~~~~a~~i~l~~~~=~e (ii:'Z·:7:!Cl!e~~~~~~~~r~~:t~~~~~~:~g

DEVELOPMENT AND PF.ARMACOLOGlCAL CHARACIERlZATIC N OF AN ACUTEMODELOF ALLODYNlA

INmEANEST1!E:'l'IZEDRAT

by

Steph en EdwardSheDnaIl

Athesis su1:xni.tted to the School ofGr a duat e Studies inpar t ial fu lfilment ofth e requirementsfor the degree of

DoctorofPhil osophy

Specialization:Neuroscience

Divi s ionof Basi cMedical Sciences Faculty of Medicine Merrorial University of Newfoundland

St. JohnIS,Newfoundland

1994

ABSTRAcr

~~~~a~~i~l~~~~=~e (ii:'Z·:7:!Cl!e~~~~~~~~r~~:t~~~~~~:~g

resemblesal.Iodynie , a svrrptcmof clinica l neura l, injur y pain. In thepresent st udy , the effects of Lt. STR were examined in urecbane -anes cbeedaedrats. Noxiouspawpinch (PP), or tail irrrnersi on (TIl in 55°C water, evoked a pronounced pressor response , tachycardia,a motor\~ i;:hd..r,Jwal reflex and desynchronization of the efec t.rc e ncecnaIcqr ern (EEG) . Non-noxious, hair deflect ion (HD), applied to the back, flanks, legs and tail of the rat,elicited onlYminor cardiovascular responses. Following Lt. STR (010 .ug ) ,an identicalliDstimulusevoked responses resemblingthos eseen with noxious stimuli: an increase in mean arueriaL blood

~~S=f~~t:tf~~st;i~hi~r~~e~e~,IOO~:d~~;~~~;li~~f~

were: 1) observed only with a light J?lane ofanesth~s ia (as determinedby EEG) , 2) reversiblewfth time (with m 15- 30 minutes), 3)observed without convulsions, 4) evoked onl y when HD was applied to cutaneous dermatomeswithInnerva cion from spinal segments near the L,e . STR injection site (s egment al), and 5) i.t STR dose-dependent <lO-SOlIg} . I.t.

glycine produced dose-dependentinlubition of allindice s of STR-dependent allodynia with ~o and 95%C.!. values of 609 (429-865),694(548-8 7 8)and 54!)(45 8- 65 8)/19fo r inhibition of heart rate (HR.) increases, M.A .P . increases and rrccor-

d~~~~~) re:ls~xe~os~~~~~;~~~~ry i~ibfte~i~~'~deg;~~~~~

allodynia,possiblythrough meta..OOlism to glycine. The E:Dso and 95% C.I.valueswere 981(509-1889), 1045 (7 4 0- 14 76 ) and 10 83(843-13 91) IJ.g for inhibition of HR, M.A.P. and motor withdrawal responses, respectively. EEGsynchrony was unaffected by i.t. glycine or i.t. betaine. Neonatal capsaicin (25 rrg/kg, s.c., post-natal day (PNDl1, and 50

~~~Ua~ed'~e::n;es ~~t~l~ ~~i~f

^(8p5J)

,s~~~i(~ir

~ff~~t~:~e~%~~~l aIT;cf;la .n°tif~~~":~~' s~~~e~~~d~~

allodyniawere also unaffectedby1.e.rrorphine at a dose (50 /1g, Lt.) which completely al:olishe d responses evokedby noxious TI or PP. S'IR-dependent allodynia was dose-

~~~~~gI~l~:CUf£:t~~~~~' ~~IdD[tiifa:~~~~l~~) (~d f~~~

~~Ad~~~-~t~~~~iit-)S~l=:o~n~f~x~~r.a~~~~~~~~

CGG against HD-evoked, STR-dependentHR, M.A.P . ar:d motor - i i~

re sc ons es ·....er e 15.6 (11.3 -21.6 ), 16.9 (11.7-24 .3) and 8.1 (5.2-12.5; p.g,re s pe ct i ve l y . The ccrres pondm qvalues for NEQX·....ere 1.2.2 (6.8 -21.8 ) , H.4 {8 .6-24 . 0 Jand 10.-1 (5. 5 - lS . 6ip.g. EEQsynchronywasunaf'fected byLt.OC";:;orLt.

NBQY.. The resuft;sof thepresentsu:...dyIncac at;e that: glycine pl a ys an importan t rol e incne spina l mcduLat Lcn of non- nc cd cep trveinputand supportsthe hypot hes is that a lossof glycinergicmodulat ion may underlieallcdynia. Thefailure

~~pe~d~~tmo~~~~~d i~~i~~;~ Ctfu.Stic t~iS

^to

pJ:;;:e~ns~~

initiated~primary afferent neuronsnot norrrallyinvolved

a~p~~d~~~Pt~il;f~i~urr:lY~hf\~. ~ s~g~~~tt~~;e~:;

antagonists, and the failure of Lt. STR to produce hweralgesia to mechanical, thermal or chemic al noxi ou s atimulL,confirm the independence of nociceptive pathways and s'ra - sens dt.tveinput inthi s model. TheLt. STRmodel allows theinvestigationof an important synptom of neural injury pain (opi o id-r e s i s t an t allodynia) inanest he tizedanirrels, wi thout having to inflict inju;y or toexpose a conscious animal to aver sive painful conchtions, andmight providea usefu l alternatdve to chronic conscious animal modelsof allodynia .

KeyWords:

- iii -

I wish. to express my sincere apprec f.a ti.cn tomyresearch supervisc r ,D'!.'. Olriscopher Loonus, for his expert; guidance and encouragement throughout myresearch. His patience.

insight and sense of humourhave beengre3.t l y appreciated . I amalso gratefu l for histno r ouqh revt ev.... of this thesis.

I would also like to extendmygrati tude to the follO\V'ing individuals:

Dr. Richard Neuman for his advice about recording and interpreting the electrcencephalograms, his act ive participation onmy thesis ccenu.tcee,and for his prompt and effectivereview of this thesis.

Dr, David Bryant for many helpful discussions of the statisticalmethods. His open door, his patienceandhis manyinsights are greatly. appreciated.

Dr. Detlef Biegerfor letters of support onmybehalf.and gentlereminders of how muchrrcr eI need tole a rn. Dr. Paul Redfern for takingti me from his busy scheduleto serve onmy thesis comnittee.

Dr. James Reynolds for his open door, his exampleand hi s insightfulparticipationin mycomprehensive exam.

Dr.caroline Harley for her thought-provokingparticipation in myseminars and comprehensive exam.

Dr.Gerald Duncan for generously availing to me the space and services of the School ofPharmacythroughoutmyresearch.

Mrs.EleanorDuncan for makingmefeel welcome atmanysocial occasions.

Dr. Dongyuan Yao for inj ecting innumerable animals onmy behalf, not to mention several patientlydeliveredOuneee lessons.

Ms. Sue Ellen Maher for her expert assistance with the histology, andher good company.

Ms. Margaret Miller, Mr.John Bautista, Mr. Robert Gilbert and. Mr.Jacques Desousa for friendship andmanydistractions tomakethis degree less painful.

-iv-

Ferdos Rcbe z-t.s for sec r ecarfaf Dr. Lenk a Husa and the Animal Care staf f for the i r coope r at i onand exee l.I enccare ofour animals .

I wish toacknowledge the use of operat ing funds for this research , which were furnished in the fonn of grants co Dr. Loomis from theMedical ResearchCouncil of canadaand the ResearchandDeve l opment Comnittee,Facul t yof Medicine, MemorialUniversity of Newfoundland.

I am grateful to the Phannaceutical Manufacturer's Association of canada - Health Research Poundacdon, andthe Faculty of Medicine of Merrorial University for persona';

fundingduringlll'.1degreepr ogram.

r would also like to thank Novo Nordisk for providing a sample ofNBQXfor1.1Sein these experiments.

EorNagwa

- vi-

TABLE OF CONTENTS ABSTRAcr•.. ..•.•.•.•..•

ACKNOWLECG1Er-rr s..

J~ISTOF F'IGURESANDTABLES ... . .•. .. •.. • .. .. .. ..

LISTOF ABBREV'IA': IQNSAND S':r.(.!50LS... ... .. ... . ...• •. . 1.0GENERALINIRODucrrON...

1.1 Statementof th e Res ea r chProblem . 1.2 AnatomyandPhysiolcgy ofTactileandPain

sense.eden .

1.:2 .1 Peripheral Recep torsand Primary

Aff e r ent Neuron s .

1.2.:2Organizationof theSensorySpinal

Cord .

1.2.3Spinal Sensory Neurotransmissionand

Neurorrodulat i cn .

1.2 . 4Supraspinal Pro jections . 1.2.4 .1Dorsal Col umn- Med i a l

1..e:'utis ca l System . 1. 2.4 . 2 The Anterolateral Sy s t em . 1.3

~~fil~~~~ ~;~~~s: ::::::::::::::::::::

1.3.2 CUrrentTreatmentsfor Neural Injury Pain... . . ... ... . ....•.. . . ... .. .

1.3.3 ~~~~ht~~~~~~.~~~~~~.~: ...

1.3.3. 1PostulatedMechanisms of Allodynia.. .. • .... . ... ... . 1.3.3.2TheRo l e of Inhibitory

Neurotransmittersin Allodynia.. ... . . . ... ..•.

1.4 The Strychnine Model of Allodynia . 1.4.1Experimental Basis fortheSt rychnine ModeL.. . ... . .. .. ...• ...

1.4.2 Development of an Anesthetized Animal Model of Strychnine-Dependent

Allodynia .

1.5Rationaleand SpecificResearch

Objecti ves .

-vii -

it Iv :d :dii

11 15 15 17 21 21 23 27 28 32 36 J6 37 43

2.0 Q1A.q}l."CI"ERISTICS OF" STRYC-:NlNE-DEPENDENr ALLODYNi.;'INTI-!EANEST.~!Z E:DRAT .

2.1Introduction.

2..2Methods....... . .

2.2.1Ani mal s .

2.2.2 Implantationof Intratheca l catheters

2.2.3Drug Adrnin istra eion .

2.2.•4AcuteAnesthetizedAnimalPreparatio n

2.2.5Application ofStitwli .

2 .2.6Experimental Protocols .

2.2.7 DataAnal y s is .

2. 3Resu l t s .

2.3.1General Obs ervat i ons Followi ng Intrathec al Stryclmi ne . 2.3.2 Effec t sof the Dept hof Anesthesia . 2.3 . 3Ti me-Course ofStrychnine -Dependent

Hai r Deflection-EvokedRespons e s..... 2.3.4Effec tsof Int rat hecalStrychnine

Doseon Res ponsesEvoked by Hair

Defl ec tion .

2.3. 5 Effec ts of Intrathecal Glycineand

Int rathecal Betaine .

2.4 Discus s i on .

2.4.1 Int rathe calStrychnineProducesAcut e

Revers ibleAllod~a .

2.4 .2

6~~~~~fR~~~~~i~o~a~~rr~hnine-

St~Jm.l lation .

2.4 . 3Time-Course andSegmental Loca lizat io nof Stry chnine-Dependent

All odynia .

2 .4. 4 ~~=~~::D~~~~~tS~:~~~ .

2.4.5

~~*~~~;:D~~~~~;tSill:~~

^.

2.4. 6Surrmary .

-viii -

45 45 47 47 48 48 49 50 52 54 56 56 59 60

61 66 69 69

70

73 75 77 79

3.0 $PHlALPP.AF.MA.CXl:GlOF 57?YC^{H : N}INE-DEPE.'1CENT ALLODYNIA•••

3.1 rncrcducticn .

3.2Methods .

3.2.1Animals .

3.2.2implantationof Int ra thecal

catheter-s .

3.2.3 DrugAdminist:::-ation ..

3.2 .4 Acu t eExper.imen-.s . 3.2.5Imnunohis t och e mis try .

3.2.6 DataAnal y s i s .

3.3 Results .

3.3.1 Eff ec t sof Neonatal caps aicinand AcuteIntra t h ecalSt rychnine on 3.3. 2

~F~~e~f~~~~t~ ~~~a~~f~e~~ion

^..

Re soo ns e s to Noxic:..:.sStirmlli.. 3.3.3Effec t s ofAcuteIn tra t heca l

Strychnineon Responses to Nox ious

Stirruli .

3 .J.4EffectsofIntrathe c alMorphine . 3.3.5 Effect s of Int r a thecalOOCand

In t rathecalNBQX•• •• •• • •••.• •••• • ••••

3.4Di s cu ssion .

3.4.1Neonatal Cap saicinandIntrathe c a l Morp hi ne did not Affect STR-Dependent 3.4. 2

~~=~~~6~p~~~;t 'Aii;,ctY;i~' i~ ""

Dose-Dependen tly Suppres s e dbyExcit- atoryAminoAcidRe ceptor

Antagonis ts .

3.4.3 Neonatal capsaicinReducedResponse s to Noxious Stimuli...•... . . .... . 3.4.4 IntrathecalSt rychnineReduced Some

Responses to Noxiou s Stimuli..•.. ....

3.4.5Conclu sion s .

-Lx-

80 80 82 82 82 83 83 87 88 90

90 94

98 98 99 104 104

110 113 11 6 118

4.0 GENERAL DISaJSSION •.••.•. .••..••.

4.1surrmary of Results.

4 .2 r~i:iS~t~~~ ~~~t;d~~~~~t~f ' th~' ROi~'

~~~~rCi~ju~ ~~~~~~~:~~~~~.~ ....

4.2.2 Investigationof the Physiologyand Pharmacology of M-Mediated

Allodynia .

4.2.3 Variations of the Anestheti::edModel ofSt rychn i n e-Depend e n t Al1odynia ... . 4.3 Significance oftheResults •....•.. .••••• •.

4.3.1Significance of SpecificFindings.. . 4.3.2Significance of anAcute Modelof

Allodynia .

5.0 REFERENCES••••• ••.•• • •.•. • • •• . • • • • . .• •

119 119 122

122

131 132 136 136 138

141

LIST OF FIGURES AND TABLES

Tab le 1.1 Defini tionsof terminologyus edco

des cr ibe pai n .

Figure1. 1 Thedorsalcolwm · med ial lemniscal

system.. . . . 16

33

58 57 18

62

63

64 Figure1.3

Figure1.2

Figure2.1

Figure2.2

Figure2.3

Fi gure 2.4

Figure 2.5

Theanterolateral system . Schemati cdiagram of the spinal dor:sa l ho rn illustratingthe postulatedroleof

glycine .

~~~\l~;~~s~~c~s~ ~a~~~e_

anesthe tizedrats .

Time-courseof int rathecal (Lt . )

~rcl;~~i ;~~~~~e:c~~mo~°hai~

deflection (lID) as determinedbythe area ofabnormal cutaneous sensitivity . Relati onshipbetweenEm synchronyand responsesevokedbyirmocuoushair deflection(lID) after40/Jogof intrathecal strychnine(i.t. STR)•••••• •• Ti me- cours e of cardiovascular and ncccr re s pons e s evokedbyhairdeflection(HO) following intrathecal (Lt. ) strychnine

(STR) inlightlyane s thet i ze d rats . Effect of the levelof anesthesiaonthe relationship between intrathecal strychnine(L t. STR) dose and respcceee evokedbynon-noxious hair deftectdon

(HD) in anesthetized rats .

Figure 2.6 1Dgdos e - r espo ns e relationship for in t r athe c a l (i . t.)gl yc ine inhibitionof responsesevokedbyhair deflection(HO) after1.t.strychmne (STR) .... ... 67

-xi-

Figure2.7

Figure 3.1

Logdos e-r-espons erelationshipfor intrathecal (i.t.)betaine inhibit ionof re s pons e s evokedby hair deflection (lID) after1. t .stry-chnine (STR)....•..• • • •••.

~~~~~aifp;:~~~t:a ~=~e~a~~ (:f

evoked bycutaneousstimuli . 68

89 Fi gure 3.2 Effects of neonatal capsaicinandacute

~~~~~~~a~~t~~·~/~rr;~~~ ~e~~~~

^on

(HOI... .. 93

Fi gure3.3 Effects of neonatalcapsaic inandacut e int rathecalstryclmine(i.c,STR) on responses evokedbypawpinch(PP ) 95 Fi gure 3.4 Ef f e cts of neona ta l capsa icinandacute

intrathecal strychnine (i.ti.STR)on re spon s es evokedby tailimners i o n (TI).. 96 Figure 3.5 Effects of neonatal capsaicinandacute

intrathecal st r ychnine (L.t; •8m) on re s ponses evokedby topi ca l xylene

(XYL).. . . ... .... . .... .. ... .... 97 Figure 3.6

Figure3. 7

Figure3.B

Comparis onof the ef fectsof intratheca l (L.t)mcrpldneonres~esevoke dby either non-noxi oushafrdeflection(HD) in thepr esenceofi.t.strychnine(STR), orbynoxious stimul iintheabsenceof S'IR•...• •..• •.•..•.•• •...• •• ... ••..•• ••..

logdose-responserelat i on s hipof intrathecal (Lt.)garrm:l-D- glutamylg lycine (CGG)inhibitionof respons e s to hair deflect i o n(HO)in the pr esence of L,t. str-ychnine {SIR) . logdos e - re spon s e relations hipfor int rathe c al (i.t .l 2,3-dihydroxy-6-ni t ro- 7- s u lfamoyl -benzo(F)q ...ti noxaline (NBQX) inhibitionof re sponses to hair deflect ion(HO)after 1.t . strychnine (STRI •... ••... .... . . ... ...•••

-xii-

100

102

103

0700 h

3~F£OL

3,4-TAZA

4,5~TAZA

.

B

!

-c

PH l\Jl M_ A ANOVA APS bet aine C

c.r.

CllRP ci- en

rnox

015 Co.

LIS T OF ABBREVIATIONSANDSYMBOLS 7 0Iclock intherroming

5~(3-pyrrolidinyll-3·is 0 2010 1

2, 5,6,7~tetrahyd=0 · lH- azepine -4~carbo:<ylic acid

2, 3, 6,7~t e trahydro- 1 H- a zepine- 4- carboxyli c acid

~lg~~1~a~~~ke~~~e~e

beta, aGr e eklet.ter decc-eesCelsius

delta,aGr eek letter,asubtypeof opieid receptor

equal (s)

garrma.,a Greek let t er greaterthan

kappa,a Greek letter, a subtypeof opieid receptor

les s than

mu, a Greekletter,usedto indi ca t e microin met r ic units,"1.150a subtype of opioid receptor

::;;~~~(~; , l~;·~sr~~~s~~~f

^or-:lume

micrometer(a), ro- metres,unit of distance mtcr-crrolez-, 10-⁶molesper litre,unitof concentrat ion

mi..::rcxrole {slI lO -~rroles, numberof molecu les percent

plus , in combinat ionwith rho , aGr'eek le t ter

hydrogenatomicwei ght thr ee (trit iated) A-beta,a cl ass ofpr dmaryaf f erent neurons

A~delta. a classofpr ima ryafferentneurons amino-3 -hydroxy- S-methyl -4-isoxazol e propionic acid

analys is ofvariance D-2-amino-S-phosphono -va lerate corrmon name for N,N,N-trimethylglycine

~o~~!~~~ hf~la1=gtJi:=)

calc i t oni n gene-relatedpeptide chlorideion

centimeter(s)

s-cyano-? -rutrc -qui noxat.Ioe-z ,3·dione cer.tralnervous system

company

-xi i i -

Corp. CSF D DC-G DL DREZ DRG e.g.

EM!s) ED..

et:al. EEG Fig.

i!-

GABAer gic h H202 HCl HD Hg HR Hz

i.e.

Lp.

Lt. Lv.

IgG rASP

Ie..

II

III IV IR Inc. in vitro invivo iso-THAZ kg

corp o rat ion cer e b r osp i nal fluid

~:~~~l~~~f~YCine

~i~~~tatoryandlevorotatory(r a c emic dorsal root; entry zone

dors alroot: ganglia

~xempligratia, (f or example) excft.atoryamino acddta)

eff ect:ivedose forSOpercent response eealia (andothers)

eleccrcenc ephalogram(s) /gra ph(sl figure

gram(s)

y-aminobutyricacid,y-aminobutyrate usi ngGABAas a neurotransmitter hour(s)

hydrogenperoxide hydro chl or i c acid hairdeflection elemental symbol formercury heartrate

hertz,S·l, reciprocal seconds. unit of frequency

Reman numeral one, used to denoteoutermos t spinallamina

idest,thatis intraperitoneal(ly) intrathecal(ly ) int r a venou s(l y ) inm.mog l obu l in

Inte rnat i onalAssociationfor the Study of concentration for 50Pain percent inhibition Roman numeralt'NOI used to denote spinal lamina

ROlT'aIlnumeral three,usedto denote spinal lamina

Roman numeral four,usedto denote spinal lamina

irmn.moreactivity incorporated in glassware in theLi.ving body

5,6,7,8-tetrahydro-4H-isoxazo lo[3,4-d]aze p i n- 3-01

kilogram{s), 10³grams - xdv>

kPa K, L L1 LC log mm/s M,A.P.

m1

:In

mm mm' M MDL27, 551 MK-801 N N N.I.P . NBQX 11M NMDA NS oBIT IlIl101 PM PAP PE-1 0 PpH PP pSIT RU 5135 s s .c . SAL S.E.M.

SI

agcnisc dissociat.ion(Michael i s-Me n t on) ccnscan t

kiIc p u scaI (s),uni t of pres s ure antagonist. di s s ociationconst ant levorotatory

lumbar vertiebranumber one locu s coeruleus lcg a r ithm (base10) meter(51

meter/second, unitof velocity meanarter i a l (bloocil pressure milligram(s) ,lO' Jgrams minut. e(5), (usedinfiguresonly) milliliter{ s), 10-³liters millimete r(5)Iunitof di s t anc e milli mete r(s) squared, uni tof area molar (mol e s/lite r)

~;~~~1;~3-meth y l s ul phonyl - S-phenyl - 4H-l ,2,4 - (+)-S- methyl -1 0,l1.-dihydro-SH-dibenzo(a,d) cycle hepte ne -S/IO -imin e maleate number ofdeterminations

~~~~e~njw:y

^{Pa in}

~I~~~~XY-5-nitro-7-sulfamoYl-benzo{f)

=~:~ti~~·9moles per liter, unit of N-met h yl -D- a sparta t e

no at.drnilua neosp i not ha l amic tract nanomole(s) , 10'9rmles

peria~eductalgray percx ddaee - en t dpe r ccdda s e

size 10polyethylene tub ing (diameter"0.61 mm)

probabilityoferror

ne'::!'at i ve logof thehydrogenion concentration pawpinch

paleospinothalamictr a c t

3-(I;-hydroxy-16-imino-SiS-1? -eaa-endroetan- ar-cne

second(s) subcu t aneou s (ly) sal ine

st andarderrorofthe mean senso ry receiving area one of the somat o sens o ry cortex

SII SKF10047 SMr SRT STTSTR T THAZ TIlIA TIllP TIvide infra w/v V

WDR

X

sensory reeeivi.-:g area two ofthe somatosensorycortex

SmithKlineand.French ccmpound10047 (~

antagonist)

spi.nanesencephalictract spinorec.icular trace st rychnine SJ?inothala'ltic er aee tune

5,6,7,8- t.~trahydro- 4H-isoxazolo(.,I,S-d}a::epin- 3-01

5,6 ,7,8-tetrahydro-4H-isoxazolo [5 , -i-cleaepin- 3-01

4,5,6,7· c.e trahydroisoxazole(5,4-c]pyridin-) - 01tailirtmers i on

se e below weight per velure

Rcmmnumeralfive.used to denote spinal lamina

~~s~fp~eby).

^{als o Rcxranmrteralten}

- X'n. -

1.0GENERAL mrn.oDUC'I'ION 1.1 Statement ofthe ResearchProblem

The most ccnmon form of clinical pain is nociceptive pain, the type of pain causedbyinflarrmation, a superficial injury, cr a cancerous growth confined to somatic tissue.

Nociceptive pain is generally regarded as a state of cont; inuous stimulation of nociceptors, resul ting in the sustained transmission of nociceptive irrpulses via specific neural pathways from the injury site to the somatosensory cortex. 'Theonset of nociceptive pain is ircmediate, and directlyrelated to tissue injury or exposure to a noxious stimulus.Int e rrup tion ofthe nociceptivestimulation orits transmission,bymeans of analgesicdrugs(e .g., apioids),or bysurgical interruptionof theneural pathway, arrests the pain(Ta s k e r . 1990).

Neuralinjurypain (N.I.P.) re f er s to a syndrome that developsin a sub:Jrcup of patients with traumaticinjury to, or pathology of, the nervoussystem. It is a rare and idiosyncratic outcomeof neural darraqe (Noo rde nbosandWall, 1981 ; Arnerand Meyerson, 1986; Tasker, 1990). In thos e patientsaffected,however, itis anextremelydebilitating and often intractable condition (Rowbotham etal., 1991;

Baron and Saguer, 1993).

Thecharac t e ris t ics ofN. I. P. arequit e different from those of nocicept i ve pain. Painoftencoexistswith senso ry los s (Ea ron and Saguer, 1993) , and patients frequently describe their pain in terms of physical injury, such as burning, ripping, tea ring, pressingor twisting. It may be difficult to identify or locate the inciting st imulus (Lindblom andVerri llo , 1979) . In addition , the onsetof N.I.P.isusuallydelayedforweeks orevenmont hs following the causativeevent (Ta s ke r , 1990).

One of the most disturbing feat uresof N.I. P. is its poor response to treatment. Pharmacotherapywi th opioid analgesics, tricyclic antidepressants, anticonvul sants, barbiturates, local anesthetics or use-dependent sodium channel blockersis ofteninadequate(Shiba sak i andKuroi wa, 1974; ArnerandMeyerson, 1988; Tasker, 1990 ; Rowbothamet al.,1991; TriggsandBeric,1992). Indeed, cer t aintypes of N.r.p. appear tobe corrpletelyrefractory to current drug therapy (Trigg s and Berie, 1992; Baron and Saguer, 1993).

Similarly, surgical int enrentions intended to alleviate N. I.P. usually provideonlytemporaryandoftenincomplete relief,with paineventuallyre t urning (Tasker, 1990; Tasker et al ., 1992) .

In addition to spontaneous pain ,which canbe steadyor intennittent, N.I.P.is often associatedwith variousforms of dysesthesia (s e e Table 1.1), including allodyn i a,

- 3 -

hyperalges ia cr hype rpe.eh i.a (Noo r de nbos end NaIl , 1981; campbel lata1..1988; Nurmikko and Hiet3l'...==:;l.:., 1992;Tasker eeai.,1$092). Allodyn i a is a particularlydistxessfnq form of pain, -,;hie:: arises from stimuli that do not nonnally provoke pain (;'le r s key,1986). 'rhus, a colddraft; or a light tactilestnmrlus , such as the couchof clcthing, can evoke excruciating painin patientswithN.I.P. Thermally-evoked allodyniaor hy;:eralgesiaareinf r equ ent symptcns of N. I. P., while mecr.anically-evokedallodynia isthemo s t; ccorrontype, occurring in 48% of patientswithperipheral neuropathic pain and 54%of patients with central pain (Nurmikko and Hietaharju, 1992). Opioid-insensitivepain, evokedbyan innocuoustactile stimulus, is a major clinical problem.

N.I.P. is believed to involve multiple pathophysiologicalmechanisms(tcese x , 1981;Campbelletal., 1988; Priceeetil ., 198?; Priceet ai., 1992 ;Dubner, 1991;

Gracelyec ai., 1992; Koltzenburget ar.. 1992; Baron and Saguer, 1993). Unfortunately, the precise mechanisms of N.I.P. remai n 1J.I'1k.no\oIn. The abilit y of a ligh t tactile stimulusto tr:"ggerthepe r c ep t i on ofpainimplicateslarge diameter myelinated (AB) af ferent fibers in initiating allod yn i a (Campi::ell ec al. ,19 88 ; Koltzenbu-org et al.,1992;

Price etai., 1992; Baron and Sague r ,1993). Experimen t al evidence suggests that abnorrral central processing of somat o s en so ry infcrnat Lon in the spinal dorsal horn

- 4 -

Tab l e 1..1 Definitions of Te rmi nol ogyus ed to Describe Pain

Te", Definition

Pain Anunpleasantsensory and emot ional experience associated withactualor potent ialtissuedamage, or described in terms of such.

Dysesthesia Anunpleasant abnormal sensation, wheth e r spontaneousor evoked.

Allodynia Pain due to a stinulus that does not nortrally provoke pain.

H:rperalgesia An increased response to a st illU1us which is normallypainful.

Hyperpathia Apainful. syndrome,characterizedby increasedreaction to a stdmilua, especiallya repetitive stdmulus , as well as an increasedthr es hold .

Hyperesthesia Increased sensitivityto stimulation, excluding special senses.

Ce ntr a l Pain Painassociated with ales i o n of the central nervoussystem.

Theabovede f i n i tions are taken from the classification systemout l ine dbythe International Associationforthe Studyof Pain (!ASP) as presentedbyMerskey (1986 ).

(or higher centers) results:"~ strer.gt:1':e::ed syr.apt:'c c Les betweenAtprir.'ar,/ aftere.'1.::' fi.i:::en (whic~::lc=rrally rredi.eue touch sensation)and pain- s ig:.allingpat±..aysintheCNS.

Low threshold. A& pr:''r'ary affere."lc fibe~s re l ay Lnfcrm a tIon co secondorder wide dynamic nnge (l</DR) neurons inthe spinal dorsal hom. 'lhe seWDR neuron s also rece i ve input from nocicepciveprimaryafferent fibers. Why then does AB input notnormallyprovokepai n? It appea r s that th e perceptionof a stimulus as being tactile or noxious is governedbyth ebalance of excit a t ory (e.q, glu t amate) and inhibitory (e.g.glycine)inputsinfluen c!r.g thedis c hargeof these second- o rder neurons (Henry,198 9;Ski lling ec a~., 19 90J. Local glyc inergic neuronsin the spinal coniare vulnerable to da:rag e as a result of pbys.Lcal, traurra or is chemia C'I\1reen, 1936; Davidaf-: et:a.l.•1967). Ifthes e spinalLntemeuronsnormllyregulatet:hedis charge ofsecond order WDR neurons, theloss of thisglycine rgicinhibitory regulat ion couldresultinabnormal AS-evokedfiringofWDR neurons. leading to theinappropri at eperce ptionof pain.

Studies using the gl ycine recepcor ant agonist. strychni ne (STRI. support the conc e pt of glycinergic dys fun ctionas a mechanism of allodynia. The tenporary removal, of glycinergicinhibitionwith intrathecal(1.t. )STR induces an allodynia-likestateine:<perimentalanimals,and nay serveas a usefu leodeI for inves t igat ing changes in

- 6 -

scroauosenscry processing which resul t fr cm glyc:' ne rgic dys f ur.c: ion. Theproblem addressedbythepresent.thesis was the adaptation of the 8TR rrodel of allodynia to an aneetihec dzedanima lpreparation . This modelwas thenused. to ccmpa re thespinal pharmacology of allodynia wi t hthat of nociceptivepain.

1.2AnatomyandPhysiology ofTa c t ile and Pain Sensation sensory systems consist of serial neural pathways, linking t-..he periphery with the spinal cord, braanscem . thalamusandcerebral cortex. The sensatio nof pain is more complex than othersensorymodalities,since it involvesboth a sensory cceponent as well as an affective, or emotional, component (Henry, 1989) . To distinguish the sensory component from the associated affective qualities.

neurotransmission induced by noxious et imn.Lwas termed nociceptionbySherrington (1947).

1.2 .~Peripheral ReceptorsandprimaxyA£tere:atNeurons Neuronsrela ying sensoryinformation,whether peripheral or central, often exhibit remarkable specificity for a particular type of stamil.us • There are fi ve types of peripheral receptors that reg i st e r i.nfcnrati.cn regarding

-7 -

tacti le stimuli and txaneduce this in: or:<'at:'c:> in t o eleccr:' caleventsin neurons . In glabro usskin.Meissner' 5 corpuscles endMerkel'5 receptor-s are fcundin thedennis (near the skin surface), while sacfruan and Ru:fini corpuscles are deeper wi thin the skir:. (l-'!a:::t ::n, 1985b ).

Meissner's corpusclesand Pacinian corpuscles arera pi dly- adapt ingmechanoreceptors; they respond brieflyat the onset (andsometimes at the termination)of a prolonged stirm.l1us.

but are di st i nguishedbytheirdifferentialresponsivenessto variations rn stimulus frequency. In hairy skin, th e Meissner's corpuscle is replacedby the hair receptor, a rapid!y-adapting receptor that responds to hair deflection.

Merkel receptorsandRuffini corpuscles r ...«pond continuously to an enduring stirrulus, and are termed slowly adapting mechanoreceptors (Marti n ,1985b). When tissue is exposed to darraging, or potentially damaging stimuli, this nociceptive infonnation is transduced into electrical signals by the least differentiated receptors of the skin: free nerve endings. Besides the skin, nociceptors are located in skeletal muscle, joints, the cornea, tooth pulp, heart, gut andcerebral blood vessels(Henry, 1989). Although each of these receptcrs responds to particular stimulus qualities, natural stimuli seldom trigger only one type. It is the combined activation of different groups of pedpheral receptors that ultimately provides the brain with a rich and

-8-

deta iledpi ctureof each perdpheral sensory event . A var i ety of primarjaf fe r ent neurons convey somato- sensory infomation fromthe site of stimulation to the cent r al nervous system (CNS). ~iith some excepttons (Kurraza wa, 1990), the cla s s of afferent nerve fiber is relatedto the type of sensoryreceptorit inne rv-at es , and ult i matel y th e type of information conveyed. Mos t low thresholdmechanoreceptors relayinformationto theCNS via large diameter (12 - 14 .urn), mye l inate d, M prirrary afferent fibers. These neuronscond uc t impulsesatvelocitiesfrom80 to85 m/s (Collins etai., 1960). AO-and C-fibersrelay different typ es ofinf onna tion about the source, intensity and qualityof noxious stimuli. Small diameter (1- 9 p.mlI

myelinated, A5 axone have conductionve l ocit i es of 5to 30 m/a, and are prinarily involved in signalling strong mechanical pressure (He nry, 1989). Other less comron noc t c ep t c r s , with AlJ axcns , can beactivatedbyintense heat or chemical algogenicsubstances (Kelly, 1985; Besson and Chaauch,1987; Henry,1989).M fibers conveyrrore detailed informationabout the quality ofthe stimul us than C-fibers (Adr i a e ns en eeai., 1983),andtheiractivation is associated withsharp, well-localized pain (Chaprran and Bonica,1985).

C-fibershave srrall diameter (0.5-2 ,urn), unmyelinated axons and conduct impulsesmor e slowly thanot he r primary af ferent neurons (1-2.5m/s; Collins, 1960; Henry, 1989). They are

-9 -

usually polymodal, meaning that they canbe acctva ted bya range of stimuli, includir..gno:dousheat,intensemechani ca l pressure or chemical mediators of inflarrmation such as bradykinin, serotonin, histamine or potesatumion s (Levi ne, 1984; Kelly, 1985; srenry, 1989 ; Dickenson, 199 1). Sane C- fibersalso re spo nd tonoxious cold stimuli (Henry, 1989 ) . Pain associated with C-fiber activation is not well- localized, and is characterizedas a perai.scent; stinging, burning or aching sensationfChapmmand Bonica, 19851. An impor t ant characteristicof C-nociceptors is that they can be sensitizedbya previous exposure to noxious stimuli. This sensitization is rranifest as a reductioninthreshold and an enhanced level of responsiveness that can persistfor hours following the inciting event (Be s sonandChaouch, 1987).

.j,.2.2 Organizationofthe Sensory Sp:i..na.l Cord

All primary afferent neuronshave cell b:xiies in the dccsat root gangliaandproject exonsei t he r to the spinal dorsal hom, or through the spinal cord to the brainstem.

A6 - andC-fibero, along withsomeAf,afferentfiber s , enter the spinal cord, primarilyin the dorsal hom, where they form synapses with secondorder neurons. These second order neurons relay nociceptiveinfonnation from the spinalcord to supraspinalstructures. Themajority ofAf,axons involved in

- 10-

low thresi:oldtactile sensacccnprojectaxonedi r ectly tothe dorsalmedulla,although sere also havecollateralaxonethat form synapses within the spinal cord.

The graymatter of the spinal ce rd is compos ed of10 cytoarchitecturallydistinct.layers orla minae (Rexed, 1954z Steiner and Tw:ner, 1972). The most superficial la ye r is lamina I, while lamina X lies adjacent tothe central canal of the spinal cord. High threshold M-mechanoreceptors terminate mainly, rot not exclusivelyinlamina I, with some terminations in lamina II and V (Light and Perl, 1979;

MaxwellandRethelyi,1987). Unmyelinated C·fibers terminate mainlyinlaminae IandII, although some have projections to deeper layers of the spinal gray matter (Maxwell and Rethelyi, 1987). lamina III and IV corrprise the nucleus proprius, which contedns numerous large diametermyelinated axons . Cellsinthis region are activated predominantlyby innocuous mechanical stimuli, includinga major input from hair mechanoreceptors (Henry, 1989).

Second order spinal neurons are either relay cells, with axons projecting to the brainstem or thalamus, or intemeurons that transfer sensory inforrretion to other intemeurons or relay cells. Although a variety of classification schemes exist, second order neurons may be divided into 3 general categories,non-nociceptiveneurons, noc i..:ep t i ve specificneurons and nociceptive non-specific

- 11 -

neurons (Besson and Olaouch , 198 7; Henry, 19891. Non- noc i c eptiveneuronsarisemainl yfrcmtheaucL eus pr cprdus, have la rge recept ive field s and respond optimally to innocuous stimulationoftheskin. Nocic e pcive non- spe cifi c neuro ns , someti mes called WDRneurons, carr-y informat ion regardi ng noxious and non-noxiousstimuli ; the i r fi ringrate iscorrelatedwith stimulus intensity (Sorkin, 1991). WDR neurons arethe rrosc comron cell typeinthe spinal dorsal horn, and are foundmainlyinlaminae I, IIandV(Henry, 1989) . As the name implies, nociceptive specificneurons respondonlyto signalsarisingfrom noxious stinuli. Thes e neurons emanate nainly from the superficial zones of the dorsalho:rn.

1.2.3Sp i nal Sensory' Neurotransmissionand NeurclllXXiulati on Avariety of substanceshave beenproposed as possible neurotransmitters or neuromodulators in primary sensory neurons. Putative afferent neurotransmitters include: substance P, neurotensin, cholecystokinin, vasoactive intestinal peptide, calcitonin gene-related peptide, somatostatin,glutamate and aspartate (Bessonand'Chaouch, 1987;Gibson et; al., 1981;CousinsandMather,1984:Sa ltand Hill, 1983). The precise roles ofmanyofthe s e substances have yet tobefully elucidated.

- 12 -

Conside rable evidence sugges ts that excitat ory ami no acids (EAAs),suchasglutamateor aspartate,are impor tant neurotrans mit ters in sensory pat hway s. Gl utama te irmnmor eac tivity has been identified in primary sensory neurons in roth dorsal root ganglia (DRG) end trigeminal ganglia of the rat (wanaka etal., 1987). 'The unique subcellulardist:dbution of glutamicoxaloacetictransaminase isozyme sin sensory ganglia, combined wit helevat ed levels of glutaminase (Cangr o et al., 1985) , would provide the enhanced production and turnover of glutamaterequired for a rteurotransmitter role. FUrthermore,re l eas e of anpart.at.e andglutamateintothe spinal extracellularspace ofthe rat spinal dorsal hom is evoked by peripheral noxious stimulation with intradermal formalin (Skilling er al., 1988).

The postsynaptic actions of EAAsprovidefurthersupport fora neurotransmitter role. Iontophoretic application of L-glutamate, L-aspartate, DL-horrocystei ne or N-methyl-D- aspartate (I'l-IDA) to the spinal cord of anesthetizedratsor cats produced depolarization resulting in increased interneur on firing rates (Engberg andRyall, 1966; Anis ee al., 1982; Biscoe et al., 1976). rnvestdqatdone using specific exc itatory amino acid receptor antagonists and extracellular singleunit recordingsreve a Ied that bothNMDA and non-NMDAreceptorsmediate non-noxious afferent input,

- 13-

whileonly NMCA receptors are invo l ved intr ansmis sion of nocdcepmve Inf onrataon (Radhakrishnan and Henry, 1993).

Tne se obs e rva c aona ar e supported by behavior a l data inddcat inqthat ~Arec eptorantagonis ts produc e analgesia followir.g i.t. admini str ation to mice (Nasstram eeal., 1992 ). However , thesame studyderronstrated antinociceptive effec t s wi th selective non-NMDA receptor antagonists (Nasstrometal.,1992 ),andother studiessuggest that NMDA antagonism pre v en t s hyperalgesiabut not nocicepti ve pain (Ya marroto and Yaksh, 1992 ). Thus, while EM receptor agonists appear to serve as neurotransmitters insensory pramary afferents , the specific roles of the receptor subtypes involvedrequire further clarification.

Substance P is the most extensively studied. neuro- tr'eusmft.ter-cenoidatefor nociceptivepathways. The highest densi ty of substance Pinmunor ea c t i vi t y (IR) is found in regions of the spinaldorsal homwhich are known to contain te rminals of primary afferent neurons (Ljungdahl ee el., 1978; Gibson et: al., 1981). In addition, substance P is present in primary afferent neurcns, since it is depleted following rhizotomy and released with antidromic nerve stimulation (Jessel1etal., 1979; Levine , 1984; Lembeck and Holtzer , 1979). Furthernore,subs tanceP release is evoked byelectricalnerve stirm.tlationat intensi tiesgreat enough to recruit M- or C-fibers (Yak s h er al., 1980), and

- 14-

fOllOWing noxious but not non-no xious mechani cal stimuli (Kura i shi ecei. . 1985). Substances that pr oduce spi na l analgesia, including morphine and norep i nep hr ine , inhibit roththerel easeof endogenous substanceP(Pan g andVa sko . 1986) and the effects of exoqenou s subs t ance Ponanimal behaviour (Hylde n and Wilcox , 1983). Although thes e cbservacLonehave led to the proposal thatsubs tanceI?isa neurotransmitter subserving nocicept i on , a rror e accurate descriptionwouldbeneurorrodulat or . In electrophysiological studies, i.t .substancePenhanced theexcitabilityof spinal cord neurons in response to noxious stimuli (Wie senf e l d - Hallin.1986). Theslow onset and termination of the effect (s eve r a l minutes) relative to classical neurotransmitters, andthe indirect facilitatingaction, sugge stthat substance P is a neurorrodulator rather than a neurotransmitter in nociceptiveprimaryafferentneurons.

Synapses betweensens ory primary afferent neurons and second order neuronsare subject to inhibitory rrodulationby anumberof endogenous systems. Local inhibitionof both ecmatcsensoryandnociceptiveneurotransmissioninthespi na l dorsal horn is meddaced by intemeurons rel easing y-aminobutyricacid(GABA) or glycine (Game and Lodge,1975;

Dt§sarmenienetal.,1984;Duggan and Foong, 1985 ) . '!his type of local inhibitionwillbediscussedindetailin a later section. Endogenous opioidssuch as enkephalin are present

-15 -

inspinal tnce rn eurons and mediateselective inhibitionof nocicept i ve input (Lamot te ee al., 1976; Ruda, 1982;

Willcockson ec al., 1984b). In addition, synapses in nociceptive pathways aresubject to descending inhibitory modula t ion from supraspinal sites . Examples of descending inhibitorysystems includea serotonergic system arisingfr om the nucJ.eus raphe magnus (Hamrondet a1., 1980; Duet a1., 1984; HanmondandYak s h , 1984; Harrmondet a1., 1985),anda ponto-spinal noradrenergicsystem whosemajor originis the locus coeruleus (Le i Seg a l andSan dberg, 1977i West lund

ec

al., 1982;Mokhaetal., 1985).

1.2 .4Supraspinal Projections

1.2. 4.1 Dorsal Column-Medi al Lermi:~calSys tem

The dorsal column-medial lemniscal eyseem (Fig. 1.1) mediates tactile sense andlimbproprioce ption. Thedorsal columns consist mainly'.)f the centralbranc he s of DRGcells whi chas ce nd,withoutsynapsing, tothe dorsalcchamnuclei of the medulla (cun eate and grac ile nucle i ). Asmall percent age ofthe se axons arisefr omsec ond ord e rneurons of the spinal dorsal hom (Martin, 1985a ) . Afferent neurons from theca uda l extremit ies synapseinthegracile nucl eus, wt..il ethos e of the rostralextremiti es formsynaps e sinthe cuneat e. Within ea ch of thes e tracts, somat ot opy is

- 16 -

Fig. 1.1 The dorsal column-medial lemniscal system.

Information about somatic sensation is carried through the spinalcord bythe long axons of dorsal root ganglion cells whichterminate in the dorsal column nuclei of the medulla.

In the medulla, the cells of the dorsal column nuclei project ax o n s across the midline with terminations in the contralateral ventralposterolateral nucleus of the thalamus. Th i s nucleus, in turn, sends an extensive projection to the primary somatosensory cortex. Source: Modified from Kandel, 1985.

- 17 -

pres erved ,si nce fi be r sareadde dto the lateralportionsof eachtra ct.inanor de rlymanne rIandthe locationof a fiber in a tract. is related to the region of the body it innervates. Thepostsyr.apticfibersfromthedorsalcol umn nuclei cro s sthe midlineat thelevelofthemedulla , as the internal ar cuate fibe r s,andasce nd throughthe brainstemto the vent ra l pos t e r i or thalamus, as the medial lemniscal sys tem (Marti n , 1985a ) . 1hird order neurons of this tract proj ectto several discrete receivingareas of the primary somatosensory cortex in the postcentral gynIs. Here,further processing occursthat ultimately contributes to conscious perceptionof the features of the stinulus. Thus, under normal conditions, information about innocu ous tac t i l e stimulation is conveyed to specific sites inthebra i n by discrete neural pathways, which are not believed to be involved in pain perception.

1.2.4.2111eAnterolateral System

Second order neurons thatcarrynociceptive information are situated in several ascending tracts of the spinal cord.

The anterolateral system (Fig 1.2)is the most extensively studiedandwell-characterizednociceptive pathway. It is also invelved in thermal and etude touch sensation, but discussion of thesefunctions is beyond the scope of this

Fig. 1.2 The Anterolateral System. The neospinothalamic

~~~de~ :e ~~~~;reJ~lo:sra;i ~rean~~~i;~:~r

system. This system conveys i ..1formationabout; pedn to a number of regionsinthe bradnszernanddiencephalon. Source:

Kelly, 1985.

- 19-

thesis. The anterolateral system has cla ssical l y been sul:::divided in t o t·.~,::lmajo r ccrrconents , the neo sp inccbakami c tract (nSIT) and thepaleospinothalamictract (p57:'; Kelly, 1985). Although thedi v i s ionof the antero l ateral system int o nSTI' and pSTI' is ecrrewnat;artificial and rrairuy of hi s torica l interes t , it isused hereas a re minde r of the multiplicityof afferentnociceptivepathways.

Thece llbodiesof nSlTneurons are found prirrarilyin lamina I and. IV-VI of the dorsal horn, and receive the i r principal input from ArJ primary afferent fibe:rs with terminals in the nucleus proprious (l a minae III and IV).

F\Jnctionally, theseneurons arebotih WDRandnociceptive - specific (He my, 1989 ). nS'ITaxons ascendinthe lateral part oftheventrolateral funiculus. Most ofthe s e fibers cross infront of thecentral canalandascenddirectlyto the thalemrs (Chapnan and Eonica, 1985). This cro s s i ng results in a rough somatotopic organization , in that the lower partsofthebcx:lyare representedbylateralascending fibers and the upperpartsbymedialascendingfibers. Of the several thalamic nuclei receiving these inputs,rroat; go to the ventralposterolateralnucleus,the medialpartoftl:e posteriorcompl ex , the intralaminar nuclei andthe sJ.bmedian nucleus(Henry,1989). Cellsfromthevent r a l posterolateral nucleus ofthethalamus project. ina somatotopic mannerf to corticalareas 5I and5II, whichare important for sensory

- 20 - disc:::-iminat i on (Ke lly , 1985).

ThepSTI is.3.mult i s ]'!'.apt i c system\...ithterminationsin the Int za l .arrdna r andmedialnuclei ofthethalamus (Olapman and Bonica , 1985). Since thesenuclei proj ect to limbic regi ons , inaddfziontothe frontaland parietalcortex ,the pSTr isbelieved to be important in initiating the affective component of peen (Sor k in, 1991). This pathway is also thought to mediate diffuse, slow, burning pain(Ke lly ,1985) . Since many pST! axons project to the brainstem reticular forrnatdon and themidbrai n periaqueductal gray (PAG), the paleospinothalamicsyste:,:\ has often subdivided Into the pSTI proper, spancretfcul.ar tract (SRT) and spinomesencephalic tract (SMr). SRT cells are mainly nociceptivespecific,and arise predominantly from lamina V of the dorsal horn(Henry, 1989). The terminations of the SRT include the gigantocellular nuc l eus , the nuclei reticularis pontis, caudalis and oralis, the nucleus subcoerulus, and the cuneiform nucleus(Zem1anetal.,1978). The cells of origin of the SMT are found primarily in laminaI and include nociceptive specific, WDR and non-nociceptive neurons.

Fibers of this tract ascend ipsilaterally and contralaterally and terminate in the PAG, the cuneiform nucleus and other mesencephalic regions (He nry, 1989).

In addition tc the nSTI andthe pSTr, several other spinal tracts arebelieved to playa role in nociceptive

- 21 -

transmission, and a number-of di f f erent pai n modulating syst emshavebeen described. The s e have beenthe subject of several extensivereviews (Dubner andBennet t , 1983; Besson andChaouch,19 87;Henry,1989;Sorkin,1991). Itis evident fro mthis curs ory des crap t ccn, that the pr oc e s si ng of somatosensory and nccdceptive info rmation is compl ex, involvingsevera lparallel butdis cretesy st emswhichextend throughout the neuraxis, multiple neurotransmitters and several.levelsofregu l a t i on .

1.3 Neura l Inj uryPai n 1.3.1General Features

Neuralinjury pain referstoa syndrome thatdevelopsin a sul:groupof patientsfollowingneural injw:yor pathology.

ExamplesofN.r.p. inc l udephantom limbpa in; central poat.- stroke pain ; diabetic,alcoholic, nutritional, traumaticor cance rousneuropathy;anterior spinalarterysyndrome;post- herpetic neura l gi a; reflex sympathetic dystrophy; pl exus avulsion; postcordotomydysesthesiaandpainful conditions associated withparapleg..i.aand multiplesclerosi s (Sh ibas ak i and kurcdwa, 1974; Boivie et a1., 19 89; Tasker, 1990 ; Port enoy ec a1., 1990i TanelianandBrose, 1991; Price et a1., 1992 ; TriggsandEerie , 1992 ; Baronand Saguer, 1993) .

N.: .? develops in 1 ot 15, 000 strokes, 7~40% of craurratrc spir.alIes tcn s,a~;:-:::-oximately10%of herpesccster- infec t i cns and approxtrracel.y 17%of multiple sclerosis ca s e s (Shibasaki andKuroiwa,1974; Tasker,1990;Rowbotham er301. , 1991). 'rhus, it is a relatively rare and idiosync ratic outcere ofneuralinjury (Nco rd enbo s and Wall, 19 81; xrner and Meyerson , 1988; Tasker, 1990 ). In those patients affected , however, it is excrerrejy debilita tingand often intractable (Rowbotl'.amee al. , 1991 ; ArTIerandMeyerson, 1988; Baro n and Saguer, 1993) .

N.I. P . is frequentlydescribedbypatientsasablrning, ripping , te a ring , pressingor twistingpain; terms associated with physicalinjury. Patients are often unable to identify or loca tethe inciting stimulus, and radiation of sensation, abncrmaf temporal sumratd on, and after-sensations are frequent sequelae of this syndrome (Li ndb l om and Verrillo, 1979 ; NoordenbosandWall,1981; Price et al.f 1992). Pain oftenccexf.at.a with sensory lossinthe same cutaneous region (Bar on and Saguer, 1993). Unlike nociceptivepain,the onset is usually delayed for weeks or months following the causativeev en t (Tasker,1990 ) . For example, 82% of patients withepinaI cord lesionsexpextenceda delayinthe onset of pain, which ranged from less than a month to more than one year afterinjury(Ta ske r et al. , 19 92) .

- 23

1.3.2CUrrent Trea t::nentsfor NeuralInjury Pain N.I. P. responds poorlyto all currentlyavafLabl .epain therapies. A number of clinical studies have repc rce c insufficient pain control, using a variety of opioid anal ge sics,at dosessui .cabje fornccicepti vepain(Arner and Meyerson, 198 8; Dubner, 1991; Triggs and Beric, 1992).

However, the lack of opioidefficacy in the management of N.r.p. iscon t rover s i a l (Arner and Meyerson, 1988 ;1991; Do Pen and Williams, 1991; Portenoy ee al., 1990; 1991;

Rowbotham et al. , 1991), with some authors arguing that significantanalgesiacanbe obtained i fopioid doses are carefullytitrated. untilth e appearance of either analgesia or I..I1'lIla1'la.geable side effects (Portenoy er el;, 1990).

Interpretation of studies like these is complicated. by the absence of appropriate control groups (Dubner, 1991), disagreement over a suitable clinical analgesic endpoint (AIner and Meyerson, 1991; Do Pen and Williams, 1991 ; Portenoy ec al., 1991) andan extremely high incidence (=60%") of placebo responders in the N.I.P. patient population (Ve r dugo and OChoa, 1991; OChoa, 1993). The elusive nature of this preble!!': is exemplified by a double- blind, placebo-controlledstudyinwhichpatieJ..~ :;were asked to rate their pain along sensory and affective dimensions.

Mo:r:phine dose-dependentlyreversedthe painaffect.scoresin the neuropathic pain groups, while pain sensory ratings

- 24 - remai ne dunchanged (Kupe r s et: al.,1991) .

pespi.te t.he controvers y , a reasonable consen su s has developed conc e rr. in gcer t a in aspects of N.I.P.andthe role ofop ioid analgesics: 1) Ascientificbasisexistsfor a rightwardshiftof the opioi ddos e-res po nsecurve in N.I.P.; neuropathicpain is generallymor-eseverethanother forms of pain (Arner andMeyerson , 1988; Tasker, 1990;Rowbothamee al. , 1991).2) There is evidenceind i ca t ing thatsometypes ofN.I.P. are mediated by neural substratesnot noneal.Ly Lnvokve dinnociception (Campbell etal. ,1988;Portenoy et al., 1990) . 3) N.L P . is be lieved to l:ave mult iple pathophysiologicalmechanisms,whichcanexistseparatelyor invariouscombinationsindifferentpatients (Loeser, 1981;

':a.mpbell etai., 1988; Price etal., 1989 ; Priceet al., 1992;Dubner,19 91;Gracelyetal.,1992; Koltzenburgetal., 1992; BaronandSaguer, 1993) . As a consequence, it is diff icultto predicthow individualpatients wi llrespondto opioids. 4) Most investigatorsnowagreethat opioids should be tested on a patient-to-patient basis , and that doses should be titrated appropriately before labelling pain as

"refraccory", andthereby denying a patient the possible benefitsofopioid therapy(Arn er andMeyerson,1988; 1991; DuPenandWilliams, 1991; Partenoy eeal., 1991; Rowb:ltham et al. ,1991 ) . The fact remainshowever, that many patients with N,I.P. are not helpedbyhigh doses of opioids ..whic h

- 25 -

caus esedation ,confusionanddys pho ria(DuPenand l'1illiams , 1991; Portenoyeca.l.•1991 ). Moreo~Jer, veryhig hdos es of apioidsnay exacerbate, oreven cause,dysesthesia(Sj "'Sren etai., 1993).

Membe r sof severalother dxug classes havebeenuse din attemp tsto controlN.!.P.,with varyfnqdegreesofsuccess.

Tricyclicantidepressants,anticonvulsantsandbarbiturates are rooderatelyeffectiveinsome forms ofN.!.P. (Shibas ak i andKuroiwa. 19 74; Tasker, 1990; Rowbotham ee ai .• 1991), however, the response is often only temporary(Wa t son etai. , 1988) , and certain types of N.!. P. appear tobe cOll;Iletely refractoryto these agents(Tr i ggsandBeric,1992; Baronand Saguer. 1993). Temporary relief is usually obtained with local anestheticbl ocka de,butnotbysurgicalinterruption at the same site (Watson ee al., 1988; Tasker. 1990). Administration of acu te 1.v, lidocaine or chronic oral mexiletineto pat ientswithderronstratedCNS lesionsresulted in improved pain ratings and a reduction in mechan i ca l hype r alges i a (Marchettini eeal., 1992; Edrrondso nee ai .•

1993). 'The site of actionof these agents is postulated to beinthe CNS ratherthanthe peripheral nerve since the dose used does not affect peripheral ne rv e transmission. and direct inj ectianin toperipheralnerves abolishedevokedpain and sensoryfunction, but did notreJ.ieve spontaneous pain (Marchettinietal.• 1992) . However, adequate control groups

- 26-

werenot included,andsome patientsreportedpa in relieffor up to 25 days aft er a single 1. v. lidocaine tr eatment (EdJronds on et al. , 1993) . The acti ons of lidoca i ne . carbamazepineandmexiletine inN. I.P .arebelievedtobedue to use-dependent sod i um channel blockade. Use- dependent sodi umchannel bl oc ke r s arethought to target nerveswith abnormal spontaneous act ivity, while sparing conduction mechanisms in nonnal nerves (Tanelian and Brose, 1991)..

Considering the high incidence of placebo responders, the la ck of appropriatecontrols,andthe absence of long-term.

carefullycontrolledtrials, the trueef f ec t i vene s s ofthese putative therapies cannot be adequately assessed at the present time.

Surgical interventions intended to alleviateN.I.P. , including neurectomy, rhizotomy, cordotomy. cordectomyand other destructiveprocedures applied to the neuraxis.usually provide temporary, incomplete pain controlat best,andthe pain eventually returns (Tasker, 1990; Taskereeal., 1992).

The short termbene fi t of some forms of surgery canbevery misleading,but after sometimethe ineffectiveness of this approach becomes apparent (Noordenbo s and Wall, 1981).

Successful surgical interventions have been reported in patients withcertain typesof N. I.P. (Tas ke r etal., 1992 ). However, steady pain, the most corrrnon form of pain suffered by patients with spinal injury, responded pcorly to

-27-

desnructive surgeries such as cordotomy, cord ecacmyand dorsalreo': entry zcne(DREZ)surgery(Ta sker eeal., 1992) . Thus , a numbe r of approaches co treati ngN.r .p .have been att empt ed with some success , but the vase maj crity of pacdencsare tnadequat efycontrolledbyavailable thera pi e s (Ta ske r,1990; Dubner,1991;Triggs and Berie, 1992) .

1.3.3 Path oph ysiol o gi calMecban1 sms o£NeuralInjuryPain

The etiology of the somatosensory dysfuncticn which leads to the development of dysesthesiae is unknown.

Sensitization of prlnary nociceptors, ephaptic neural connect i ons , spont aneous activity in a neur omata or regenerating axons , central disinhibition (du eto lossof local or descending modulatory systems) and possible secondary sensitizationof central nociceptive pathwayshave been proposed (Loe s e r , 1981; Wall, 1984 ; Ochoa, 1993). Growing evidence supports the involvement of rrultiple pathophysio1cgical mechanisms (Loeser, 1981; campbell ecal., 1988;Priceet al ., 1989; Price ecal., 1992; Dubne:-, 1991;

Gracely ec al., 199 2.; Koltzenburg etal., 1992;Baron and Saguer, 1993).

In some pacients, there isan inappropriate exaggeration of respcnsestriggered by nociceptive afferent fibers leading to hyperalgesia. This abnorwality may involve sensitized

-::9 -

nocicep t ors and/or ana.bnom-a~centralfaci litatory mechanism (Pr i ceezal., 1992) .Other;-at:ien~se."'Ch.ibital loiyr.i a....'hich appearsto beinit iat edby.3£pr frraryaf f erent fiber s lvide infra). Alterati ons interr;:ora l sur.rnation of reperItfve stimuli appear to be Invcf ved in both dyse sthes i a and spontaneous ongoing pain (F=:'ce eeaI. , 1992 ). ~reover, thesegeneralmechan i sms may notbemutuallyexclusive,si nc e AE,- fibe r -initiated allodynia, hea t hyperalgesia and slow tempor al surrrnat ioncoexi s t in somepatients or appea r in various permut a t i onsinothers (Priceeeal., 19921.

1.3.3.1 Postulated Mec.hani.smsof Allodynia

The ability of light tactile stirruli to trigger perception of pain i~lica~es large diarreter mye.iinated af ferent fibersininitiatingallodynia (Pri ce eeal.,1992). nus conceptis supportedbydirectevidence francl inica l studies (Can'pbellee al. , 19 88 ; Kol tzenburget aI. , 1992; PriceeeaI., 199 2 ;BaronandSaguer,1S93l. Folla...ing nerve bloc k by is chemi a or nerve compres sion, allodynia was eliminated concurrently wi~h light to uch sensation on adjacentnormal skin (cal1ilbelletal.,1988;Koltzenburger al., 1992), suggestingthatbeth sensa tionswere medi a tedby the same neural elements; na:r.ely AS-fibers. Terrpe ra t ur e

- 29~

dis:::=,:"~,i:"..3.t ionwas unarfecc edin the se pat i e nts , si gnify ing cba c :\.:....ict ioneI.l\~-an d C-fi 1::er s didnot media t e the allodynia (QilT";loell etal.,1988) . Response la t encies for de t ec t i onof mechanfcaIstimuli indica tedthat theconduc tionvelocity for de ee ceccnofpainin thenerv e- injured limbwas aimi.Lazto that for det ection of touch in the norroal, limb. acen latenci e s wer emuch fast er chanthe latencyfordetectionof pai n in the normal, limb (Li ndbl om and Ve rri llo, 1979;

Campbell etal., 1988; Gracelyetal., 1992 ). Additional evidence for the involvement of AB fibers inallodynia is derived from the observation that high-frequency, low- in t ens i t yel ec t rical nerve st i mulationexacerbatesallodynia, rather than eliciting the analgesic effe ct seen with nociceptive pai n (Pr i c e etal., 1992). FUrthermore, the degree of inpainnent of C-fiber function is positively correlated withthe intensityof allodynia (Baron and saguer, 1993 ) .

Several mechanisms have been proposed to explain allodyniaevoked bylighttouch. The existenceof myelinated nociceptorswith conduction velocitiesinthe AB range has led tospeculat ion that these nociceptorsmightbeinvolved in the pr oduc tion of allodynia. However, nociceptorsdonot normallyhave suf fic i e nt l y low thresholdstobeactivatedby light tactile stimuli (campbell et al., 1988). Another possible mechani sm was suggestedbya study of pa tientswith

- 30 -

AB-mediated allodynia in cutaneous regions that; were phys i ca lly sepa rated fr em sites of spontaneous pain (Gracely et al. , 19 92 ) . These investigators proposed that AB allodynia was rradnradneddynamicallybyongoing activity from nociceptiveaff e r ent: inputthatproducedspontaneouspain in a separatecut an eous region . However, this mechanism does notaccountforcbse rvatdonthat AB-mediated allodynia can be evoked at a time whenM- andC-fibers are differentially blockedwithlocaleneechecdc (campbell ecal., 1988), nor canit explainthe occurrenceof allcdynia in neuropathic painpatient s wi th normal C-fibe r function (Price et al., 19 89 ). While this mechanism maybeactive in some cases of allodynia, it is clearly not a gen e rally applicable mec hanism. Anot her pos s ibilit y is that allodynia is the result of cross -activat i on (vcross - tafk " ) between large diameterprimary aff e r ent fibersandnociceptorsat the site of injury. However, beca u s ethe latencyfor detection of painis short, the possibilityof alink betweenABand C fibers within the peripheral nerve trunk is unlike l y (campbell etal., 1988).

Anotherpropos ed.mechanism of M allodynia, whichis su pported by cons i de r able experimental evidenc e, is that central change s occur inthedor salhomofthespinal cord (or high er center s ) following nerv e injury, lead i ng to strengthen ed synapt i c ties between low-thre shol d mechano-

-31-

cecepc ive aff'erent; fibe r sandpain-sigr.allingpathwaysin the eNS. AB affe -rent; neuronsterminat einlaminaeIII to Vof thespina lco rd where they form synapses with second-order . WDRneurons. WDRneurons alsoreceiveconvergentexc f catory inputf:.-cmsmall diameter myelinated (All ) andunmyelinated , (C-fiber)noci ceptiveafferentneurons,andarethoughttobe invo l ved in che transmissionofpain(Maye r et al., 1975 ). Thepe rception of a stimulus as being tactile ornoxious appears tobe gove rn edbythe balance ofexcitatory (e. g . glutama te ,substanceP,calcitoningene relatedpeptide)and inhibitory(glyc in e,GABA, taurine)inpu t s influencing the dischargeof these second-orderneurons (Skilling et al., 1990, Henry, 1989; Kangrga ec al. , 1990). Disturbances in synapticactivitybetweenlow thresholdafferent fiber s and spinalWDRneurons could lead to the miscoding of thisAf, afferent input and ultimately to the perception of pain.

'rhese synaptic links might ordinarily be present but net effective eno ugh to activate central pathwaysunder normal conditions (carrpbellet al., 198 8). Alternatively, changes in dorsalhorn circuitry have been observed following both central and peripheralnerve le s i ons (Woo lf, 1983; Woolf ec al.,1992; BennettandXi e, 1988; tureen. 1936;Davidoff et al. , 1967) , and this new circuitry might provide an inappropriate link between low threshold input and pain processingmechanisms inthe Qi/S.

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1.3 .3.2The RoleofInhibi t oz y neurotrensmincexsin..:ulcd}n i.:l GABAandglycinear e inhibitoryneurotransmitterswhi c h areimportantin sensoryprocessinginthe spinaldorsal horn (Ni s t r i , 1983; Willcockson, 1984a; Desannenienet::al.,1984).

Glycine-containingcells are most abundant in lamina IIIand IV of the rat spinal cord, whilesomear e distributed in laminaeIIand V. Glycine appears toco-exist withGABA.in many, but not all,GABAergiccells inthe dorsal hom(Todd and Sulli van , 1990). It has been dem::mstrated tha t glycinergi cneuronsinlaminae II and IIIoftherat spinal cord receive a maj or m:mosynaptic input fr om myelinatedlOll!

threshold mechanoreceptiveprinaryafferentneurons (Todd, 199 0),prov i ding anatomical evidenceforthe hypothesisthat the sefibersacti va t e local gl ycinergic neurons in the dorsal horn (Fi g. 1.3). If glycinergi c int erneurons normally regulatethedischargeof second orderWDRneurons, theloss of this inhibitory regulation could resu lt in abno rna l Af,-fi.berevoked firingofthes e WDRneurons.

Exog enous lyapplied glycinehas been shown to inhibit the discharge ofidentified spinothalamic tract (SIT )neurons in lamina V (Willcockson ecai., 1984a , Gameand Lodge, 1975) , consistent with the ident ification of gl ycinergic terminal s inthedorsal hom (Ribeiro-Da-SilvaandCoimbra, 1980). Although SIT neurons in lamina I may also be inhibitedbyglycine(Willcockson etal.,1984a ) , the glycine

- 33- A) Normal

C)Neural Injury

rlg. 1.3 Schematic Di agr am of the SpiD.al Dors a l Born Illustrat.ing the Poetulated Role of G1yc:ille. A&primary afferentneurons (medi ating touch) and AS!C primary a.fferent neu ro ns (media t i ng painl conve r geon spi nal wide dynamic ran g e (WDR Ineuronsan dthei r combined inpu t determin e s the finalmes sagerelayed to the brain. Norma lly (Al gl y c i nergic inte r neu r o ns mo dulate tact i1e-evoked af fere n t input toWDR neurons ; howeve r , fallow ing Lt. st ryc hn i n e (STRi Bl or neu r a l in jury (e.g. from is c hemia ; C) this glyci ner gic mod u l ation is los t. The re sulti ng enhan ced synaptic eff i c ac y, asindicatedbyth e numberof plus (+)signs ,migh t cau seA&affere nt inputtobe misinte rp reted as pai n.

- 34 -

antagonist , STR, had no effect on nociceptive neurons (pr e sumabl y nociceptive specific )inthi s lamina(Yoko t a et al., 1979). A difference in glycinergi c cont rol of nociceptive neurons inla mina I as comparedto laminaV was suggested (Yokot a et al., 1979). In other studies , application of 8m onto dorsal horn neurons facili tatedthe post-synaptic discharge evoked by afferent input, and enlarged the receptive fields for innocuous tactile (brus h) stimulation (GameandIA:ldge ,1975;ZieglgfulsbergerandHen, 1971). These data are consistentwith the hypothesis that activation of glycinergic neuro ns in the spinalcordbylow threshold primaryaf f e r ent fibers regulates thedischarge of WDRneurons.

Local glycinergic neurons

::.n

the spinal cord are vulnerable to ischemic and excitotoxic damage. For example, terrporary aortic occlusionreduced. the number ofint ern eurons inthe central gray matterof the cat spinal cord (Ture en, 1936). Changesinthe content of GABAandglutamineinthe spinal cord were not detected after ischemia. In contrast, the content of glycine, aspartate and glutalT'-:'lte was decreased, and the reductionsinglycineandaspartate were reported to correlate significantly with the interneuron count (Davi dof f et al. , 1967). 'These data sU3gest an association between glycine,aspartate, and the interneurons destroyed by is chemia , as well as a greater sensitivityof

- 3S -

gl,cine-, as compared to GABA-con'C.::l.i~i::.g This different ial sens i.ti.vitv may be reLa ted to the relative abundance of GABAergic neurons when ccroarec with those containing glycine in the spinal dorsal horn (Todd and Sullivan, 1990 )_ The sensitivity of glycinergic spinal neurons to is ch emia . and their probabl e role in sensory processing, is consistent with pain reportedinpatients having anterior spinal artery syndrome following infarction of the anterior spinal artery (Tr i gg s and Beric, 1992).

These patients e.'thibit painful buming dysesthesia , which develops below the level of the spinal cord lesion. andis refractory to opioid, anticonvulsant and antidepressant therapy.

A localized ischemic injw:y can be produced experimentallyinth e rat spinalcordbyinjectingthedye, Erythrosin B, int rav enou sly and focusing a tunable argon laser on a discretespinal segmen t. The laserirradiat.i on ca u s es a localized phot ochemica l reaction with the dye, leading toafoca l ischemic spinal injur/_ Thisprocedure results inne cro s i s of lami na e I-Vof the affected spinal segments, hypersensitivity of WDR neurons, and cutaneous mechamcaLa'l.Lodyni.a which is resistant to roorphine, but sensitive to baclofen (Haoet al., 1991ci 1991d).

Following peripheral nerve tiransection, histological evi.dence of post-synaptic necrosis is also present inthe

- 36-

dorsalhorn(Su g i mot o ecal .•1987; 1989; 1990). This effect has been act:::ibuted to massive injury discharge and subsequent exc':'totoxicity to postsynaptic cells. Injury discharge is a high frequency burst. of activityin sensory fibers followir'.g acute injury (Wall et al., 1974) , the post- synaptic effects of which are blocked by NMDA-receptor antagonists (Se l t ze r et ai., 1991). EAAsare known to rise to toxic concentxati.ons following injuryto the rat spinal cord (Liu ee al. , 1991).Thus, there is ample evidence of prominent rrorphological changes in the dorsal horn after nerve injurywhichreflect trans-synaptic changes associated with prirrary afferent te rmina l degeneration (Kapadia and LaMotte, 1987).

Collectively, the above studies derronstrate that experimentally-inducedspinal cord or peripheral nerve injury inanimalsresultsin allodynia. neuropathic pain behaviour andmorphological damage in the dorsal hom, including the loss of interneurons and reduction in glycine content (Davidoff etal., ^196';'; Sugirrotoetal., ^1990; Haoet al., 1991C,dl. Injury to the spinal cord or peripheralnervesis the rrost; conmon eventpr ec eding cl inic a l N.I.P. (Ta s ker , 1990).

- 37 · 1.4 The StrychnineMedel of Allodynia 1.4.1 ExperimentalBasis for the Strychnine Model

Studies using the glycine receptor antagonist, STR, provide evidence forglycinerg i c dysfunctdonin allodynia.

Microinject ion of STR into the nucleus caudal.Ls of anestheti zed catsresulted in augmentedresponses ofsecond orderneurons to non-nox ious , tac t ile stimuli (Khayyat, ee al.,1975 ) . In naivecons cious rats, light tactile stimuli suchas hair deflection elicitedno rrccrethanan orientation respons e. Following i.t. STR,however , lighttactile stimuli evoked vigorous scrat ching andbiting of the stinulation si t e , vocalization, at tempts to escap e and aggressive behaviour(Beyeree ai., 1985;Beyeree al.,1988;Sosnowski andYaksh, 1989 ; Yaksh , 198 9). Theseobs ervations sugge s t tha t the temporary rerroval of glycinergic inhibitionwith 1.c. STRresul t sinanallodynic st ate ,andprovidea basis for the use of i.t. S'IR as an experimental nodef of allodynia.

1.4 . 2 Developnent of an Anesthe t i zed Animal MJdel of Strychnine -Depen dentAllodynia

TIleeff ec tivenes s ofLt.S'IRinelicitingallodyniain consc i ous animals is well-documented (Beyeret ai., 1985;

1988;SosnowskiandYak s h ,1989 ; Yaksh, 1989) . In contrast, thesens ory effectsofi.e,STRinanesthe t izedanimalshave