5 spinal cord- Neuropatbic Pain Model CGRP binding sites in the spinal eord (L4-L5) following constriction of the sciatie nerve or sham surgery. Sections were incubated in the presence of 50 pM l:!sI_ CGRPu. Non-specifie binding was detennined in the presence of 1JlM CGRP. • CGRP SINDIHG SITES • AT L4-L5 SPINAL CORD LEVELS

, . , ': "

.' ~""t,. -; -" L ._ "è _.;' _~ ~. .. ~". -.

ln the presence of 1 uM CGRP Control

Figure 13a: Inflammatory Pain Model •

Oll: 4 da,. post-injectlon FCA: 4 clay. postooÏnjection

Figure 13b: Neuropathic Pain Model

~. - . ..:. .~~...~ ~.""<' . ;~r t~~f'~ -f'

• SMAM: 14 daya post..urgery CUFF: 14 days post..urgery Chapter III Results 71

a) Inflammatory Pain Model • A significant increase in CGRP binding was observed in laminae III and IV following injection ofFreund's complete adjuvant (Figure 14).

C251]-CGRPa Binding 20 .

= ~ ~ o

.20-l-La~m-:-in-a-:-I-~L-a-m-:-in-a-:-:II:-----:-L-am-i~na---IlI-~La-m-:i-na-1-:-V~--:-L-a-m-:-in-a":"":V:---~La-m-:i~na-X~

~OIL I ...... ! FCA 4 days rimZI FCA 7 days _FCA 14days

Figure 14: Calcitonin gene-related peptide binding sites in the L4-L5 spinal cord - InOammatory Pain Model CGRP binding sites following • injection of 40 J.11 of fCA (lmg/ml) or minerai ail (vehicle). Sections were incubated in 50 pM I:!SI-CGRPa. Results are expressed as·~the percent difference between the ipsilateral and contralateral sides, mean ± S.E.M. of5-14 animais.

Neuropathic Pain Model

A significant decrease in CGRP binding was observed in lamina III at 14 days

following constriction of the sciatic nerve (Figure 15). Although not reaching

significance, apparent decreases in CGRP binding were observed in lamina II at

aIl timepoints. • Chapter III Results 72

125 • [ 1]-CGRPa Binding 20- 1 l~~' ~IT' 6~ç, 0_ 1•• TI" T1T'

-20- * • n:icaCes ~.05 coopndte SNlm-opel'ateé ann.1s

Lamina 1 Lamina Il Lamina III LamIna IV Lamina V Lamina X

c:::=::l Sham ...... , Cuff 4 days ŒI!Z!l Cuff 7 days _ Cuff 14 days

Figure 15: Calcitonin gene·related peptide binding sites in the L4-LS spinal cord - Neuropathic Pain Model CGRP binding sites following sciatie nerve constriction or sham-surgery. Sections were incubated in the presence of 50 pM 115 I-CGRPa. Results are expressed as the percent difference bet\'..een the ipsilateral and • contralateral sides, mean ± S.E.M. of5-11 animaIs. 2) Galanin Binding Sites

125I_galanin binding sites were concentrated in the superficial dorsal homo

Moderate Ievels of binding were observed in deeper layers of the dorsal hom

(Figure 16).

Following page:

Figure 16a: Galanin binding sites in the L4-L5 spinal cord - Innammatory Pain l'Iodel Galanin binding sites in the L4-L5 spinal cord following injection of 4°111 of FCA (1 mg/ml) or mineraI oil (vehicIe). Sections were incubated in 50 pM l:!sI-galanin. Non-specifie binding \Vas determined in the presence of 1J.LM galanin.

Figure 16b: CGRP binding sites in the L4-L5 spinal cord- Neuropathic Pain Model CGRP binding sites in the L4-L5 spinal cord following constriction of the sciatic nerve or sham surgery. Sections were incubated in 50 pM 12SI_galanin. • Non-specifie binding was detennined in the presence of ll-LM galanin. GALANIN BINDIHG SITES • AT L4-L5 SPINAL CORD LEVELS ."'.

~ "~~;<.: ... ~,.. or. -' ,..·.1· ... '~;:~~~ffj~f~J.~-

:-.~ L •

ln the p.....nce of 1 uM galanin Control

Figure 168: Inflammatory Pain Mode' •

Oll: 4 day. post-injection FCA: 4 day. post-injection

Figure 16b: Neuropathic Pain Mode' 1-...., .•

• SHAM: 14 _ys post..urgery CUFF: 14 clays post-surgery Chapter III Results 74

• a) Inflammatory Pain Model 125r_galanin binding was found to he increased in lamina II and V at 4 days

foIlo\\ing induction of inflammation (Figure 17). Significant increases were

also observed in lamina 1, IV and V at 14 days. A small increase in binding \vas

also observed in deeper laminae at 4 and 7 days, although not reaching

significance.

[1251]-Galanin Binding ;;... 40 ~ ~ 30 -tS ... uQ ~§~ 20 o ~~ ~- ~O 10 ~ ~ 0ü;- 0 1 ~ y T ·10 • lamina 1 Lamina Il Lamina III Lamina IV Lamina V Lamina X

e::z::::::lOIL i...···-! FCA 4 days ti:liCIa FCA 7 days - FCA 14 days

Figure 17: Galanin binding sites in the L4-L5 spinal cord ­ Inflammatory Pain l'Iodel Galanin binding sites following injection of 40 J..lI of fCA (1 mg/ml) or minerai oil (vehicle). Sections were incubated in 50 pM mt-galanin. Results are expressed as the percent difference bet\\'een the ipsilateral and contralateral sides. mean ± S.E.M. of4-16 animais per group.

• Chapter ID Results 7S

b) Neuropathic Pain Model • Significant iacreases were observed in laminae III, IV and X at 7 days post- surgery (Figure 18). A significant decrease was observed in lamina II and III at

14 days.

[1251]-Galanin Binding * 40 *

• Lamina 1 Lamina Il Lamina III Lamina IV Lamina V Lamina X [==:J Sham 1...··-..) Cuff 4 days CIl:!lZi:I Cuff 7 days _ Cuff 14 days

Figure 18: Galanin binding sites in the L4-L5 spinal cord ­ Neuropathie Pain Model Galanin binding sites following sciatic nerve constriction or sham-surgery. Sections were incubated in 50 pM 125I-Galanin. Results are expressed as the percent difference between the ipsilateral and contralateral sides, mean ± S.E.M. of 4-8 animaIs per group.

• Chapter III Results 76

3) Substance P Binding Sites • 125I-BH-substance P binding sites were observed in high densities in the superficial layers of the dorsal horn. Moderate to low levels of binding were

observed deeper into the dorsal horn (Figure 19).

•

Following page:

Figure 19a: Substance P binding sites in the L4-L5 spinal cord- Inflammatory Pain l'fodel Substance P binding sites in the L4-L5 spinal cord following injection of 40J.!1 of FCA (1 mg/ml) or minerai oil (vehicle). Sections were incubated in 50 pM 125I-BH-substance P. Non-specifie binding was detennined in the presence of 1J.1M substance P.

Figure 19b: Substance P binding sites in the L4-LS spinal cord - Neuropatbic Pain Model Substance P binding sites in the L4-L5 spinal cord following constriction of the sciatic nerve or sham surgery. Sections were incubated in 50 pM 1:25I-BH-substance P. Non-specifie binding was detennined in the presence of 1!-lM substance P. • SUBSTANCE P BINDING SITES • AT L4·L5 SPINAL CORD LEVELS

.~.. .-

ln the p.....nce of 1 uM SP Control

Figure 198: Inflammatory Pain Mode•

. .:,; • "~;..

.,...•. ~. Oll: 4 c:tays post-injectlon FCA: 4 daya post-injection

Figure 19b: Neuropathic Pain Model

• SHAM: 14 _ys post..urgery CUFF: 14 .18 post..urgery Chapter III Results 78

a) Inflammatory Pain Model

• No significant differences were observed al4 and 7 days following the injection of Freund~s complete adjuvant (Figure 20). Significant differences were

observed in laminae II, ID and IV at 14 days post-injection.

r2sl]-BH Substance P Binding • 40 *

20 ~H:1,:·11 ~~i·:· o [:j ... l;j ...

-20~L-a-m-jn-a-I--L-am-I-·na-J1--La-m-i-na-III--La-m-i-na-IV--La-m--=-in-a-V--La-m"""'":j-na-X-

• EZ:ZI Oll e··..··3 FCA 4 days ZZl FCA 7 days _ FCA 14 days

Figure 20: Substance P binding sites in the L4-L5 spinal cord ­ Inflammatol1' Pain Model Substance P binding sites following injection of 40 J.lI of fCA (lmgiml) or mineraI oil (vehicle). Sections were incubated in 50 pM 1251-BH­ Substance P. Results are expressed as the percent difference between the ipsilateral and contralateral sides. mean ± S.E.M. of6-13 animais.

b) Neuropathic Pain Model A sÏl:mificant...... increase in binding sites was observed in lamina l at 14 davs- and in lamina II at aIl timepoints (4, 7 and 14 days) follo\.ving constriction of the

sciatic nerve (Figure 21). 1:!5I-BH-substance P binding sites were found to be

significantly increased in the deeper laminae at 7 and 14 days (laminae III-V) • post-surgery. No change was observed in laminae X. Chapter ID Results 79 •

[1251)-BH Substance P Binding

* * 40- *

-20...L...------Lamina 1 Lamina Il Lamina III Lamina IV Lamina V Lamina X

~ Sham Im_.•••! Cutf 4 days !'ZZZa Cuff 7 days _ Cuff 14 days

Figure 21: Substance P binding sites in the L4-L5 spinal cord ­ Neuropatbic Pain Model Substance P binding sites following sciatic nerve constriction or sham-surgery. Sections were incubated in 50 pM mI-BH-substance P. • Results are expressed as the percent difference berween the ipsilateral and contralateral sides, mean ± S.E.M. of4-1 1 animaIs per group.

• • Chapter IV Discussion

•

• Chapter IV Discussion 81

There is increasing evidence that the neuropeptide CGRP plays an important • raie in sensory mechanisms. In fact~ CGRP is highly expressed in areas known to be involved in sensory processes ( Skofitsch and JacOb\\11Z., 1985; Franco­

Cereceda el al., 1987b; Wimalawansa et al., 1987a). CGRP is also found to be

highly colocalized with the nociceptive neuropeptide substance P in dorsal root

ganglia (\Viesenfeld-Hallin el al., 1984; Lee el al., 1985) and superficial dorsal

horn (plenderleith et al., 1990; Ribeiro-da-Silva, 1995). In acute pain models,

Ménard et al. (1995a) have reported a modulation of CGRP binding sites and

CGRP-like immunoreactivity following the development of tolerance to

morphine. In addition, the CGRP antagonist, CGRPS-37, was shown to prevent

the development of tolerance to morphine (Ménard et al., 1996). However~ in

the clinical setting~ tolerance is normally observed following prolonged • administration of opiates for the treatrnent of chronic pain. Thus, the aim of this thesis was to study possible alterations in CGRP markers in neuropathic

and inflammatory models ofchronic pain. Additional neuropeptides believed to

be in\'olved in nociception, such as substance P and galanin. \Vere studied to

assess the specificity ofthe changes observed with CGRP.

In the present study, no changes were observed in the expression of any of the

neuropeptides examined consequent to the chronic pain inj unes. However,

there was a significant increase in CGRP binding sites at the L4-L5 levels ofthe

spinal cord in parallel to the development of chronic inflammatory pain. • Conversely, there was a significant decrease in CGRP binding sites following Chapter IV Discussion 82

the development of chronic neuropathic pain. These changes are specific to • CGRP and do not reflect general changes in the density of binding sites for other neuropeptides in the spinal cord. Changes in galanin and substance

PINK-l receptors were distinct from those seen for CGRP receptors.

A) Chronic Pain Models

Chronic pain observed in severa! clinical situations is known to be associated

with long·lasting pathological neural mechanisms, such as alterations in gene

regulation and heightened excitability, which strongly differ from those

observed in acute pain situations (Calvino et al., 1987; Dray and Urban, 1996).

Experimental models have been developed pennining the study of mechanisms • of inflarnmatory and neuropathic pain. An understanding of the pathophysiology in these experimental models may lead to an improvement in

our understanding of comparable human states and hopefully improved

therapies. In this present srudy, two chronic pain models were used: the

Freund"s complete adjuvant inflammatory pain model and the sciatic nerve

constriction mode!.

1) Inflammatorv Pain Madel

The injection of complete Freund's adjuvant in the hindpaw of rats is

considered to be a reliable model of inflammatory pain (Butler et al., 1992; • Donaldson et al., 1993). In the present study, the injection of Freund's Chapter IV Discussion 83

complete adjuvant in the hindpaw of male Sprague-Dawley rats produced a • significant and long-lasting inflammatory response compared to both saline­ and oil-treated animaIs. FCA-treated animals showed significant mechanical

allod)nia and thermal hyperalgesia, although ofdifferent duration.

The differences observed in the duration of thermal hyperalgesia compared to

mechanical allodynia suggest that different fibre subsets may mediate the

distinct modalities studied during the course of these experiments.

HyperaIgesia is described as a lowered threshold for the activation of primary

afferent nociceptors (Levine et al., 1993). By comparison, it is believed that

allod)nia arises through "wind up" or central sensitization induced by C-fibres

(Dickenson, 1995). \Vind up is responsible for the enhanced responses, up ta • 20 foid in duration and intensity, of dorsal horn neurones despite a Iack of change in peripheral input. This hypersensitivity leads ta innocuous stimuli

becorning painful.

Butler et al. (1992) have reported a modei of monoarthritis showing

inflamnlation of the FCA-injected paw for the 6 week observation periode

During this observation period, the animaIs aiso demonstrated lower paw

withdrawal thresholds in the Randall-Selino test (paw pressure test used to

rneasure mechanical hyperalgesia) (Butler et al., 1992). The Butler's model

appears to be a more robust model and this cao be explained by the difference in • the dose administered. Butler et al. used a FCA solution (300 ~g) over 5 times Chapter IV Discussion 84

more concentrated than the preparation used in the present study (40).1g). It is • expected that a larger dose of fCA would produce more pronounced behavioural responses as it has previously been shown that small alterations in

the dose or route ofadministration ofthe adjuvant produce major differences in

the extent and duration ofthe responses (Donaldson et al., 1993). Donaldson et

al. (1993) showed that the induction and more particularly the spread ofarthritis

was dependent on the dose of adjuvant used. Vehicle and small doses of

adjuvant produced only a minimal local inflammation while higher doses

caused rapid initial local inflammation. The highest dose (>250).1g

l'vf.tuberculosis) used in their study even resulted in a bilateral inflammation.

The injection of fCA in the hindpaw of rats produces a long-lasting • inflammatory and allodynic response even at small doses. The injection of large doses of FCA producing a polyarthritic syndrome poses ethical concems.

The appearance of severe systemic effects prevents any comparison with the

monoarthritic rat mode!. Inflammatory pain induced by the injection of

formalin (Abbon et al., 1995), carrageenan (Sluka and Westlund, 1992; Garry

and Hargreaves, 1992) or urate (Coderre and Wall, 1987) seem to be considered

as more acute inflammatory pain models lasting a few hours to a few days.

Hence. the model used in the present study both in terms of dose of adjuvant

and duration of effects has been weIl validated in earlier reports (Butler et al., • 1992; Donaldson et al., 1993). Chapter IV Discussion 85

2) Neuropathic Pain Model

• The constriction ofthe sciatic nerve produces hyperalgesia and allodynia which are prominent symptoms accompanying peripheral neuropathies observed in

humans (BeIUlett and Xie, 1988).

In the present study, the encapsulation of the sciatic nerve using polyethylene

tubing produced a significant and long-lasting mechanical allodynia and thermal

hyperalgesia. The increase in thennallatencies observed in both groups, sham

and cuff animaIs, from day 14 ta 56 post-injection can be explained by an

habituation ta the test and by the appearance ofa thicker keratinous cell layer on

the hindpaws of the animaIs as they age. Although still significantly different, • the latencies observed at later timepoints suggest a recovery. Contrary to the thermal hyperaIgesia, mechanicaI allodynia did not show any signs of recovery,

Iatencies ofcuff-operated animaIs being significantly Iower than sham-operated

animaIs. This suggests that different fibre subsets may respond ta thennal and

mechanical stimuli.

In their report of the "cuff moder', Mosconi and Kruger (1996) observed 5

categories of behaviour or appearance ofthe animais: general appearance ofthe

skin and digits, gait, posture, response ta mechanical stimuli and cold. The

behavioural assessment scores were maximal between 10 and 14 days post·

surgery and pain scores suggest recovery by day 28. The present study showed • that mechanical allodynia and thermal hyperalgesia lasted for longer than 28 Chapter IV Discussion 86

days. This could he explained by the fact the animaIs were slightly bigger than • the animaIs used in Mosconi et al. (1996) possibly leading to a more pronounced constriction ofthe nerve.

The standard sciatic nerve constriction injury model was developed by Bennett

and Xie (1988). As in the present study, Bennett and Xie (1988) observed

more pronounced allodynia and than hyperalgesia suggesting central

sensitization.

The neuropathic constriction model provides longer-lasting allodynia and

hyperalgesia compared to the Freund's complete adjuvant-treated animaIs. This

could be explained by the presence of the constrictive stimulus, the • polyethylene tuhing, throughout the experiment preventing recovery of the nerve fibres. It is a1so possible that a higher dose of FCA would provoke

longer-lasting thennal hyperalgesia and mechanicaI allodynia as shown by

Donaldson et al. (1993) studies. Distinct mechanisms and systems may be

involved in different types of injuries, such as inflammation and neuropathy,

and therefore exhibit unique sets of sYmptoms. In fact, Malmberg el al. (1997)

have shown that mice with a mutation of the type 1 regulatory suhunit of

cAMP-dependent protein kinase respond differently than wild-type mice 10

tissue-injury associated with significant inflammation. The mutant mice show a

decreased response to inflammation while no differences are observed in their • response to neuropathic pain (Malmberg et al., 1997). Chapter IV Discussion 87 • B) Plasticity of Spinal Cord Neuropeptides 1) Immunohistochemical Staining

Immunohistochemical staining was perfonned to determine whether

inflammatory and neuropathic chronic pain models would modulate the

expression of neuropeptides in the spinal cord. No changes were observed in

any of the neuropeptides studied. The reason for the lack of changes is not

believed to he of a technical nature as the antibodies used in the present study

are weB characterized and have been used in severa! other reports ( Kar et al.,

1993; Szabat el al., 1994; Ma and Bisby, 1997). Furthennore, strong

immunostaining was observed for ail three peptides and the lack of background • staining suggests that the experiments succeeded. Small changes in immunostaining may not be observed without appropriate quantitative

measurement. However, preliminary semi-quantitative analysis of CGRP

immunostaining sections did not reveal any significant changes but more

elaborate semi-quantitative studies should be performed for all three

neuropeptides.

In the inflammatory pain model~ it is possible that the dose of fCA was not

large enough to cause and sustain heightened excitability and consequently

cause changes in the expression ofcertain genes. Authors observing changes in

the CGRP, substance P and galanin immunostaining typically use doses offCA • much higher (75-275 ~g) than that used in the present study (40 I-lg) (Donnerer Chapter IV Discussion 88

et al., 1992; Donaldson et al., 1992; Nahin and Byers, 1994; Seybold et al., • 1995). In these experiments, the dose of FCA was kept as 10\\' as possible to avoid unnecessary discomfort to the animals and the induction of systemic

effects. In addition, the dose of fCA was sufficient to significantly modulate

densities of binding sites for all three neuropeptides observed in this study. In

fact, the dose administered was judged sufficient since it pro\'oked a long­

lasting inflammatory response without the induction ofsystemic effects.

a) CGRP

In the present study, CGRP immunostaining was not found to be altered by

FCA-induced inflammation at any of the timepoints studied (4, 7 and 14 days).

These findings are in contrast to a report by Seybold el al. (1995) which • demonstrated an initial decrease in CGRP immunostaining at 2 days after the injection of fCA followed by an increase at 8 days. Donnerer and Stein (1992)

also observed an increase in CGRP content, measured by RIA, 5 days following

the injection of FCA. Furthennore, increases in CGRP immunostaining (Smith

el al., 1992; Nahin and Byers, 1994) and mRNA (Iadorola and Draisci, 1988;

Galeazza el al., 1995) in the dorsal root ganglia of FCA-treated animais have

also been observed. However, this newly synthesized CGRP appears to be

predominantly transponed to the periphery. Thus, Nahin el al. (1994) reported

an increase in the number of CGRP-immunoreactive peripheral afferent fibres

in deep tissues of ankle and digits following fCA treatment. This is in • accordance with the findings of Donnerer and Stein (1992) showing increased Chapter IV Discussion 89

CGRP axonal transport ta the periphery in response to FCA-induced • inflammation. Peripheral transport of newly synthesized CGRP couId explain the absence of central changes in CGRP immunostaining found in the present

study in addition to the above comment on the dose ofadjuvant used.

Similarly to the inflammatory pain model, no change in CGRP immunostaining

was observed during the development of neuropathic pain. Changes in CGRP

immunostaining in neuropathic pain models are controversial. The results of

the present study are in accordance with work from Garrison et al. (1993) but

are not supported by other reports showing a decrease in CGRP

imrnunostaining follo~ing Iigation of the sciatic nerve (Bennett et al., 1989;

Kajander and Xu, 1995). These discrepancies may be a consequence of • differences in the techniques used. One important difference between the present study and previous reports is the intensity of the neuropathic insult.

Polyethylene tubing used in the sciatic cuff technique provides a standard

constriction of the sciatic nerve across animaIs. By comparison, there is

considerable inter-animal variability in the intensity of the constriction with

nerve ligation which may result in greater variability in the changes of

neuropeptide markeTS (Mosconi and Kruger, 1996). The nature of the material

used to create the constriction of the sciatic nerve has been sho\\n ta have

differential effects on the development of neuropathy (Maves et al., 1993; Xu et

al.. 1996). In a study conducted by Xu et al. (1996), chromic gut suture • constriction appears to produce a modulation of CGRP while polyglactin and Chapter IV Discussion 90

plain gut sutures had no effect (XU et al., 1996). The authors postulated that the • material might induce sorne effect by itself or that different material would produce different timecourses in the observed changes. No data is available as

to the effect of polyethylene tubing on the development of neuropathy.

Mosconi and Kruger (1996) have aIso studied the effects of chromic gut

compared ta silk and de},:tron sutures and found no significant differences in the

effects produced.

b) Substance P

Substance P immunostaining was not found to be altered in the dorsal hom

follo\\'ing FCA-induced inflammation. This finding does not appear ta be

consistent with reports showing increases in substance P rnRNA (Donaldson et • al.. 1992) and immunostaining (Smith et al., 1992; Ahmed et af., 1995) in the DRG of fCA-treated animals. Moreover, an increase in substance P release

was also observed (Oku et al., 1987; Garry and Hargreaves, 1992). However,

immunostaining is limited to the labelling of \\ithin the nerve

tenninais. Thus, it is possible that the apparent increase in substance P

synthesis is coupled to a concomitant increase in the release. This would lead

to an apparent lack of change in immunohistochemical staining. This

hypothesis is supported by reports of increased substance P release from central

terminaIs in fCA-treated animaIs (Oku et al., 1987; Garry and Hargreaves,

1992). • Chapter IV Discussion 91

The lack of changes in substance P immunostaining observed in the present • study does not correspond with other reports ofsubstance P immunostaining in chronic constriction model of neuropathic pain (Bennett et al., 1989; Garrison

et al., 1993; Kajander and Xu, 1995). This discrepancy may reflect a more

deleterious insult incurred from chronic constriction in comparison to the

application of the sciatic cuff despite the similarities in hyperalgesic and

allodynic responses. Our finding that the sciatic cuff modulated nociceptive

behaviour without a concurrent increase in substance P immunostaining

suggests that the increase in immunostaining observed following CCI is not

directly related to the behavioural responses.

c) Galanin • The absence of changes in galanin immunostaining 0 bserved in the spinal cord of monoarthritic rats is consistent with a report by Ji et al. (1995) showing no

change in the expression of galanin 3 days following the injection of

carrageenan.

There were also no differences in galanin immunostaining in the dorsal horn of

neuropathic animais. This finding does not agree \.\.'ith data from Ma and Bisby

(1997) demonstrating an increase in galanin immunostaining in the superficial

dorsal horn of the spinal cord fo11o\o\ling chronic constriction of the sciatic

nerve. Similarly to substance P, this discrepancy may reflect a more deleterious

insult incurred from chronic constriction in comparison to the application ofthe

sciatic cuff. The suggested role of galanin in nerve regeneration may account • for the increased immunostaining ofthis peptide fo11owing chronic nerve injury Chapter IV Discussion 92

(Burazin and Gundlach, 1998). In fact, there is no direct evidence in the • literature supporting a raIe for galanin in models of chronic pain in conscious animaIs.

2) Binding Sites

Binding site levels were studied as marker of pathophysiological changes that

may occur following chronie pain injury. Modulation of receptor populations

following chronic pain insults can be used to predict which neuropeptides may

have a role in chronic pain syndromes or neuroplastic events related to the

pathophysiology ofchronie pain.

• a) CGRP Significant increases ln CGRP binding sites were observed following the

injection of fCA. This inerease in binding sites is most probably due to

changes occurring at the cellular level rather than compensatory rnechanisms in

response to the availability of endogenous peptide. This hypothesis is

supported by the lack of differences in immunostaining. This finding was not

previously reponed. Sorne groups reported a small decrease in CGRP binding

sites in the superficial dorsal hom at 4 days post-injection (Galeazza el al.,

1992) while others reported no change at 2 and 8 days (Seybold el al., 1995).

However, small increases in binding were noted in lamina V at 4 days and in • lamina X at 2, 4 and 8 days post-injection (Seybold et al., 1995). Chapter N Discussion 93

• InterestinglY9 CGRP binding sites were shawn to he decreased in the dorsal horn of neuropathic rats. This decrease in CGRP binding sites is consistent

\vith the presence of CGRP receptors on primary afferent terminaIs and

degeneration of these neurones following constrictive injury. Decreases in

CGRP binding observed in this study were not reponed by Gany et al. (1991)

follo\ving the constriction of the sciatic nerve. This difference may he

explained by a greater number of degenerating prirnary afferent terminaIs

follo\\ing the procedure used in the present study. Moreover. Garry et al.

el 991) used a different approach in the analysis of binding densities, measuring

only four areas of 27.5J.U112.

• Hence, CGRP binding is differentially altered depending upon the type of chromc pain studied. This may improve our understanding of the role of CGRP

in chronic pain syndromes observed in the dinic. It is still unclear as to which

ofthe t\\"o CGRP1 or CGRP2 receptor subtypes is involved in chronic pain as no

evidence is yet available regarding their functional significance.

b) Galanin

Small increases in galanin binding sites densities \Vere noted following the

injection of FCA. This finding was not observed by Ji et al. following the

injection of carrageenan (Ji et al., 1995). This could he explained by • differential effects ofcarrageenan versus Freund's complete adjuvant. Chapter IV Discussion 94

Galanin binding sites were decreased in lamina II at 14 days following surgery

• and increased in deeper layers of the dorsal horn at 7 days post-surgery. This

shift in binding observed over time suggests plasticity in galanin binding, one

week being sufficient for G-protein coupled receptors tum over. Increases in

galanin binding sites may be due to cellular changes rather than compensatory

mechanism in response to changes in peptide content since we failed to observe

alterations in galanin immunostaining. Hence, increases in galanin binding sites

could result in increased inhibitory response and may contribute to endogenous

antinociceptive systems (Wiesenfeld-Hallin et al., 1992).

c) Substance P • Significant increases in substance P binding sites were observed following the injection of Freund's complete adjuvant. The increase in substance P binding

sites observed in the present study confirmed previous reports showing

increases in substance P~l(-1 receptor immunostaining (Abbadie et al., 1996)

and substance P binding densities (Stuck.)' et al., 1993).

Substance P binding sites were aIso sho\\n to be increased in the dorsal horn of

neuropathic rats. The increase in substance P binding sites is consistent \.Vith

previous work from Basbaurn's group reporting that substance PINK-l receptor

immunostaining was increased (Abbadie et al., 1996).

These results also support studies suggesting that substance P receptors are • mostly located post-synaptically (Helke et al., 1986). Chapter IV Discussion 95 • C) Conclusion The injection ofFreund's complete adjuvant produces a stable long-lasting paw

oedema and mechanical allodynia as well as a short period of thennal

hyperalgesia. This animal model provides a tools for the study ofinflammatory

pain. The constriction ofthe sciatic nerve using polyethylene tubing produces a

standardized nerve injury and led to long-lasting mechanical allodynia and

thennal hyperalgesia and may be usefui in the study ofneuropathic pain.

The changes in CGRP binding levels observed following the injection of

Freund~s complete adjuvant or constriction of the sciatic nerve were not

mirrored by similar changes in substance P and galanin binding sites. This

• finding indicates differential involvement of these neuropeptides and their

receptors in chronic pain mechanisms.

• • References Abbadie, C., Brown, J.L., Mantyh, P.W., and Basbaum, A.I. (1996) Spinal cord

substance P receptor immunoreactivity increases in both inflammatory

and nerve injury models ofpersistent pain. Neuroscience 70, 201-209.

Abbott, F.V., Franklin, K.B.J., and Westbrook, R.F. (1995) The fonnalin test:

scoring properties of the first and second phases ofthe pain response in

rats. Pain 60, 91-102.

Ahmed, M., Bjurholm, A., Schultzberg, M., Theodorsson, E., and Kreicbergs,

A. (1995) Increased levels of substance P and calcitonin gene-related • peptide in rat adjuvant arthritis. A combined immunohistochemical and radioimmunoassayanalysis. Arthritis and Rheumatism 38,699-709.

Aiyar, N., Rand, K., Elshourbagy, N.A., Zeng, Z., Adamou, J.E., Bergsma, D.J.,

and Li, Y. (1996) A cDNA encoding the calcitonin gene-related peptide

type 1 receptor. J.BioI.Chem. 271 , 11325-11329.

Amara, S.G., Jonas, V., Rosenfeld, M.G., Ong, E.S., and Evans, R.M. (1982)

Alternative RNA processing in calcitonin gene expression generates

rnRNAs encoding different polypeptide products. l~lature 298, 240-244. • References 97

Bates, R.F.L., Buckley, G.A., and MeArdle, C.A. (1984) Comparison of the • antinoeiceptive effects of centrally administered calcitonins and calcitonin gene-related peptide. Br.J.Pharmacol. 82,295.

Bauer. F.E., Ginsberg, L., Venetikou, M., MacKay, D.1., Burrin, 1.M., and

Bloom, S.R. (1986) Growth honnone release in man induced by galanin,

a new hypothalamic peptide. Lancer 2, 192-195. .,

Bell, O. and McDennott, B.l. (1996) Calcitonin gene-related peptide in the

cardiovascular system: characterization ofreceptor populations and their

(patho)physiological significance. Pharmaco/.Rev. 48, 253-288.

Bennett, G.l., Kajander, K.C., Sahara, Y., Iadorola, M.1., and Sugimoto, T. • (1989) Neurochemical and anatomical changes in the dorsal horn of rats with an experimental painful peripheral neuropathy. In: Processing of

sensory information in the superficial dorsal horn ofthe spinal cord,

463-471. Edited by Cervero, F.~ Bennen., G.l., and Headley, P.M.~ New

York, Plenum Press.

Bennett. G.l. and Xie, Y.-K. (1988) A peripheral mononeuropathy in rat that

produces disorders of pain sensation like thase seen in man. Pain 33,

87-107. • References 98

Benne~ M.M. and Amara, S.G. (1992) Moleeular mechanisms of celI-specifie • and regulated expression of the calcitoninla-CGRP and J3-CGRP genes. Ann.NYAcadSci., 657, 36-49.

Berkley, K.J. and Hubscher, C.H. (1995) Are there separate central nervous

system pathways for touch and pain? Nature Medecine 1~ 766-773.

Bharga\'~ H.N. (1994) Diversity of Agents That Modify Opioid Tolerance,

Physical Dependence, Abstinence Syndrome, and Self-Administrative

Behaviour. Pharmacol.Rev. 46,293-324.

Bharga\'~ H.N. and Gulati, A. (1990) Down-regulation ofbrain and spinal cord

mu-opiate receptors ln morphine tolerant-dependent rats. • Eur.J.Pharmacol. 190, 305-311.

Brady~ L.S., Herkenham, M., Long, J.B., and Rothrnan, R.B. (1989) Chronic

morphine increases J-l-opiate receptor binding in rat brain: a quantitative

autoradiographie study. Brain Res. 477, 382-386.

Brain, S.D. and Cambridge, H. (1996) Calcitonin gene-related peptide:

Vasoactive effects and potential therapeutic role. Gen.Pharmac. 27,

607-611. • References 99

Brain, S.D., Cambridge, H., Hughes, S.R., and Wilsoncroft, P. (1992) Evidence • that calcitonin gene-related peptide contributes to inflammation in the skin and joint. Ann.NYAcadSci. 657,412-419.

Brain, S.D. and Williams, T.J. (1985a) lnflammatory oedema induced by

synergism between calcitonin gene-related peptide (CGRP) and

mediators ofincreased vascular permeability. Br.J.Pharmacol. 86, 855­

860.

Brain, S.D., Williams, T.J., Tippins, J.R., Morris, H.R., and MacIntyre, I.

(1985b) Calcitonin gene-related peptide is a potent vasodilator. l'la/ure

313,54-56.

• Bunker. C.B., Terenghi, G.. Springall, D.R., Polak, J.M., and Dowd, P. (1990) Deficiency in calcitonin gene-related peptide in Raynaud's phenomenon.

The Lance/ 336, 1530-1533.

Butler. S.H., Godefroy, F., Besson, J.M., and Weil-Fug~ J. (1992) A limited

arthritic model for chronic pain studies in the rat. Pain 48, 73-81.

Calvino. B., Crepon-Bernard. M.-O., and Le Bars, D. (1987) Parallel clinical

and behavioral studies of adjuvant-induced arthritis in the rat: possible

relationship \\ith chronic pain. Behavioral Brain Research 24, 11-29. • References 100

Carlton, S.M. and Coggeshall, R.E. (1996) Stereological analysis ofgalanin and • CGRP synapses in the dorsal horn of neuropathic primates. Brain Res. 711, 16-25.

Chang, M.M. and Leeman, S.E. (1970) Isolation of a sialogogic peptide from

bovine hypothalamic tissue and its characterization as substance P.

J.BioI.Chem. 245,4784-4790.

Chaplin! S.R., Bach, F.W., Pogril, J.W., Chung, J.M., Yaksh, T.L. (1994)

Quantitative assessement of tactile allodynia in the rat paw. J. Neurosci.

Alethods 53, 55-63.

Chatterjee, T.K. and Fischer, J.A. (1991) Multiple affinity forms of the • calcitonin gene-related peptide receptor In rat cerebellum. J\foI.Pharmacol. 39, 798-804.

Chatterjee, T.K., Moy, J.A., Cai, J.J., Lee, H.C., and Fisher, R.A. (1993)

Solubilization and characterization of a guanine nucleotide-sensitive

form of the calcitonin gene-related peptide receptor. Mol.Pharmacol.

43,167-175.

Chanerjee, T.K., Moy, J.A., and Fisher, R.A. (1991) Characterization and

regulation of high affinity calcitonin gene-related peptide receptors in • cultured neonatal rat cardiac myocytes. Endocrinology 128,2731-2738. References 101

Coderre, T.J. and Wall, P.D. (I987) Ankle joint urate arthritis (AlUA) in rats: • an alternative animal model of arthritis to that produeed by Freund's adjuvant. Pain 28, 379-393.

Collin~ E., Mantelet, S., Frechilla, D., Pohl, M., Bourgoin, S., Hamon, M., and

Cesselin, F. (1993) Increased in vivo release of caleitonin gene-related

peptide-like material from the spinal cord in arthritic rats. Pain 54, 203­

211.

Crawley, l.N., Austin, M.C., Fiske, S.M., Martin, B., Consolo, S., Berthold, M.,

Langel, Ü., Fisone, G., and Bartfai, T. (1990) Aetivity of centrally

administered galanin fragments on stimulation of feeding behavior and

on galanin receptor binding in the rat hypothalamus. J.Neurosci. 10,

• 3695-7000.

Cuello, A.C., Ribeiro-da-Silva, A., Ma, W., De Koninck, Y., Henry, l.L. (1993)

Organization of substance P primary sensory neurons: ultrastruetural

and physiological correlates. Regul. Pepl46, 155-164.

De Koninck, Y., Ribeiro-da-Silva, A., Henry, l.L., Cuello, A.C. (1992) Spinal

neurons exhibiting a specifie nociceptive response receive abundant

substance P-containing synaptic contacts. Proc. Acad. Sei. US.A. 89,

5073-5077. • References 102

Dennis, T., Fournier, A., Cadieux, A., Pomerleau, F., Jolicoeur, F.B., St·Pierre, • S., and Quirion, R. (1990) hCGRPs-37, a calcitonin gene·related peptide antagonist revealing calcitonin gene·related peptide receptor

heterogeneity in brain and periphery. J.Pharmacol.Exp.Ther. 254, 123­

254.

Dennis, T., Fournier, .A..., Guard, S., St-Pierre, S., and Quirion, R. (1991)

Calcitonin gene·related peptide (hCGRP alpha) binding sites in the

nucleus accumbens. Atypical structural requirements and marked

phylogenic differences. Brain Res. 539, 59-66.

Dennis, T., Fournier, A., St-Pierre, S., and Quirion, R. (1989) Structure-activity • profile ofcalcitonin gene-related peptide in peripheral and brain tissues. Evidence for receptor multiplicity. J.Pharmacol.Exp. Ther. 251, 718­

725.

Dickenson, A.H. (1995) Central Acute Pain ~fechanisms. Annals ofMedecine

27, 223-227.

Ding, W.G., Guo, L.D., Kitasato, H., Fujimura, M., and Kimura, H. (1998)

Phylogenic study of calcitonin gene-related peptide-immunoreactive

structures in the pancreas. Histochem.Cell.Biol. 109, 103-109.

Donaldson, L.f., Harmar, A.J., McQueen, O.S., and Seckl, J.R. (1992) • Increased expression of preprotachykinin, calcitonin gene-related References 103

peptide, but not vasoactive intestinal peptide messenger RNA in dorsal • root ganglia during the development ofadjuvant monoarthritis in the rat. .Molecular Brain Research 16~ 143-149.

Donaldson, L.f., Seckl, J.R., and McQueen, O.S. (1993) A discrete adjuvant­

induced monoarthritis in the rat: effects of adjuvant dose.

J.Neuroscience Methods 49,5-10.

Donnerer, J., Schuligoi, R., and Stein, C. (1992) Increased content and transport

of substance P and calcitonin gene-related peptide in sensory nerves

innervating inflamed tissue: evidence for a regulatory function of nerve

growth factor in vivo. lveuroscience 49, 693-698.

• Dray, A. and Urban, L. (1996) New pharmacological strategies for pain relief. Annu.Rev.Pharmcol. Toxicol. 36, 253-280.

Edwards, R.M., Stack, E.J., and Trizna, W. (1991) Calcitonin gene-related

peptide stimulates adenylate cyclase and relaxes intracerebral arterioles.

J.PharmacoI.Exp.Ther. 257, 1020-1024.

Feuerstein, G., Willene, R., and Aiyar, N. (1995) Clinical perspectives of

calcitonin gene-related peptide pharmacology. Cano J .Physiol.

Pharmacol. 73, 1070-1074. • References 104

Fong. T.M. (1996) Molecular Biology of Tachykinins. Chapter 1, In: • Neurogenic Inflammation. Eds. P. Geppeni, P. Holzer, CRe Press, Boca Raton, New York, 3-14.

Foulkes, R., Shaw, N., Bose, C., and Hughes, B. (1991) Differentiai vasodilator

profile of calcitonin gene-related peptide in porcine large and small

diameter coronary artery rings. Eur.J.Pharmacol. 201, 143-149.

Fowler. C.J. and Fraser, G.L. (1994) fJ.-. ô-, k-Opioid Receptors and their

Subtypes. A Critical Review with Emphasis on Radioligand Binding

Experiments. Neurochem.lnt. 24, 401-426.

Franco-Cereced~ A., Gennari, C., Nami, R., Agnusdei, D., Pernow, J., • Lundberg, J.M., and Fischer, J.A. (l987a) Cardiovascular effects of calcitonin gene-re)ated peptides 1and II in man. Circ. Res. 60, 393-397.

Franco-Cereced~ A., Henke, H., Lundberg, J.M., Petermann, J.B., H5kfelt, T.,

and Fischer, J.A. (l987b) Calcitonin gene-related peptide (CGRP) in

capsaicin-sensitive substance P-immunoreactive sensory neurons ln

animais and man: distribution and release by capsaicin. Peptides 8, 399­

410.

Franco-Cereced~ A. and Lundberg. J.M. (1985) Calcitonin gene-related peptide

(CGRP) and capsaicin-induced stimulation of heart contractile rate and • force. lvaunyn Schmiedebergs Arch.Pharmacol. 331, 146-151. References 105

Galeazza, M.T., Garry, M.G., Yost, H.J., Strai~ K.A., Hargreaves, K.M., and • Seybold, V.S. (1995) Plasticity in the synthesis and storage ofsubstance P and ealcitonin gene-related peptide in primary afferent neurons during

peripheral inflammation. Neuroscience 66, 443-458.

Gale~ M.T., Stueky, C.L., and Seybold, V.S. (1992) Changes in

125 [ I]hCGRP binding in rat spinal cord in an experimental model of

acute, peripherai inflammation. Brain Res. 591, 198-208.

Garriso~ C.l., Dougherty, P.M., and Carlton, S.M. (1993) Quantitative analysis

of substance P and ealcitonin gene-related peptide

immunohistochemical staining in the dorsal hom of neuropathie MK­ • 80 I-treated rats. Brain Res. 607, 205-214. Garry. M.G. and Hargreaves, K.M. (1992) Enhanced release of immunoreactive

CGRP and substance P from spinal dorsal hom slices occurs during

carrageenan inflammation. Brain Res. 582, 139-142.

Garry, M.G., Kajander, K.C., Bennett, G.l., and Seybold, V.S. (1991)

Quantitative autoradiographie analysis of [115 I]_human CGRP binding

sites in the dorsal hom of rat follo\\ing chronic constriction injury or

dorsal rhysotomy. Peptides 12, 1365-1373.

Gibson, S.l., Polak, l.M., Bloom, S.R., Sabate, LM., Mulderry, P.K., Ghatei, • M.A., Morrison, l.F.B., Kelly, l.S., Evans, R.M., and Rosenfeld, M.G. References 106

(1984) Calcitonin gene-related peptide (CGRP) immunoreactivity in the • spinal cord ofman and eight other species. J.Neurosci. 4,3101-3111.

Girgis, S.L, Macdonald, D.W., Stevenson, J.C., Bevis, P.J., Lynch, C.,

Wimalawansa, S.J., Self, C.H., Morris, H.R., and MacIntyre, I. (1985)

Calcitonin gene-related peptide: potent vasodilator and major product of

calcitonin gene. Lancer 2, 14-16.

Giuliani, S., Wimalawansa, S.1., and Maggi, C.A. (1992) Involvement of

multiple receptors in the biological effects of calcitonin gene-related

peptide and amylin in rat and guinea pig preparations. Br.J.Pharmacol.

107,510-514.

• Gouardères, C., Jhamandas, K., Cridland, R., Cros, J., Quirion, R., and Zandberg, J. (1993) Opioid and substance P receptor adaptations in the

rat spinal cord following sub-chronic intrathecal treatment with

morphine and naloxone. Neuroscience 54, 799-807.

Habert-Ortoli, E., Arniranoff, B., Loquet, 1., Laburthe, M., and Mayaux, J.-f.

e1994) Molecular cloning of a functional human galanin receptor.

Proc.Natl.AcadSci. USA 91, 9780-9783.

Hannar. A.J., Hyde, V., Chapman, K. (1990) Identification and cDNA sequence

ofdelta-preprotachykinin, a fourth splicing variant ofthe rat substance P • precursor. FEBS Letl. 275, 22-24. References 107

Heinrich, P.C., Behnnann, 1., Muller-Newen, G., Scaper, F., Graeve, L. (1998) • Interleukin-6-type cytokine signalling through the gp130/JakISTAT pathway. Biochem. J. 334, 297-314.

Helke, C.l., Charlton, C.G., and Wiley, R.G. (1986) Studies on the cellular

localization ofspinal cord substance P receptors. Neuroscience 19, 523-

533.

Henry, l.L. (1976) Effects ofsubstance P on functionally identified units in cat

spinal cord. Brain Res. 114, 439-451.

Hëkfelt, T., Arvidsson, U., Ceccatelli, S., Cortés, R., Cullheim, S., Wiesenfeld­

Hallin, Z., lohson, H., Orazzo, C., Piehl, F., Pieribone, V., Schalling, • M., Terenius, L., Ultbake, B., Verge, V.M., Vilar, M., Xu, X.-J., and Xu.Z (1992) Calcitonin gene-related peptide in the brain, spinal cord,

and sorne peripheral systems. Ann.NYAcad.Sci. 657, 119-134.

H6kfelt, T., Kellerth, J.-O., Nilsson, G., and Pemow, B. (1975) Experimental

immunohistochemical studies on the localization and distribution of

substance P in cat primary sensory neurons. Brain Res. 100,235-252.

H6kfelt, T., Wiesenfeld-Hallin, Z., Villar, M.J., and rvlelander, T. (1987)

Increase in galanin-like immunoreactivity in rat dorsal root ganglion • cells after peripheral axotorpy. Neurosci.Lett. 83,217-220. References 108

H611t, V., Oum, J., Blasig, J., Schubert, P., and Herz, A. (1975) Comparison of • in vivo and in vitro parameters of opiate receptor binding in naive and tolerant/dependent radents. Lift Sei. 16, 1823-1828.

Hughes, J.J., Levine, A.S., Morley, J.E., Gosnel1, B.A., and Silvis, S.E. (1984)

Intraventricular calcitonin gene-related peptide inhibits gastric acid

secretion. Peptides 3, 665-667.

Iadorol~ M.J. and Draisci, G. (1988) Elevation ofspinal cord mRNA

compared to dorsal root ganglion mRNAs during peripheral

inflammation. In: The Arthriric Rat, 173-184. Edited by Besson, J.-~l.

and Guilbaud, G., Amsterdam, Excerpta Medica.

• Jansen-Olesen, 1., Mortensen, A., and Edvinsson, L. (1996) Calcitonin gene­ related peptide is released [rom capsaicin-sensitive nerve fibres and

induces vasodilation of human cerebral arteries concomitant \vith

activation ofadenyl cyclase. Cepha/a/gia 16, 310-316.

Ji, R.-R., Zhang, X., Zhang, Q., Dagerlind, A., Nilsson, S., Wiesenfeld-Hallin,

Z., and Hôkfelt, T. (1995) Central and peripheral expression of galanin

in response to inflammation. Neuroscience 68, 563-576.

Jolicoeur, F.S., Ménard, D.P., Fournier, A., and St-Pierre, S. (1992) Struture

activity analysis ofCGRP's neurobehavioral effects. Ann.NY Acad.Sci., • 657, 155-163. References 109

Kajander~ K.C. and Xu, 1. (1995) Quantitative evaluation of calcitonin gene­ • related peptide and substance P levels in rat spinal cord following peripheral nerve injury. Neurosci.Lett. 186~ 184-188.

Kapas, S.~ Clark, AJ.L., and Abbadie, C. (1995) Identification of an orphan

receptor gene as a type 1 calcitonin gene-related peptide receptor.

Biochem.Biophys.Res.Commun. 217, 832-838.

Kar, S. and Quirion, R. (1995) Neuropeptides receptors in developing and adult

rat spinal cord; an in vitro quantitative autoradiography study of

calcitonin gene-related peptide, neurokinins, J.1-opioid, galanin,

somatostatin, neurotensin and vasoactive intetinal polypeptide receptors. • J. Comp.Neurol. 354, 253-281. Kar, S., Rees, R.G., and Quirion, R. (1993) Altered calcitonin gene-related

peptide, substance P and immunoreactivities and receptor

binding sites in the dorsal spinal cord of the polyarthritic rat.

Eur.J.Pharmacol. 6, 345-354.

Kawaguchi, Y., Hoshimaru, M., Naw~ H., and Nakanishi~ S. (1986) Sequence

analysis of cloned cDNA for rat substance P precursor: existence of a

third substance P precursor. Biochem. Biophys.Res.Commun. 139, 1040­

1046. • References 110

Kawai, Y., Takami, K., Shiosaka, S., Emson, P.C., Hillyard, C.]., Girgis, S., • MacIntyre, L, and Tohyama, M. (1985) Topographie locaIization of calcitonin gene-related peptide in the rat brain: an immunohistochemical

analysis. lVeuroscience 15, 747-763.

Kawamur~ M., Kuraishi, Y., Minami, M., and Satoh, M. (1989)

A.ntinociceptive effect of intrathecally administered antiserum against

calcitonin gene-related peptide on thermal and mechanical noxious

stimuli in experimentaI hyperalgesic rats. Brain Res. 497, 199-203.

Kelly, 0.0. (1985) Somatie Sensory System IV In: Central representations of

pain and analgesias.199-211 .

• Khan, L, Collins, S.M. (1994) Fourth isomer of preprotaehykinin messenger RNA encoding for substance P in rat intestine. Biochem. Biophys. Res.

Commun. 202, 796-802.

King, S.C., Slater, P., and Turnberg, L.A. (1989) Autoradiographie localization

of binding sites for galanin and VIP in small intestine. Peptides 10,

313-317.

Kotani. H., Hoshimaru, M., Nawa, H., and Nakanishi, S. (1986) Structure and

gene organization of bovine neuromedin K precursor. Proc .Narl. Acad. • Sei. USA 83, 7074-7078. References 111

Krahn~ D.D., Gosnell~ B.A., Levine, J.D., and Morley, J.E. (1984) Effects of • calcitonin gene-related peptide on food intake. Peptides 5, 861-864.

K.rause, J.E., Bu~ J.-Y., Taked~ Y., Blount, P., Raddatz, R., Sachais B.S., Chou~

K.B., Takeda, J., McCarson, K., DiMaggio, D. (1993) Structure,

expression and second messenger-mediated regulation ofthe human and

rat substance P receptors and their genes. Regul. Pept. 46, 59-66.

Kumar, V.~ Cotran, R.S., and Robbins, S.L. (1992) Disease ofhlood vessels. In:

Basic Pathology, Fifth Edition Ed., 293-294. Edited by Mitchell, J.,

Philadelphia, W.B. Saunders Company.

Kuraishi, Y., Nanayama, T., Ohno, H., Minami, M., and Satoh, M. (1988) • Antinociception induced in rats by intrathecal administration of antiserum against calcitonin gene-related peptide. lVeurosci.Lett. 92,

325-329.

Kurz. B., von Gaudecker, B., Kr~ A., Krish, B., and Mentlein, R. (1995)

Calcitonin gene-related peptide and its receptor in the thymus. Peptides

16, 1497-1503.

Lai, J.P., Douglas, S.D., Rappaport, E., Wu, J.M., Ho, W.Z. (1998)

Identification of a delta isofonn of preprotachykinin mRNA in human

mononuclear phagocytes and lymphocytes. J. Neuroimmunol. 91, 121­ • 128. References 112

Le Greves, P., Nyberg, F., Hokfel~ T., and Terenius, L. (1989) Calcitonin gene­ • related peptide is metabolized by an endopeptidase hydrolyzing substance P. Regul.Pept. 25, 277-286.

Lee, Y., Takami, K., Kawai, Y., Girgis, S., HiIlyard, C.J., MacIntyre, L, Emson,

P.C., and Tohyama, M. (1985) Distribution of calcitonin gene-related

peptide in the rat peripheral nervous system with reference to its

coexistence with substance P. lveuroscience 15, 1227-1237.

Levine, J.O., Fields, H.L., and Basbaum, A.I. (1993) Peptides and the primary

afferent nociceptor. J.Neurosci. 13,2273-2286.

Libe~ f., Pannentier, M., Lefort, A., Dinsart, C., Van Sande, J., Maenhaut, C., • Simons, M.-J., Dumont, J., and Vass~ G. (1989) Selective amplification and cloning of four new members of the G protein­

coupled receptor family. Science 244, 569-572.

Luebke, A.E., Dahl, G.P., Roos, B.A., and Dickerson, I.M. (1996) Identification

of a protein that confers calcitonin gene-related peptide responsiveness

to oocytes by using a cystic flbrosis transmembrane conductance

regulator assay. Proc.lVatI.Acad.Sci. USA 93, 3455-3460.

Ma, W. and Bisby, M. (1997) Differentiai expression of galanin

immunoreactivities in the primary sensory neurons following partial and • complete sciatic nerve injuries. Neuroscience 79, 1183-1195. References 113

MacDonald, M.R, Taked~ J., Rice, C.M., and Krause, J.E. (1989) Multiple • tachykinins are produced and secreted upon post-translational processing ofthe three substance P precursor proteins, alpha-, beta-, and

gamma-preprotachykinin. Expression of the preprotachykinins in AtT­

20 celIs infected with vaccinia virus recombinants. J.Bio/. Chem. 264,

15578-15592.

MacIntyre, 1. (1992) The calcitonin farnily of peptides. Ann.l\'Y Acad.Sci. 657,

117-118.

Maggi~ C.A., Santicioli, P., and Giuliani, S. (1995) Role of cyclic AMP and

protein kinase A in K'" channel activation by calcitonin gene-related

peptide (CGRP) in the guinea-pig ureter. J.Auton.Pharmacol. 15, 403­

• 419.

Malcangio, M. and Bowery, N.G. (1996) Calcitonin gene-related peptide

content~basal outflowand electrically-evoked release from monoarthritic

rat spinal cord in vitro. Pain 66, 351-358.

t\1almberg~ A.B., Brandon, E.P., Idzerda, R.L., Liu, H., McKnight, G.S., and

Basbaum, A.I. (1997) Diminished inflammation and nociceptive pain

with preservation of neuropathic pain in mice with a targeted mutation

of the type 1 regulatory subunit of cAMP.dependent protein kinase. • JNeurosci. 17, 7462-7470. References 114

Mao, J., CogghilI, R.C., Kellstein, D.E., Frenk, H., and Mayer, D.J. (1992) • Calcitonin gene-related peptide enhances substance P-induced behaviors via metabolic inhibition: in vivo evidence for a new mechanism of

neuromodulation. Brain Res. 574, 157-163.

Maves, TJ., Pechman, P.S., Gebb~ G.f., and Meller, S.T. (1993) Possible

chemical contribution from chromic gut sutures produces disorders of

pain sensation like those seen in man. Pain 54, 57-69.

McDonald, TJ., Dupre, J., Tatemoto, K., Greeberg, G.R., Radziuk, J., and

Mun, V. (1985) Galanin inhibits insulin secretion and induces

hyperglycemia in dogs. Diaberes 34, 192-196.

• ~1cLatchie, l.M., Fraser, NJ., Main, M.J., Wise, A., Brown, J., Thompson, N., Solari, R., Lee, M.G., and Foord, S.M. (1998) RAMPs regulate the

transport and ligand specificity of the calcitonin-receptor-like transport.

.Nature 393, 333-339.

t\.1elander, T., Hokfelt, T., and Rokaeus, A. (1986) Distribution of galanin-like

immunoreactivity in the rat central nervous system. J Comp.Neurol.

248, 475-517.

t\.1énard, D.P., van Rossum, D., Kar, S., Jolicoeur, F.B., Jhamanda, K., and

Quirion, R. (1995a) Tolerance to the antinociceptive properties of • morphine in the rat spinal cord: alteration of calcitonin gene-related References 115

peptide-like immunostaining and receptor binding sites. • J.Pharmacol.Exp_ Ther. 273, 887-894.

Ménard, D.P., van Rossum, D., Kar, S., and Quirion, R. (l995b) Alteration of

calcitonin gene-related peptide and its receptor binding sites during the

development of tolerance to J.L and cS opioids. Can.J.PhysioI.Pharmacol.

73, 1089-1095.

Ménard, D.P., van Rossum, D., Kar, S., St-Pierrre, S., Sutak, M., Jhamandas,

K., and Quirion, R. (1996) A calcitonin gene-related peptide receptor

antagonist prevents the development of tolerance to spinal morphine

analgesia. JNeurosci. 16, 2342-2351 .

• rvtiyoshi, H. and Nakaya, Y. (1995) Calcitonin gene-related peptide activates the K~ channels of vascular smooth muscle celIs via adenylate cyclase.

Basic Res. Cardiol. 90, 332-336.

Morley. J.E., farr, S.A., and Flood, J.f. (1996) Peripherally administered

calcitonin gene-related peptide decreases food intake in mice. Peptides

17,511-516.

Mosconi. T. and Kruger, L. (1996) Fixed-diameter polyethylene cuffs applied to

the rat sciatic nerve induce a painful neuropathy: ultrastructural • morphometric analysis ofaxonal alterations. Pain 64, 37-57. References 116

Mulderry, P.K., Ghatei, M.A., Spokes, R.A., Jones, P.M., Pierson, A.M., • Hamid, Q.A., Kanse, S., Am~ S.G., Bunin, J.M., and Legon, S. (1988) Differentiai expression of aIpha-CGRP and beta-CGRP by

prirnary sensory neurons and enteric autonomie neurons of the rat.

Neuroscience 25, 195-205.

Nahin, R.L. and Byers, M. (1994) Adjuvant-induced inflammation of the rat

paw is associated with altered calcitonin gene-related peptide

irnrnunoreactivity within cell bodies and peripheral endings of primary

afferent neurons. J.Comp.Neurol. 349,475-485.

Nakajima, Y., Tsuchida, K., Negishi, M., Ito, S., Nakanishi, S. (1991) Direct

linkage ofthree tachykinin receptors to stimulation ofboth phosphotidyl

• hydrolysis and cyclic AMP cascades in Chinese hamster ovary

cells. J. Biol. Chem. 267,2437-2442.

Nakanishi, S. (1991) Mammalian Tachykinin Receptors. Ann. Rev. lveurosci.

14, 123-126.

Nawa, H., Kotani, H., and Nakanishi, S. (1984) Tissue-specifie generation of

two preprotachykinin rnRNAs from one gene by alternative RNA

splicing. Nature 312, 729-734. • References 117

OkLl, R., Satoh, M., and Takagi, H. (1987) Release of substance P from the • spinal dorsal horn is enhanced in polyarthritic rats. Neurosci.Lett. 74, 315-319.

Parsons, A.M. and Seybold, V.S. (1997) Calcitonin gene-related peptide

induces the formation of second messengers in primary cultures of

neonatal rat spinal cord. Synapse 26, 235-242.

Pecile, A., Guidobono, F., Neni, C., Sibilia, V., Biella, G., and Braga, P.C.

(1987) Calcitonin gene-related peptide: antinociceptive activity in rats,

comparison with calcitonin. Regul.Pept. 18, 189-199.

Pert. C.B. and Snyder, S.H. (1976) Opiate receptor binding-enhancement by • opiate administration in vivo. Biochem.Pharmacol. 25,847-853.

Plenderleith, M.B., Haller, C.J., and Snow, P.J. (1990) Peptide co-existence in

axon tenninals within the superficial dorsal horn of the rat spinal cord.

Synapse 6, 344-350.

Portenoy, R. K. Tolerance to opioids analgesics: cIinicaI aspects. (Volume 21

Palliative Medecine: Problem ..c\reas in Pain and Symptom

Management), 49-65. 1994. Imperial Cancer Research Funds. Cancer

Surveys. • References 118

Post~ C., Alari, L., and Hokfelt, T. (1988) Intrathecal galanin increases the • latency in tail-flick and hot-plate tests in mice. Acta Physiol.Scand. 132, 583-584.

Quirion~ R., Dumont, Y. (1998) Multiple receptors for CGRP and related

peptides. In: CGRP'98, D. Poyner (Ed.) Academic Press.

Quirion~ R., van Rossum~ D.~ Dumont, Y.~ St-Pierre, S., and Fournier, A. (1992)

Characterization of CGRP1 and CGRP2 Receptor Subtypes. Ann.NY

AcadSci. 657, 88-105.

Ribeiro-da-Silva, A. (1995) Ultrastructural features of the colocalization of

calcitonin gene-related peptide with substance P or somatostatin in the • dorsal hom ofthe spinal cord. Can.J.Physio/.Pharmacol. 73, 940-944.

Rea, M. and Changeux, J.-P. (1991) Charaterization and developmental

evolution of a high-affinity binding site for calcitonin gene-related

peptide on chick skeletal muscle membrane. IVeuroscience 41, 563-570.

Resenfeld, M.G., Emeson, R.B., Yeakley, J.M., MerilIat, N., Hedjran, F., Lenz,

J., and Delsert, C. (1992) Calcitonin gene-related peptide: a

neuropeptide generated as a consequence of a tissue-specific,

developmentally regulated alternative RNA processing events. Annals • l'/ew York Academy ofSciences 657, 1-17. References 119

Rosenfeld~ M.G.~ Mermod, J.-J., Am~ S.G.~ Swanson, L.W., Sawchenko~ • P.E., Rivier, J., Yale, W.W., and Evans, R.M. (1983) Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific

RNA processing. Nature 304, 129-135.

Rossler, W., Gerstberger, R., Sann, H., and Pierau~ F.K. (1993) Distribution and

binding sites of substance and calcitonin gene-related peptide and their

capsaicin-sensitivity in the spinal cord of rats and chicken: a

comparative study. Neuropeptides 25,241-253.

Rassowski, W.J., Jiang, N.Y., and Coy~ D.H. (1997) Adrenomedullin, amylin,

calcitonin gene-related peptide and their fragments are patent inhibitors • ofgastric acid secretion in rats. Eur.J. Pharmacol. 336, 51-63. Rothman, R.B., Long, J.B., Bykov, V., Xu, H., Jacobson, A.E., Rice, K.C., and

Holaday, J.W. (1991) Upregulation of the opioid receptor complex by

the chronic administration of morphine : a biochemical marker related to

the development oftolerance and dependence. Peptides 12, 151-160.

Routh. V.H., Helke, C.J. (1995) Tachykinin Receptors in the Spinal Cord.

Chapter 6 In. Progress in Brain Research, Nyberg, F., Sharm~ H.S.,

Wiesenfeld-Hallin (eds), 104,93-108. • References 120

Santicioli, P.~ Morbidelli, L., Parenti~ A., Ziche, M., and Maggi, CA. (1995) • Calcitonin gene-related peptide selectively increases cAMP levels in the guinea-pig ureter. Eur.J.Pharmacol. 289, 17-21.

Seybold, V.S., Gale~ M.T., Garry, tv1.G., and Hargreaves, K.M. (1995)

Plasticity of calcitonin gene-related peptide neurotransmission in the

spinal cord during peripheral inflammation. Can.J.Physiol.Pharmacol.

73, 1007-1014.

Sigrist~ S., Franco-Cereced~ A., Muff, R., Henke, H., Lundberg, J.M., and

Fischer, J.A. (1986) Specific receptor and cardiovascular effects of

calcitonin gene-related peptide. Endocrinology 119, 381-389.

• Skofitsch, G. and Jacobwitz, M.D. (1985) Calcitonin gene-related peptide: detailed immunohistochemical distribution in the central nervous

system. Peptides 6, 721-745.

Sluka, K.A. and \Vestlund, K.N. (1992) An experimental arthritis in rats: dorsal

hom aspartate and glutamate increases. .NeurosCÎ.Leu. 145, 141-144.

Smith~ G.O., Harmar, A.J., McQueen, O.S., and Seckl, J.R. (1992) Increase in

substance P and CGRP, but not somatostatin content of the innervating

dorsal root ganglia in adjuvant monoarthritis in the rat. Neurosci.Letr. • 137, 257-260. References 121

Stuc~:, C.L., Galeazza, M.T., and Seybold, V.S. (1993) Time-dependent • changes in Bolton-Hunter-Iabeled 125I-substance P binding in rat spinal cord following unilateral adjuvant-induced peripheral inflammation.

Neuroscience 57, 397-409.

Sun, Y.D. and Benishin, C.G. (1995) Effects of calcitonin gene-related peptide

on cyclic AMP production and relaxation of longitudinal muscle of

guinea-pig ileum. Peptides 16, 293-297.

Szabat, E., Salo, A., Vinanen, 1., Uusitalo. H., and Soinila, S. (1994) Production

and characterization of a monoclonal antibody against human calcitonin

gene-related peptide (CGRP) and its immunohistochemical application • to salivary glands. Histochem.J. 26, 317-326. Taga. T., Kishimoto, T. (1997) Gp 130 and the interieukin-6 family of

cytokines. Ann. Rev. Immun. 15, 797-819.

Takahashi. T. and Otsuka, M. (1975) Regional distribution ofsubstance P in the

spinal cord and nerve roots of the cat and the effect of dorsal root

section. Brain Res. 87, 1-11.

Tatemoto, K., Lundberg, J.M., Jômvall, H., and Mun, V. (1985) Neuropeptide

K: isolation, structure and biological activities of a novel brain • tachykinin. Biochem.Biophys.Res.Commun. 182, 947-953. References 122

Tatemoto, K., Rokaeus, A., Jornvall, H., McDonald, T.J., and Mun, V. (1983) • Galanin-a novel biologically active peptide from porcine intestine. FEES Lett 164, 124-128.

Tong. Y., Dumont, Y., van Rossum, D., Clark, A., Shen, S.·H., and Quirion, R.

(1998) Discrete expression of a putative calcitonin gene-related peptide

(CGRP) receptor mRNA in the rat brain and peripheral tissues:

comparison with the autoradiographie distribution ofthe CGRP receptor

binding sites. In: CGRP'98, D. Poyner (Ed.), Academie Press.

Torii, H., Hosoi, J., Asahina, A., and Granstein, R.D. (1997) Calcitonin gene-

related peptide and Langerhans cell function. • J.lnvest.DermatoI.Symp.Proc. 2,82-86. Tschopp, F.A., Henke, H., Petennann, J.B., Tobler, P.H., Janzer, R., Hokfelt,

T., Lundberg, J.M., Cuello, C., and Fischer, J.A. (1985) Calcitonin

gene-related peptide and its binding sites in the human central nervous

system and pituitary. Proc.NarI.Acad.Sci. USA 82, 248-252.

van Rossum, D., Hanisch, U.-K., and Quirion, R. (1997) Neuroanatomical

Iocalization, pharmacological characterization and functions of CGRP,

related peptides and their receptors. Neurosci.Biobehav.Rev. 21, 649­

678. • References 123

van Rossum, D., Ménard, D.P., Fournier, A., St·Pierre, S., and Quirion, R. • (1994) Binding profile of a selective calcitonin gene·related peptide (CGRP) receptor antagonist ligand, (125I]hCGRPg_37, in rat brain and

peripheral tissues. J.Pharmacol.Exp.Ther. 269, 846·853.

van Rossum, D., Ménard, D.P., and Quirion, R. (1993) Effect of guanine

nucleotides and temperature on calcitonin gene·related peptide receptor

binding sites in brain and peripheral tissues. Brain Res. 617,249-257.

von Euler, V.S. (1981) The bislory of substance P. Trends in Neuroscience 4,

IV-IX

\Vang. 5., Hashemi, T., He, CG., Strader, C., and Bayne, M. (1997) Molecular • cloning and phannacological characterization of a new galanin receptor subtype. Alol.Pharmacol. 52,337-343.

\Ve Il man, G.C., Quayle, J.M., and Standen, N.B. (1998) ATP-sensitive K+

channel activation by calcitonin gene-related peptide and protein kinase

A in pig coronary arterial smooth muscle. J.Physiol. 507, 117-129.

\Verling, L.L., McMahon, P.N., and Cox, B.M. (1989) Selective changes in J.L

opioid receptor properties induced by chronic morphine exposure.

Proc.Natl.Acad.Sci. USA 86, 6393-6397. • References 124

Wiesenfeld-Hallin~ Z.~ Bartfai, T., and Hokfelt, T. (I992) Galanin in sensory • neurons in the spinal cord. Frontiers Neuroendocrinol. 13, 319-343.

Wiesenfeld-Hallin, Z.~ Hokfelt, T., Lundberg, J.M., Forssmann, W.G.~

Reinecke, M., Tschopp, F.A., and Fischer, J.A. (1984) Immunoreactive

calcitonin gene-related peptide and substance P coexist in sensory

neurons to the spinal cord and interact in spinal behavioral responses of

the rat. Neurosci.Lel/. 52, 199-204.

Wiesenfeld-Hallin, Z., Xu, X.-J., Hao, J.-X., and Hokfelt, T. (1993) The

behaviouraI effects on intrathecal galanin on tests of thermal and

mechanicaI nociception in the rats. Acta Physiol.Scand. 147,457-458.

• \Viesenfeld-Hallin, Z., Xu, X.-J., Hokfelt, T., Bedecs, K., and Bartfai, T. (1992) Galanin-mediated control of pain: Enhanced role after nerve injury.

Proc.Narl. AcadSei. USA 89, 3334-333ï.

\Viesenfeld-Hallin, Z., Xu, X.-J., Villar, M.J., and Hokfelt, T. (1990) Intrathecai

galanin potentiates the spinal analgesic effect of morphine:

electrophysiologicaI and behavioural studies. .Neurosci.Lett. 109, 217­

221.

\Vimalawansa, S.J. (1992) Isolation, purification, and biochemical

characterization of calcitonin gene-related peptide receptors. Annals • New York Academy ofSciences 657,71-87. References 125

\Vimalawans~ S.l. (1993) The effects of neonatal capsaicin on plasma levels • and tissue contents ofCGRP. Peptides 14,247-252.

\Vimalawansa, S.l. (1996) CaIcitonin gene-related peptide and its receptors:

molecular genetics, physiology, pathophysiology, and therapeutics

potentials. Endocrine Reviews 17, 533-585.

\Vimalawansa, S.l. (1997) Amylin, calcitonin gene-related peptide, calcitonin

and adrenomedullin: a peptide superfamily. Cril.Rev.iVeurobiol. Il,

167-239.

\Vimalawansa, S.l. and EI-Kholy, A.A. (1993) Comparative study of

distribution and biochemical characterization of brain calcitonin gene­ • related peptide receptors in five different species. lVeuroscience 54, 513-519.

Wimalawansa, S.l., Emson, P.C., and MacInt)Te, 1. (1987a) Regional

distribution of calcitonin gene-related peptide and its specifie binding

sites in rats \Vith particular reference to the nervous system.

:Veuroendocrinology 46, 131-136.

\Vimalawansa, S.J. and MacInt)Te, I. (1987b) The presence of calcitonin gene­

related peptide in human cerebrospinal fluid. Brain 110, 1647-1655. • References 126

Wimalawansa, S.J. and Macintyre, 1. (1988) Calcitonin gene-related peptide and • its specifie binding sites in the cardiovascular system of the rat. Int.J.Cardiol. 20, 29-37.

\Vimalawansa, S.J., Morris, H.R., and Maclnt)Te, I. (1989) Both alpha- and

beta-calcitonin gene-related peptides are present in plasm~

cerebrospinal fluid and spinal cord in man. J.Mol.Endocrinol. 3, 247-

Xu, J., Pollock, C.H., and Kajander, K.C. (1996) Chromic gut suture reduces

calcitonin gene-related peptide and substance P levels in the spinal cord

following chronic constriction injury in the rat. Pain 64, 503-509.

• Xu, X.-J., Wiesenfeld-Hallin, Z., Villar, M.J_, and Hëkfelt, T. (1989) Intrathecal galanin antagonizes the facilitatory effect of substance P on the

nociceptive flexor reflex in the rat. Acta Physiol.Scand 137,463-464.

Yaksh, T.L., Jessell, T.M., Gamse, R., Mudge, A.W., and Leeman, S.E. (1980)

Intrathecal morphine inhibits substance P release from mammalian

spinal cord in vivo. Nature 286, 155-157.

Yashpal, K., Kar, S., Dennis, T., and Quirion, R. (1992) Quantitative

autoradiographic distribution ofcalcitonin gene-related peptide (hCGRP

alpha) binding sites in the rat and monkey spinal cord. J.Comp.Neurol. • 322, 224-232. References 127

Yousufzai, S. and Abdel...Latif, A.A. (1998) Calcitonin gene-related peptide • relaxes rabbit iris dilator smooth muscle via cyclic AMP-dependent mechanisms: cross-talk between the sensory and sympathetic nervous

system. Curr.Eye.Res. 17, 197-204.

Yu, L.C., Hansson, P., Brodda-Jansen, G., Theodorsson, E., and Lundeberg, T.

(1996) Intrathecal CGRPS-37 induced bilateral increase in hindpaw

withdrawal latency ln rats with unilateral inflammation.

Br.J.Pharmaco/. 117,43-50.

Yu, L.C., Hansson, P., and Lundberg, J.M. (1994) The calcitonin gene-related

peptide antagonist CGRPs-37 increases the latency ta withdrawal • responses in rats. Brain Res. 653, 223-230. Yu, L.C., Hansson, P., and Lundeberg, T. (1996) The calcitonin gene-related

peptide antagonist CGRPs-37 increases the latency ta \\'Îthdrawal

responses bilaterally in rats with unilateral experimental

mononeuropathy, an effect reversed by naloxone. .Neuroscience 71,

523-531.

Zaidi, M., Bevis, P.J., Abeyasekera, G., Girgis, S.I., Wimalawansa, S.J., Morris,

H.R., and MacIntyre, 1. (1986) The origin ofcirculating calcitonin gene­

related peptide in the rat. Journal ofEndocrinology 110, 185-190. • References 128

Zhang, X., JI, R.R., Nilsso~ S., Villar, M., Ubink, R., Ju, G., Wiesenfeld­ • Hallin, Z., and Hokfelt, T. (1995) Neuropeptide Y and galanin binding sites in rat and monkey lumbar dorsal root ganglia and spinal cord and

effect ofperipheral axotomy. Eur.J.NeurosCÎ. 7,367-380.

•

•