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A STUDY OF THE ANTlNOCICEPTlVE AND TOXICOLOGICALEFFECTS OFINTRATHECAL DEXMEDETOMIDINE AND METHOXAMINE IN THE RAT
by
SueEllenMaher
Athesissubmitt edto theSchoolofGraduate Studies in partial fulfilmentoftherequirementsfor the degree of
Master of SCience Specialization:Toxicol ogy
SchoolofPha rmacy Memorial UniversityofNewfoundland
StJohn's,Newfoundland 1998
. +.
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ABSTRACT
Alpha(a )-adrenerg ic agonistsrepresenta novel drugclass ofspinal analgesicsand are mostCOf'MlOI'llyused incombinationwithlocalanesthetics and opioids.Spinalnoradrenergic antinodception ismediated primarilyby a2- adrenoceptors,although0l -adrenoceptor-mediatedennnoclceptlonhasnever beendisprov en.AsdemonstratedvasoconstJictors.however,thereare concerns regard ing thesafety ofthespinaladministrat ionof01-and02- agonists.aloneandincombi natio n.
The puposeof thisstudy wasto determineifintrathecal(i.t) metho xamine (MX)(aC"a gonis t)potentiatesi.t.dexmedetomidine (OX) (a z- agonist)-induced antinociception.ifl.t,
O x..
aloneandincombinationwithMX,inject edtwice dailyforfo..rdays,causesspinalneurotoxicity,and theeffect ofa
sub-toxiccombinationofLt.OX and MXonLt.dynorp hin-induce d neurotoxicity.
Male,Sprague-Oawleyrats(300-400g)implanted withi.Lcatheters(l l termination)wereusedthrooghoot..Thei.ti~eclionofdexmedetomidine (0.01. 1 1J9)produceddose-de penden t antinocicepti onin thetailflick (TF) andpaw pressure(PP)tests(EOso=45 and252 ng,respective ly).The additionofafixed doseofMX(10 ).lgi.t),whichproduced<5%maximum percenteffect(MPE)in the TFtestand was inacti vein thePP test,significantl y shifted theOX dose responseccveto the left(EOso=8.1n9;TFtestand 10ng;PPtest) but didnot
pro longOX'sdur ation of action.A fixeddose combinatio nofOX (0.0251J9)+ MX(101J9)producingnearmaximalantinociception in theTFtest and intermed iateaetivltyin thePPtest,wasnear completelyblock ed by prazosin (10 IJ9u.)orWyeth27127 (0.51J9i.t).
RepeatedLt. injectionsof highdoseOX (10 IJg)and OX+MX (10IJgeach) produced sedationbutno motor dysfunction.noinfl ammation.haemorrhage or necrosis ofthespina lcord.Immunohistochemicalstudiesrevealed nodamage to or loss of calcitonin-g ene-relatedpepti deimmunoreactivity(CGRP-IR)ineither the dorsal orventral horns orsubstance Pimmunor eactivity(SP-IR) inthe ventr al hom as comparedto vehide-treatedrats.In contrast,dynorphinA,.13 (192IJQ;120nmo l)produced:1)immediate andirreversible hindlimbparalysis;2) loss of the steppingandtailflick reflexes; and 3)delayedbladderand bowel dysfunction.Forty-eighthours after dynorphin,immunohistochemical examinationrevea leda marked depletion of CGRP-IRmotor neuronsin the lumbarventral hom.CGRP-or SP-IRwas und"langedinthedorsalhom.
Toassess theeffectofLt. OXand MX,aloneandincombination,on dynorph inneurotoxicity,ratswere pretreatedwithLt.Ox.MX.a combinationof OX+MX(10IJQeach) orsaline15minbeforeLt.dynorphinA,.13(1921J9 :12O nmo l).In salinepretreatedrats,dynorphinproduced the same effects as descri bed above.Pretre atmentwithi.t.Ox. MX orOX+MXattenu ated the hindl imbparalytic effect of dynorphin.Twenty..fourholXSafterinject ion.motor
iii
reflexeswere preserved and accel erating rotarod(RR) scores were similar to controls (nodynorphin)in4outof 5OX-,3outof 5MX-pretreatedand 4outof 4 OX+MX-pretreatedrats.Histolog ical evaluationofthespinalcon::Isobtained from theseratswere consistent withthesefunctionalresults,indicativeofa neuroprotective effect.For OX and DX+MX pretreated rats,therewas a correspondingdecre ase in recta l temperature (upto3.4°Cfor OXalone ).MX didnotalter rectal temperabse.These resultsofthisstudyindicatethat1)a thresholddoseofi.t.MX potentiates az-rnediated antinociception intherat; 2) repeatedspinal administration ofthis drug combinatio n has no detectabl e neurotoxic effect and3)pretreatmentwiththese drugs effect s neuroprotection againstLt.dynOrphinintherat.
iv
For Mom and Dad Labor Omnia Vincit
ACKNOWLEDGEMENTS
I wishto eceeess my sincere appreciation to my research supervisors,Drs.
ClYistopherloomisandDevhuti Vyas,fortheirexpert guidanceand encou"aQementttYoughoutmy research.
Dr.DetletBeiger for allowing methe useof his laboratory and expertisein my immunohistochem icalstudies.Also sincerelyappr eciated are hiskindnessand
...
Dr.BruceVirgoforhisguidancein my programandhis opendoor. Dr.KanthaArunaChalamforhertech nica ladvice andsurgica ltraining during the initialstagesofmy thesis research.as wellas someofthe preliminary data for thisresearch.
Dr. Stephen Sherman forhisgenerous help,constant support andfriend sh ip throughoutmygraduateprogram.
JanetRob insonforher skillf ul tech n icalassi stan ce and kindness.
Dr.Dale Corbettforallowing metheuseofhis photograph icequipmenlAlso to SueEvans.Drs.Suzanne NurseandFred Colbourne fortheirtech nical assistance.
Mr.JohnBautista,Mr.Dong yuan Yaoand Ms.HemalKhandwa la for theirkind friendshipandalwaysgood company.
Iwould like tothankOr.MUTaY Brooker forhisbelief in me and constant enco uragement
Or.Gerald Duncanforava ilingtome the servicesof the School of Pharm acy throughoutmy research.
The staffofAnima lCare Services fortheirexcellen tcareof our animals.
Iamgratefulto theToxicologyProgramfortheirpersonalfinancial support during mydegree progra mand especiall yDr.Jean Finney-Crawl ey forher effortsonmybehalf.
MedicalAudiovisual,especiallyJohnandTerry,fortheiradviceand helpwith vi
the immunohistochemicalphotographs.
Iwouldalso liketothankOrion Corporation Farmos forproviding the dexmedetomidineHCL fortheseexperiments.
vii
TABLE OF CONTENTS ABSTRACT ACKNOWLEDGEMENTS TABLE OF CONTENTS. USTOF FIGURES..
UST OF TABLES ..
LIST OF ABBREVIATIONS .
.... .. ..ii vi ..viii .... xii
••••'AN
1.0Introduction•.. • .. . •... ...•• •.•.•... ..•. ....•... .• •.••.• .••.1 1.1Statementofthe ResearchProblem . 1.2a-Agonists as AdjuvantstoLocalAnesthetics 1.3Spina lPharmacologyda-AdrenergicAntinodc:eption 1.4Neurotoxicity of Spinally AdministeredDrugs. 1.5Pathophysiology of Lt,Peptide- InducedNeurotoxicity. 1.6TheVaso..rlarEffectsofa..A,gonists
1.7Combinatio n DrugTherapy.
1.8Rationaleand SpecificObjectives
.... ..1
.7 . ..14
...26 .33 ... 3S
2.0 Methods and Material s•••••••••••••. .•.•••••. ••.•.•••••• ••••31 2.1 Animals
2.2Surg icalProcedures..
viii
... .37 ...38
2.2.1ImplantationofIntratheca l Catheters . 2.2.2Imp lantationofIntravenou s Catheters 2.3 Antinociceptive Testing . 2.4 Motor Function
2.5Immunohistochemistry 2.6Drugs and DrugAdminis tration
2.6.1Drug s. 2.6.2OnJgAaninistration. 2.7ExperimentalProtocols.
___38 _______________ 39
40 42 _42
__44 ____45
46
2.7.1DoseResponse Experiments. 46
2.7.2Single and Repeated I.T.Dosing with DX and
~+~. . ... . . . . 46
2.7.3Acute IntravenousStudy. . 47
2.7.4Pre-Treatment Study. . ... .47
2.7.5Hypothermia Study.. . ..48
2.7.6Neurotoxicity Study 2.8DataAnalysis
. _ 49
49
3.0Results...•.•.•. .. ..•...•.• ...•.•. .• ...•• ... .•.. ..•....•51 3.1Dose-ResponseRelationships of OX andMX inthe
TailFlick andPaw Pressure Tests
3.2Effectof SingleandRepeated Dosingof DX+MX ix
_____51
on Rota-Rod Performance and TailFlicklatency .52 3.3Effectofi.v.Dexmedetomidine+Methoxam ineonTailF1K:k
Latencyand Rota-RodPerformance . 61
3.4EvaluationofSpinalCords FolIO'NingRepeatedl.t.
De:wrnedetomidine andMethoxamineAdministration . . ...64 3.5Effectof
ox
orox
+MXon
l.t,Oynorphin-lnducedNeurotoxicity. . 67
3.6ImfTUl oh istochemistryofDynorphinTreatedRatsPretreated WithDexmede tomidineandMethoxam ine .72
4.0 Discussion •••••• ••••• •• ••••••• ••••••••.•••••• •••• • •. •••.• 75 4.1 Methoxamine Potentia tesDexmede tomidi ne- IndueedSpinal
Antinociception . . ... ... . . .... . ...75 4.2 Repeated DosesofIntrathecalOx,AloneandinCombination with
MX..DoesNotCauseSpinal Neuroto xicity 81 4.3 PretreatmentWith IntrathecalOx,MXandOX+MXProtects
AgainstOynorphin-lnducedSpinal Neurotoxicity. . ....87 4.4 Absence of NeurotoxicityWithIntratheca lOXand MX 89 4.5 Mechanisms of Dynorphin-Ind ucedSpinalNeurotoxicity 93 4.6 PossibleMechanismsoftheNeuroprotecttonof1.1.OXandMX
AgainstOynorphin-InducedSpinal Neurotoxicity 98
4.6.1Hypothennia.. .. ...98
4.6.2 Hype rpolariza tion . .. ...100
4.6.3 ImidazolineRecep torMechanism(s) ...101 4.6.4 Poss ibleNet.roprotective Mechanismsof MX . _102
4.7 Summary.. . _104
5.0References•••.• ....••.••.•.•... .••..•. . .••.••..•...106
xi
UST OF AGURES
Figure 1 ArterialSupplytotheSpinalCordIBlood Supply oftheSpinal Cord,HorizontalDistribution. . . ... . . ..17 Figure 2 ProposedCycle of Isdlemia41ed iated Neuro toxicity... ...23 Figure 3 Oose-Re sponseCurves for Oexmedetom idine , aloneand
InCombinatio n WithMethoxamine... . 53 Figure4 TheEffect ofPre-Treatmentwitha-AntagonistsInjected 10 min
Beforei.t,Oexmedetomidine and Methoxamine.. . . .54 Figure 5 The Effectofi.t Dexmedetomidineon RotarodPerformance.. .51 FtgUf86 The Effect ofi.t. OexmedetomidineonTailFlickLatency . . .. .58 Ftgur97 The Effect ofi.t Dexmedetomtdineand Methoxamineon
RatarodPerformance. . .. ... _59
Figure 8 TheEff ect of U.Oexmedetamidine and Metho xamine on Tail
FlickLaten cy. . 60
Figure 9 TheEffectof Lv.Saline or Dexmed etomidineandMethoxamine on TailFlickLatency..".. . . 62 Figure 10 TheEffect ofi.v.SalineorOexmedetomid ine and Methoxamine
onRatarodPertonnance. . .63
Fig168 11 Photom iaographs ofSubstancePand calciton inGeneRelated Peptide ll'MlunoreactivityinSpinal Cord Sectionsin RatsTreated With Saline,Oexmedetomidine and Methoxamine.Alone andin
Combination. ...66
Figure12 Effect of i.tPre-Treatment with Saline,Dexrreoetomldtoe and Metho xamine,Alone andinCombinationon i.tDynorphin-Induced
Neurotoxicity. . . 69
Figure 13 PhotomiaographsofCalciton in Gene RelatedPeptide Immunoreadivityin SpinalCordSectionsin RatsPre-Treated With Saline,Dexmedetomidine andMethoxamine,Alone andin
xii
Comb ination,Before l.t,Inject ionofDynorp hin.. .. . .73 Figure14 Photomiaographs of SubstanceP ImmLrlOread Mty in SpinalCord
Sectionsin RatsPre-Treated WithSaline,Dexrnedetomidineand Metho xamine.AloneandinCombination,BeforeLt, Inject ion of
Dynorphin.. . _.74
Figure15 Possib leMechan isms ofaction ofdynorph in ontheNMDA receptor
complex.... ... ...97
xiii
UST OF TABLES
TableI StudiesofSpinalNeurotoxicity Following IntrathecalAdministration
ofPeptides intheRat_ 10-1 3
Table II Stud iesof SpinalCordBlood Flow Following Intrathecal Admini stration ofPeptidesintheRat ..24-25 Table III StudiesofSpinal N9lSOtoxicityFollowingIntratheca lor Epidural
Admini stration ofa-AdrenoceptOl'SAgonists. .. . ...31-32 Table IV ED~and C.1.forIntratheca lDrugAdministration
ofOexmedetomidine, Alone andinCombination With
Methoxa mine.. _ 51
TableV Effect ofPre-Treatment With IntrathecalSaline,Dexmedetomid ine and Methoxamine,Alone and in Combination,on Oynorphin
TreatedRats ..70-71
xiv
,
·C a, a,....ale.ale a,
Qv.,.O a,a2C
~ 6
"
adlibitum
AMPA ANOVA
ATP
AVP Ca- [Ca' CGRP CI em CNS CSF DAOTL DAG Dl DMSD DNA DPDPE DPDT DRG OX Dyn
UST OF ABBREVIATIONS AND SYMBOLS plusand minus,inadditionto,subtractedfrom{aboutthe mean}
greaterthan equaltoorgreater than less than equal toor lessthan degree(s)Celsiu s
alpha.a Greek letter,a subtype of adrenergic receptor alpha 1,a subtype of a-adrenergicreceptor alpha 1A,Bor e. subtypes of the a,-adrenergicreceptor alpha2,asubtype of a-adrenergicreceptor alpha2A,Bor e . subtypesoftheQl-adrenergicreceptor beta,a Greekletter. asubtype~adrenergicreceptor detta,a Greek. letter,asubtype ofopio idreceptor rnu,a Greek letter.used toincheate miaoinmetric units, also a subtype ofopioidreceptor
freely
a-amino-3-tlydroxy-5-methyl-4-isoxazolepropionicacid analys isofvariance
adenosinetriphosphate arginine vasopre ssin calciumion
intracellularcalcium concentration calcitoningenerelated peptide confidenceinterv al
centimeter.10"meter,unitofdistance centralnervoussystem cerebrospina l fluid [D-Arg"0-Trp' ''.Leu"}-SP diacylglyce~
dextro rotatory.levorotatory, ra ecemic mixtureof enantiomers
dimeth yl sulfoxi de,an organicsolvent deoxyribonucleicacid
[D-Pen2,D-Pen5jenkephalin,a6-opio idagon ist [D-Pro2,D-Trp1"l-SP
dorsal root ganglion
(4 )(5}-[2.3-d imethylph enyl)-imidazo fe dynorphin
EAA excitatory aminoacid(s)
EDso effectivedoseyieldinga50 percentresponse
e.g. exempli gratia(forexample)
etat. atalia(andothers)
9 gram,unitofmass
Glu glutamate
Gi inhibitory G-protein
h hourIs )
'H tritium,radioactiveisotypeofhydrogen
W hydo"ogen;oo
Hg me«:ury
H202 hydrog enperoxide
HCI hydrochloricacid
IC~ inhibit orydosedecreasinga responseby 50 percent
i.e. Idest,thatis
IgG immunog lobulinG
invitro inglasswa re
invivo inthelivingbody
i.t. intrathecal(ly)
i.v. intravenous (ly)
I,II Romannumerals one andtwo,used to denote spinal lamina
~ imidazolereceptorsubtype
i.p. lntrapemonea[
IP3 inositol triphos phate
IR immunoreactivity
I.U. International units
K' potassusnion
KCI potassiumdlloride
Kg kilogram,1~grams, unitofmass
lC locus coerule us
-l l -likeimmunoreactivity
l1 lumbar vertebra number1
log logarithm(base10)
m meter,unit ofdistance
M molar(moleslliter),unitofconcentration mg milligram,1~grams,unit ofmass mg/Kg milligramlkilogram,unitof concentration mglml milligr amlm illili ter,unitofconcen tration
min minute(s)
ml milliliter, 10" liters,lX'lit of voIlMT18 xvi
mUh MK-801
mM
mm mmHg MPE MX N,n NA Na' ng NAn NMOA nmoI NOo,:
NOS P PAP PCP PE-10 perse pH PKC PlO pmol PP PZ RR RVlM SAl SCBF S.D.sec SEP SP SST ST TF
milliliter/hou r,rateof delivery (+}-S-melhyl-10,11-dihydro-5H-dibenzo (a,dl cydoheptene-5,1Q-irninemaleate millimo lar.1~moleslliter.lritdconcentration millimeter,1~.unitoflength
millimeters of mercury,unit ofpressure maximum possibleeffect
metho xam ine, 2-aminc>-1-{2 .5-dimethyfphenyl) ethyl) imidaz ole
number of determinations
noradrenaline.anon- selecti ve a-agonist sodium ion
nanogram,1o-"grams,unitof mass nicotinamideaden ine dinuc leotide N-me thyl-D-a spartate
nanomole(s).1 ~ ndes.l"lUT1berof molecules nitic oxide
nitric oxid e syntha se superoxi deradi cal probabilityoferror peroxidaseantj..peroxidase phenycyclidine
polyethytenetubing(diameter-0.61 mm) by orinitself
-log,,[H1 protein kinase C phospholipase0
picomole,10-12moles.IlUTIberof molecules pawpre ssu re
prazosin,an al-antagonist rotarod
rostralven tro lat eralmedulla saline
spinalcord bloodfla..v standard deviation seconds
somatosensoryevokedpotentials substance P,aneurotr ansm itter somatostatin
saint tailflick
xvii
TRIM 1-(2-trif1uoromethylphenyl) imidazole ulmL units/milli liter,ooitofconcentration IJQ miaogram(s),10--gems,lrlit of mass
~ mtcogram slKilogram.unit of concentration
~mL microgramlmillilitet".unit ofconcentration ut, micrcnterts) ,1o--litre s,unitsofvolume
\-1m miaometer(s),10--meters,unit oflength
\-Imol miaomole,10"",numberof molecules 101M micromol ar.10'"M.unitof concentation
V
YO"
VAF virusantibodyfree
W watts
WY Wyeth21121(disulpbc nammo-benzoqemcline). an a2•
antagoni st
xviii
1.0INTROOUCTION
1.1Statement of theResearch Problem
Withtheelucidation of pontospinalnoradrenergic pathwa ys ascomponents of the'endogenous paincontrol system',andthesubsequentpharmacological characterizationoftheadrenoceptorsubtypesmodu latingnociceptivetransmission in the spinalcord.itwas quicklyrealiz edthata-agonistsmightexertadirect antinoci cep tive/analgesiceffect. Indeed,theintrathecal(i.t.)administration of agonis tswithselectivityforQ2-adrenoceptorswasshownto produce adose-- dependent,behavioually~efinedelevationofnociceptivethresholdin avarietyof experimental animals. Importantly,thisantinociceptiveeffect occurswithout demonstratablemotoreffects(Kuraishiatal.,1979;Reddyat al.•1980; Yak$h and Reddy,1981;Howeet at. 1983).Incontrast, doses ofo,-agonistsnecessary to achieveantinociceptionintherat
produc:e<l
concurrent hyperr eflexia.clonicflexion of thehind limbs and serpentinemovementsofthetail (HO'Neatat,1983).The failure of i.ta1-ag onists to discriminatebetweensensoryand motoreffectshas been used as evidence against the role of a1-adrenoceptors in spinal antinocice ptiorvana Jgesia.However,thecontribution Of this a-receptorsubtype has never been disproven.IfmJltiple a-adrenoce ptor subtypeseffedantinociception, thenthe co-administrationofal-and a2-agonistsin threshold orsub-threshold dosesshouldyield additive or poten tiallysupra~addjtiveennnocrcepnon This pharmacological intera d ion has notbeeninvestigated.While segmentalandprolongedanalgesia canbeachievedwithper ispinal
aug
administration,ttWstectnicJ,Je carrieswithitanincreased risk ofdrug4nd uced neurotoxicity. Experimental studiesofselective drugs givenepidurall y or intratheca lly to the rat have frequently notedan abrupt andsustained oecreasein spinalcnrd b1oo.1 flow'priorto histopatholog ical damage inthespinalventralhom.Currentevidencesuggeststhat thisneurotoxic effect arisesfrom drug-induced isdlemicinjury.The pronounced vasoconstrictive effectofa1-anda2-agon istson central blood vessels raises importantquestionsabout theirsafety as spinal analgesics.especially'Mlen given incombinationThesafetyd suchacombination alone,orin the presence of a known neurotoxinsuc:tIas dynOrphin,hasnotbeen assessed.1herationale for such aninvestigationlies in theincreasi ngpopularity of'balanced anesthesia'inwhichamixtureof phannacologica lagents. including a-agonists,with varyingreceptorselectivities ,medlanismseXactionandadverse effecr:s,areco-administeredto achieve more effectivedinicalanes thesia\analgesia.
In the presentstudy,the effect ofi.t methoxamine(MX;a1-agonist)on
!hennal(tai l flick;TF)andmechan ical (paw pressure;PP) antinociceptionelicited by i.t.dexmedetomidine (OX;Q 2-agonist) wasfirst investiga tedin the rat.Asub- acute neurotoxicolog icalassessment using behavioura l. morphologicaland immuno histochemicalindices was subsequentlyundertaken to investigatethe safety of thisdrug combination. Lastly, theeffect ofthis combinationonl.t.
dynorphin-inducedneurotoxicitywas determined.
1.2aooAgonislsas Adjuvants to Loeal Anaesthetics
The spinal administrat ionofalocal anaestheticfor thepurposeofsurgery was firstattemptedinGennanyin 1899(Bier,1899).Bier hadnoted the success of regiona l cocaine anaesthesia in lowering the requirement for general anaesthesi a butherecognized thelimitations ofregionalane sthesia formajor surgicaloperations.In order to-renderlarge areasofthebodyinsens ibleto pain-, Bierinjected cocaineintratheCallyin doses ranging from 5to 15 milligrams(mg).
A level ofanaesthesiasufficient to allow major surgerywas achievedin allsix patients
The use of epinephrine,a non-selective adrenergicagonist,toprolongspinal anaesthesia wasintroduceddinicaJIyin1900(Braun,1914 ).Braun,Bierandother contemporaryGerman dini ciansbegan usingepineptrineto prolong the duration ofaction of1.1.cocaine,thus obviatinga significant disad van tageofspinal anaesthesia(Bier and Donitz,1904;Heinkeand Lawen,1905;Braun,1914).
Bet"Neen1900 ancl1940, theuse of epinephrine with spinalanaesthesicsenjoyed sporadicpopularitybutdidnotattain common acceptan cebecau se of an earl y report thatLt. epinephrinecaused pronouncedischemiain the spina lcordof experi mentalanimals(Biberfi el d, 1907). Using experimentalanimalsunder contro lledconditions, Prickett,Gross and Cullen(1945) demonstratedthatthe additionof1:10,000 and1:30,000(0.1mgJmLand 0.03mglmL)epineptvine could prolong the anaesthesiceffectofi.t.proca ine withoutpermanentdamageto
nervous tissue.Thisstudystimulatedthere-inYestJgatioof thistechnique.Inc:Ieed, recentclinicalstudieshaveshOwnthat the additionofepinephrine (0.1 to 0.3mg) prolongs the durationofaction of spinalbupfvacaine,lidocai neortetracainefrom 15 to44%inpatient sundergoing abdominal.hip or electi vesurgeryofthelower extremities(Leicht etal.,1966;Raele etal.,1987;Momose et aI.,1994).Inthis regard.epineptvYleisbelievedtocounteract the arteriolarvasodil at ionind ucedby local anaesthetics in the spinalcord, therebydelayingabsorption andina-easing the residence timeofthe localanaesthetic near the site of injection. Indeed, epneptvine isapaten:constrictorofcerebral(LoP acinandRudy,1983)andspinal (Partri dge. 1991) blood vessels. Whether this prolongat ion is truly a pharmacokinetic interact ion or anindepend ent pnarmecodynamic ettect of epineptYine(see section 1.3)remainscontroversial (Fink et al.,1978;Densonat aI.,1982,1983.1984;Ravindran etal.,1983).However,itisnoteworthythat non- select iveadrenergic (norep inephri ne),a1-selecli ve (phenylephrine) anda2~
selective(donidine)agonists have also proveneffectiveinprolongingtheduration of spinalanaesthes ia(Mensink etal.,1987;Racle et al.• 1987;KishikawaetaI., 1993;Fukuda et aI.,1994 ).
1.3SpinalPharmacologyofa-Ad renerg i c Antinoclceptlon
Noradrenaline (NA).released from bulbospinal neuronsduringexposure to noxious stimuli,isan important mediator oftheendogenous paincontrol system.
Thus. focal stimulation of theratlocus coerureus(l CI.a major sourced descending noradrenerg icneurons,was shown toinhibit noxiousevoked:a) neu-onalactivityinthedorsa l hom (JonesandGebhart.198Gb);b)spina l reflexes (JonesandGebhart.1986a:Janss et at,1987)andc) comptex nocifensive behaviours (SegalandSandbe rg, 1977:SandbergandSegal,1978;Margalit and Sega l.1979).The concentration ofNAandtheNAmetabolite.3-methoxy4- hydroxy..phenethylgycolwasalso significantly ina-easedin CSFfollowing electrical stimulatio noftheLC (Crawleyetal., 1979). Thel.t.administra tion ofa- ad'enoceptaantagonistsslgnificanUy blocked the antinociceptiveeffect elicitedby lCad ivation,suggesting aspinalsiteof antinocice ption(JonesandGebhart, 1986a).Among the seriesof pharmacological antagonistsused (ph entola mine, yohimbine,prazosin.naloxone,methtysergide,atropineandbiOJCUlline).onlythe non- sele ctiv e c-eoteecnrst phentolamine, or the oJ-selectiv e antagonist.
yohimbine.significantly blockedtheinc:teaseinnociceptive thresholdelicitedbyLC stimulat ion.Similarresultswerereported byMillerandProudfi t(1990).
These ear1y studiesof endogenou spainmodulation led rese archersto examinetheeffectof exogenousNAandother a-agonists.VVheninjectedi.t.,NA produceda sig'lificantdose-dependent irx:reaseinresponselatency inthe hot plate andlailflick tests (Reddy andYaksh,1980;Reddy etal.,1980:Howeetat,1983):
an effect reproduced by other adrenergic agonists including OL-a- met hylnorepinephrine;DL-epinephrine.ST-91.cicnidme,L-phenylephrine,3-4-
dihydro xytolazoline.oxymetazoline.methoxamineandcirazoli ne(Reddy et at.
1980; Reddy and Yaksh,1980.Howe and Yaksh,1982a ,b; Howe et
ar..
19B3).~ Agoni sts.suchasisoprote rencl . werewithouteffect evenat very highLt.doses (ReddyataI.,1980).While theU.administrationof either a ,-ora2..adrenoceptor agonists increasedl'llJOceptivettYeshoIdsintherat,thedoses ofa,-agonistsnecessaryto achiev e this effect produced conwrrent dose-dependent motoreffectssuchas hyperreflexia, clonic flexionof the hindlimbsand serpentinemovements of thelail (Howeatat.1983).In contrast,antinociceptive dosesofeither non-selective a- agonistsor a2-seledive agonists inhibited nociceptive behaviours without demonstratable motoreffects(Reddy et
at.
1980;Reddy and Yaksh.1980;Howe atal.,1983). largely. onthebasisof theseobservations.andon thevariable effectsof a ,..agonistson dorsalhomneurons,it was concludedthat spinal noradrenergic antinociceptionis mediatedby a2-adrenocept ors.In supportofthis daim,adrenergicagonistswiththe greatesta2\a,receptor select ivity(e.g.OX) are the mosteffectivein acute antinoclcepliv etests(fakanoand Yaksh,1993).Nevertheless, the contribution of a ,-adrenoceptors in spinal adrenergic antinociceptionl analgesiahas neverbeendisproven.
Early experiments demonstratedthat a,-selectiveagonists doeffect antinociceptionfollowing bothsystemic(Bentleyetet.,1983;Haye setal.•1986) and spinal administration (Howe et at,1983;Yaksh, 1985). In tum,
pha nnacologi ca l studiesofNA-ind ucedtherma l antinociception,showed thatthe thato,-antagonist.prazosin,wasmore effective than~-antagonistsininhi biting theantinociceptive effects ofitNA(Reddy et
at.
1980;Howeet al.,1983).These dataare supportedbytheresults ofradioliga nd bindingstudiesandquanti tative autoradiography demonstratingthepresenceof o,-actrenoceptorsttvoughout the dor salgrey matteroftheratspinalcord(Gironetat.1985;Simmo nandJones, 1988;Roud et etal.,1993 ). and areconsistentwith thehypo the sisthat spi nalal- adrenoceptors mediate atleas t somecomponentofnoradrenergicantinociception in the rat.Ifboth a,-and02-adrenergicreceptors effect spinal enuncciceptionthrough disti nd eff ect ormecha nisms,thenco-administra tionofana,-a gonistand anOz· agonistinthresholdorSlb-ttresholddoses wouldbeexpectedto yie ld an additive or potentiallyasupra..additiveantinociceptiveeffect.Such anin teractionin VIVo wouldbe consistent with thenon-selectrvenature ofNAas an agon istata- adrenoceptors.
1.4Neuroto xicityof Spin allyAdminist er edDrugs
AsthepracticerAspinal Malgesiahasgrown,there has been anincreasing appreciation ofthepot entialfor adverseeffectswiththis tech nique and theneed for systematic neurotoxicolog ical evaluation of drugs intended for spinal administration.This has provento beparticu larfy impo rtant for peptidedrug s.For
example, 100~of Lt. somatostatin,a cyclic tetradecapeptidethought tobe Involvedinthespinalprocessingof nociceptiveinformatton,produced severe hind limbdysti.n:;tionintherat,upto thetime of saaffice6dayslater.Therewasalso acorrespondi ngloss of sensitiv itytothermal andmechanica l nociceptive stimuli.
Histological (lightmiaosaJPY)examinationofthespinal cords removed from these animalsrevealedmildtosevere nudeotysis of ventralanddorsalhomneurons with localizedinflammation(GaumannandYaksh,1988).Similarty,peptideanalogues ofsubstanceP(developed as putative neurokinin receptorantagonists)giveni.t, inducedbilateral motorblockade in the hind limbs,andwidesprea dneuronal necrosis inthelumbar regionc:Atherat spinalcord(Post andPaulsson.1985;
Freed man et at,1989). Comparable outcomes have beenreportedwithi.t.
endothelin(HOkfe lt et at,1989),anendotheli~erivedvasoconstrict or peptide (Yanagisawaet at.1988),Lt.dynorphinA,anendogenous kappa opioid agonist (FadenandJacobs,1984;Herman and Goldstein,1985;Spampinatoand Oande tettl,1985;longet at.1988; StewartandIsaac. 1989)and arginine' · va sopressin(longet at,1989b). Table1summarizestheneurotoxico logical resultswiththese peptides.
Althoughtheexact IT'lE!d'la'lismsISldertyingthisneurotoxicityremainunclear, thesestudiesdemonstrate:a)the severeandoftenirreversible damagethat can
OCOJ(in the spinal cord afterthelocal administrationofbiological ly activeagents, indudingthosefoundendogenouslyin neuronsofthe spinalcord;and b) theneed
for careful toxicologicalevaluat ionof drugsintend edforspinal use.
TableI
Studiesof SpinalNeurotoxicityFollowingIntrathecal Administrationof Peptidesinthe Rat.
Drug!
no..
Tests Histology(Lishtor Immuno-) SpinalNeurotoxicityReference
Somatostatin 10,30and100 AN, 6daysafterinjection: 100~g:temporaryorpermanent·
(SST)/ .8 auto- 30~gSST:mildinflammatory hindlimb motordysfunction with flaccid Oaumannand nomic responsein 4/5ralS paralysisin mostseverecases.25%ohats Yak.h(19 88) and 100~gSST:mildor severe died 10min.withininjcction.
motor nucleolysis ofv entraland dorsal • permanent-uptoday 6 when rats were func- horns in the presenceof sacrificedandperfused
tion inflammatory reaction
Spanlidel 2.8 1,2,and3daysafter injection: Bilateralmotorblockadeof the hindlimb.
Freedmanet at, no CGRP·IRin motoneurons, that didnot recoverup to timeof (1989) cresylvioletstaining indicateda perfusion(1,2or3day.afterinjection)
completeabsenceof motoneuronsandamarked gliosis
DPDTand 2.8 AN 3 daysaftcr injcction At half·maximaldose (I~g).allanimals DADTUPost extensivenecrosi, of neuronal showedmotorweaknessofthe hindlimb, and Paulsson bodies inthe ventralanddorsal At 2~g.allanimal,hadpronounced
(1985) horns motorimpainnentofthe hindlimbsand
non-responsiveness in the tailflickor pinchingofhindlcgs.Thispersisted upto timeof perfusion, 3dayslater.
s
TableI (cont'd)
Drug! Do.. Tests Hislology (Lightor Immuno-) SpinalNeurotolcic:ity Reference
EndolhelinJ 0.03~sor 0.1 24 h after injeclion: 0.03~s:Allraushowed completeparesil HOkfelt et
u,
.8 Fullypareticratsshoweda afterS min.Allbutoneshowedfull (1989) marlc.ed1051o(CGRP-lRin recovery at 3 h.Theoneratremainedmoroneerora in(he ventralhom paralyticunlilperfu.ion. at thetip o(lheeetheter,Sinsle 0.1 .,,8:3/Srals died, 2 withinSmin.,last motoneuronsappeared rounded within3 h.SurvivinS ralsshowed withoutdeodritic processes. complete
parest.
untilperfusion, 24hlater.
Arginine'· O.S·1000pmol AN Nohislochemicaldifference Dose-relatedloISof motorfunctionin vasopressinI HD betweencontroland hindlimbs.Someratsdied withinS -10 Longer a1.• experimentalanimals min.of peptideinjection dueto pulmonary
(198%) edema.Ratsrecovered hindlimb function
within30 min.ofinjection,evenwith1.0 nmoldole.Flaccidhindlimbparalysis was eccompenied byloss offlexor or vocal responsestopinching of the hindlimbsor tail.
DynorphinAI 12.5 and25 AN 25 nmolproducedhindlimbparalysis and
Spampinatoand amol tailflaccidity laslinsscveralhours.
Candelel1i (1985)
::
TableI(cont'd)
Drug! Dose Tests Histology(Light or Immuno-) SpinalNeurotoxicity Reference
Dynorphinl 14nmolor Long-lastinghindlimbparalysis (hindlimb
Herman and greater and tailflaccidity)
Goldstein 14nrnol:paralysisworeoff in2 h
(1985) 20nmol:paralysis lastedforat leastone
week
50nmcl:paralysislasted foratleast one week
Dynorphinl Dyn( I-17): Allpeptidesproduced dose-relatedflaccid
Fadenand 30omol; hindlimbparalysis with the orderof
Jacobs (1984) Dyn(1-13): potency being DYN (1-17)>Dyn (1-13)
10,30,50 or 100 >
a
NA..Dyn(1-8).Spontaneouslyomol reversible overa 48hperiod.However,
Dyn(I -8): athighdoses (50 omol), 1/3oftheanimals
30,50or100 developedparalysisthat was irreversible.
nmcl, e-neo- endorphin:
30,50or100 nmol
;;;
TableI (cont'd)
Drug!
Do"
Tests Histology(Lightor Immuno-} SpinalNeurotollicity ReferenceDynorphinAI Dyn A(I-I)· AN DynA(I-1)25 nmol:Severe Oyn A(t-IJ):Dose-relatedlou of motor Lcngetel., J.I-50 nmol andextensive neuronal injury functionin hindlimbs and tail,loss of (1988) DynA ()-I): throogoutthelumbosacral flexororvocalresponses10 pinchingof
14·56nmol enlargement. hindlimbs.Motor dysfunctionranged from transient,mildparaparesistopersistent.
flaccidparalysis of hindlimbsand tail Recoverywasalsodose-related.wlrh low doses(3.1eod 6.2nmol),recovery complete within2 h.Slowerand more limitedrecoverywith 12.5and25 nmol.
SOnmol:rltldidnot recoverneurologic function,flaccidityoccasionallyturned to spasticityby4to 7 daysafter injection.
These animalsalso hadloss of nociceptive responsiveness,hilldlimbedema,bowel dysfunction,bladderdistensionwith infarctionand urinuy incontinence.
OynA(3-IJ):At28 and56 nmol,rats remained flaccidlyparalyzed24hlater.
Abbreviations:AN,anlinociception;HO,hemodynamics;CORP-lit,calcitoningenerelatedpeptide.inununoreactivity;DPDT(0- Pro',O-Trp"' )-subslance P;DADTL(D_Argl,D_Trp7·'). substanceP (spantide)
'"
,.
1.5Patho physiology of I.T.Pe ptidtH nd uced Neurotoxicity
Inexperiment alstudiesexaminingthe spinal neurot oxiceffect sofdiverse peptidessuchas dynorphinandendoIheIin(seeTab leI),twogeneral observations have beenconsistently reported. First,eachpeptideinduces anabrupt and sustaineddea'easeinspinalcord bloodflow(seeTab le II);an effect that precedes all behavioural,morphologicalandilTV11unohistoc:hemica lmeasu res of spina l neurotoxicity.second.cellnecrosis occurs primarilyinmotor neuronsof the ventra l horns.This latter observationis consistentwiththe difference in blood supplyto ventra l as compared to the dorsalhomsofthespinalcord(seeFigure1Aand B), and theknownvulnerabilityofventralhomneurons to disruptionsin spinal cord blOOd flow(Cousins andBride nbaug h,1988).Indeed,prolonged aorticcross- crampi ng and the failuretore-establish theblood supplyto the spinalcordisa majorrisk factor in the developmentofpost-cpereuve paraplegia in patients undergoing aneurysmrepa ir(Ma riniandCunningha m, 1993).
Studies using radiolabelledmictospherestoquantitatectalgesin bloodflow havereporteda decrease in themagnitude of spinalcord perfusionfollowing the tt,injection ofselectedneurotoxicpeptides(Helke etal.,1987;longetat,1967;
Thomehilletal.,1969).Becausethistechniq ue does not allow forthecontinu ou s measurement of bloodflow,the magnitude ofchange wasassessed at time point s chosen arbitrarilyafteri.tdrug administration.Withthe adventoflaser dopp ler technology,both themagnitUde and durationof changein spinalcord bloodflow
couldbe followedinindividualanimals.
15
16 FIGURE 1
A. Arterial Supply to theSpinalCord. Thearterial supply to thespinalcord originates from threemain longitudinalsystems:theventral spinalartery andtwo dorsalspinalarteries.These systems extend along thelengthoftheentirecord gMng011.series
at
brancI1esthatextendi1Io!hesp;naJ
cord(centra larte<y).Two additionallongitudinalarterial dlannels are found onthesurfaceofthecordwith lessfrequency:themediandorsalspinal artery,situated ator dose tothe dorsal septum,andtwolateral spinal arteries, situated aboutmidway between the att achm en t ofthe dorsaland that oftheventr alroots (Scramin,1995).(Source:Figur e 7-10, Cousins and Bridenbaugh ,1988 )
B. Blood Supply of the Spina lCord,Horizontal Distribution.The "central"
area.
suppfiedonlybytheventral spinalartery,is predominately a motor area.Thisareais most vulnerable,sincethereis only one ventralartery asopposedtothe
dorsal spinal arteriesofwhichtherearetwo(onlytheleft dorsalspinal arteryis depided).(Sotrce:Figure8-10.CousinsandBridenbaugh,1988 )
A.
B.
Central Artery 17
Internal Spinal Artery
Peripheral Central
Figure 1
/
Ventral SpinalArtery
VENTRAL
18
As shown in Table II, somatostatin.spantide,[D-Arg)-substance P, endothelin, dynorphinand arginine' -vasopressinall caused an immediat edecrease inspinalcord bloodflowfolloNingit administration.Inthecase of dynorphin A(1.
13),theobserveddecrease inlumbosacral blood flow wasalso showntobe dose- related(Longetal..1987).Usinglaser dopplerf1owmetry,Freed man at al., (1988)
~thati.tsomatostatinandspantide decreasedbloodflowto16:t: 4%and30
±6%ofcontrol,respectively.This maximaleffectOCOJrTed10 to 20 minafte r injection.Comparable reductionsinlumbo sacral bloodflOW'toIkHii ngi.t.[O-ArgJ- spantide, dynorph in andAVP werereportedusingradiola belledmicrosphere sat simitar timepoints(Helke at at.198 7;Longetat,1987;Long etal.•1989a).The rapid onset of this hemodynamiceffect wasindicat edbydeaeasesofupto 50%
ofcontroltwominafterl.t,dynorphinA(1-13) (Thomeh ill et at., 1989).
Inthecasedsomatostatinandspartide,a gradualbutincomplete recovery of spinalcord bloodflowwasobserved 60minafter injection(60t14% and53 t 20%ofthepre-iniedionbaselinevalues.respectively) (Freedman atat,1988 ).In contrast,i.lendothelinind uceda greater than50%reduction in spinalcord blood flow foratleast 3h(Westmark et al.,1995).In the only radiolabell edmicrosphere study thatlookedatduration,bloodflowreturnedtopre-treatment values60min afterLt. AVP(Long et aI.,1989a).
These hemodynamicstudies,and thebiological diversity of peptides effectingspinalneuroto xicity, suggesta commonphys iolog ical,rather than
19
pharmacological ordirecttoxicological.mechanism.That neurotoxicity isthe result ofanischemicinsultsecondary topeptide-inducedvasoconstrictioninthespinal cordis also support edbythefollowingexperimentalstudies.Intherat,transient occlusionofthedescendingaortasuppling thespinal arteriesproducedbehavioral and histopa thological changesqualita tively similartothose inducedbyi.t, somatostatin, various substanceP antagonists,dynorphinAand AVP(Marsal a and Yaksh,1994).Coadmin istrationofthevasodilatorhydralazine(1IJffiOIi.t )witha paralyticdoseofdynorphinA(20Mloli.t)blOCkedboththeelevationin lacticacid concentration inspinalCSFandtheneuropathologicaleffect of dynorphinA compared tovehicle+dynorphinA-treated controls(Lo ng etat.,1994).Lactic acid isaby-productofanaerobicglyoo/ysisindicativeofanisdlemicstate.Pre trea tment with thryotropin-r ele asing hormone(2mglkgi,v.),15 min beforeandimmedia tely
coo-
to thei.t.injectionof spa ntide,preventedboththe spinal vasconstrictiveand neurotoxiceffectofspantideintherat (Freedm an etat,1988;1989 ).Simil arly.the selective v-recectc r antagonist [1-(P-mercapt cMl. ~dopentamethylene prop'lonintcacid)2-(CHne thyl)tyrosine}-AVP blockedthedecrea se in lumbosacral perfusion(Longetal.•1989a)'and theneurotoxiceffed of i.t.AVPinthe rat (Long etat.
1989b).To testthepossibilitythat dynocphin might have adired. neurotoxicIItisimporta nt tonotethattheV,receptor antagonistdidnotalterSCBFwhen injected alone.Thisfindingarguesagainstaprominentrole ofendogenousAVPinthe norma lregulation of SCBF.However.AVPmaybeimportantin certainpathological conditi ons associatedwithvasospasm.compromisedbloodflow and ischemia
20
mechanism.Longexaminedtheeffect ofdynorphinAon cultured spinal cord neurons.Concentrationsupto 1 mMdidnotaltertheviabilityoftheseneuronsin vitro(Long etal..1994).
Vasoconstrictionperseisnotautomatically detrimentalto tissue.However, whensuch an event leads toasustained imbalancebetweenbloodflowand metabolic demand.cellula"
i1uY
andnecrosiscanOCCU".Duringnormal perfusion.oxidativephosphoryiation generatesadenos ine triphosphate(ATP), the major energysource ofthecell.Intheabsence ofadequate oxygenat ionand/orglucos e delivery,neu"OI'lSmustresorttoanaerobicglycolysis:analternate and less efficient meansofenergyproduction.As ATPstoresaredeple ted,vitalenergy -dependen t mechanis ms(i.e.ion pumps) are strained to maintaincellhomeostasis. For exarll)l e,ithas been estimatedthatthe ion gradients maintainedbythese pumps
utilize50 - 60% oftheATPsupply(Vamucci.t990).Asthesepumpsfail andas sodiumions accumulate intracellutarly.ischemicne uronsbecomesusceptibleto depolarization.This,intum,canleadto theaca.muIation of excitatory amino acids (EM)and otherneu rotran smittersintheextrace llu la r space of the ischemic field (Shimizuatat,1993;Uedaatat,1993).Indeed.aneurotoxicdoseofi.ldynorphin Aproduceda four..foldand three-fold increasein the concentrationofglutamate andaspartate,respectively.in rat spinalCSF(Longet at,1994).TheEAA-evoked influx of calciumthroughN-methyt-D.aspartate(NMDA)-gatadchannelsand SOCIilm through kalnate-and(±}-a-amino-~ydroxy-5-melhylisoxazol~-proprionicacid
21
(AMPA)-gatedchamels
010tr0w
and Coyle,1991)canf\.rtheraggravate the lIlStableconditions initiated byiscnemta Therapidinfl ux ofion smay alsocause edema and lysis of the cell membrane.The sensitivityof spinal neurons to EM receptoragonis tshasbeendemonstratedinvdro. NMDA. AMPAand kainate receptoragonistseacnind ucedtheconcentration-dependentdeathofaJltured muri nespina lcordneurons(Regan.1996).With inthenormal cell,calciumistightlyregulated by a combinationof energy-dependent ionpumps anddirectbindingtotheendoplasmicrencalum. When thepum ping mech an ismsfail,intracellular storesof calciu marereleased thereby incr easing calcium in the cytosol ([Cal) to abnOrmally high concentrations.Infact.the(Ca' isinversely relatedto ATP concentration during periods ofAT?depletion and resynthesis (Kim-Leeatal.1993).Sucharise in [Ca" canhaveseriousmetaboIicc:onsequencesforthecell (see reviewbyKrause elaI.,1988).Theactivationofphospholi pase~.triggered bya risein (Ca-.J.
initiatesthearachidoniccascade.Prostaglandins,Ihromboxanes andleukotrienes arisingfrom this pathway triggertheproduction of oxygenfree radicals thathave beenimplica tedinlipidperoxidation and nucle icaciddamage(Traystmanet at, 1991).The activationof calcium-dependent nucleasescanresu ltin direct lethal injuryto the cell'sDNA(Tulli set
at..
1982). Withinseconds of the onsetofischemia, a calcium-depe ndent, proteolytic enzyme converts xanthine
dehydrogenase from the-0"(NAD-reducingform) to the-0 ·oxidase torm (McCord
22
et at,1985).Upon repertu eton.the "0· formof thisenzymeusestwoprodu ct s of ATP degrada tion,hypoxanthine~xanthine.and oxygento produce a toxic
~radical(02
1.
Ofecorse,theinabilitytoregulate[Ca' will also promote thefurther release of EAA's(Figure2).AIthoU';tlOU"trlderstandingr:I themedlanismsofneuralinjurytriggeredby ische mia has grown considerably,the numbe randcomplexityof events follo wing ischemia has made elucida tion of the exact sequence ofevents difficu lt Nevertheless,itisdearthatanythingthatCOfl1lI'O'"isesspinal cord bloodflow,such asthei.t.injectionofthe pepudes discussedabove,hasthepotentialtoeffect spinalrle\XOtOxicity,especiallyinmargina llyperfusedregions liketheventral hom.
Ischemia- IATP- Cl+Ove~oad in Cytosol
Activate phospholipases
Activate nucleases
Conversion 01XD-XO
Release 01excitatory neurotransmitters
I
j
Figure2Proposedcycle oflscnerma-medlatedneurotoxicity.
tl
Table 2
Studies ofSpinalCordBloodFlow FollowingIntrathecalAdministrationof Peptidesinthe Rat.
\i
Drug! Do", Techniquefor ChangeinSpinal CordBloodFlow Time Course
Reference Measuring
Blood Flow
Somatostalin 30llg laserdoppler Bloodflowdecreasedimmedialely At60min.bloodflow returnedto60 (SST)/Freedman afterinjection.Themaximum :t.14%of thepre-injectionbaseline ctal.,(I 988) reduction of 16:1:4
'I.
ofthepre- value.injectionbaselinevalueoccurredal 10min.
SpantideJFreedman etaI., 2.8 laserdoppler Bloodafterinjectionand alflowdroppedimm20minwasedtetely There was a gradual butrecovery of spinalcordbloodflow.Incomplete
\
(1988) 30:1:6% of thepre-injection At60min,bloodflowwas 53±20
beseline velue. %ofthepre-injection baseline value.
0.ArgI.0.Pr02• 3.Jnmol radiolabelled Bloodflowdecreased41%inthe o-Trp"' ,Leu". micrcspheres thoracicand 17 %inlhe substanceP([D· (reference lumbose&ralspinalcord,IS1020 Argl·SP)/Heike sampling minafterinjection.
et el.,(1987) method)
AVPI 1,10, SO radiolabelled Al lOmin afteri njection, AVP Lumbosacralbloodflow remained Lcngetal., and100 microspheres reduced lumbosacralbloodflow significantlydecreased30 min after (l989a) pmol (reference from371087%with doses of10 the100 pmoldose.Recovery10
sampling andlOapmol,respectively. pre-treatmentlevelsoccurred by60
method) min.
Table 2(com'd)
~ DNg/ Do" Techniquefor ChangeinSpinalCord BloodFlow TimeCourse
Reference Measuring
Blood Flow
Endothelinl O.31mM laserdoppler Thedecrease wasrapid in onset A greaterthan SO%reduction in Weslmarketet., achievinga greater than500/0 bloodflowwas maintainedfor at
(199' ) reductionfrombaseline. leastJhwhen the animal was
anesthetisedand perfused forlight microscopic examinationofthe spinalcord.
DynA( I-IJ) 20 nmol radioillbelled Two miretesafter injection,blood
t
Thornehitletal, micospheres flowwasdecreasedby up 10SO% (1989) (reference insegments 2emcaudal and Icm sampling rostraltothetip ofthe catheter. method)
DynA(I.I3)and DynA(I- radiolabelled DynA(I·13):Dose-related Dyn A ()-lJ)I 13):12.5, microspheres reductions inlumbosacralblood Longerel., 25and50 flow,Lumbosacralbloodflow
(1987) nmol wasdecreased)]·75 0/.and
DynA (3. thoracicbloodflowwasdecreased IJ),'0 380/. at 10minpost-lniecrion..
nmol Dyn A (3-13):Therewasan
approximately 40%decrease in lumbosacral blood flow10min afterinjection.
Abbreviations: i.t., inuathecal;Dyn,dynorphin;AVP.arginine"-vasopressin
26 1.6TheVascularEffectsofa- Agonists
Inlightofthepresumed mechanismof toxicityofi.t,peptidesdesetibed abo ve,andthethefadthatperispinalroutesof administration can exposethe spinal cord to concentrateddrug solutions(mM range),itis importa nttonotethat botha,•andQ2-adrenerg ic agon istsarepotentconstrictorsofcentral blood vessels.In a studyusingisolated cerebralarteries fromthemonkey.phenylephrine andnoradrenali neproduced adose-dependentcontractionof vaSOJlar smooth mu scle (Toda.1983).Themaximumresponseswere comparableforboth drugs althoughtheED50of phenylephrine(1.11 %0.41X,Q"l' M)was5.6times great er than thatofnorepinephrine. Although,NAisa non-selective a-agonist,the contractionsinduced byNAusingisolated human and monkey cerebral arteries appeartobemediatedbyc ,-receptors.Treatmentwithprazosin (3 X 10-'0
and ' cr-
M) signiflC8ntlyattenuatedthe contractileresponsetoNA(10""'to10-&M).
Yohimbinewaswithouteffect inconcentrationsupto 10"'M(Tode,1963).
CougNanetal.(1992)showed that isolatedcaninemiddlecerebralarteries exhibit dose-relatedcontractionstothehighlyselective a2·agonist , dexmedel omidine(OX;
a2to
a.
ratioof16 20:1versus200:1for donidine;VirtanenetaI.,1988 ).Ata concentrationof 3 X1~M,themaximumconcentrationofOXthatcouldbeused withoutdetectablea1-mediatedvasoconstrictiaOXinc1Jcedaresponseequivalent to 72.8±5.7%of themaxima l effectevoked by40mMKCI.Large systemicdoses of a-agonists cancause cerebralvasoconstriction..
27
For example.'ZJ::JtroNeta!. (1990 )demonstrateda 45% cecreese in cerebral blood flawinisoflurane-anesthetizeddogsfollowing a systemicdoseof OX (10~gi.v.).
KartssonetaI.(1990) alsodemonstrated a 34% deaeaseincerebralblood flowin halothane-anesthetizeddogs usinganidentical dose of OX. The spinal administrationof0 ,•ora2.-agonists also impai rs spinalcord bloodflow.
P!lenyleplYine(0.3.0.5%)...00.,.,theIo.m>ar subaracl1noid spaceofthedog.
decreased bloodflowby 28%and44%. respectively,30 minutesafterdrug administration (Oohiat at,1984).In therat,dosesof20.100 and 400 nmolofi.t donidine each reducedSCBF 15 min afterinjection.butgrayand whitematter re sponded differently.Inthe grey matter ofthe spinal cord.the largest decreases occurred withthe20and100rmotdoses(29 - 44%comparedtocontrol).The400 nmofdose producedsignificantdeaeasesoIa lesser magnitude (12-27%).Inthe white matter,the100and 400 nmoldoses produced red uct io n s of 17-39%,while the 20 nmol dosewaswrthouteffect(Cro sby etat,1990 ).Epiduraldonidine(10 and30!J9/kg)also affected spinalcord bloodflow(SC BF )in the pig(Gordh at al., 1986a). Redu ct ions of25%to 35%were observedin thelumbar and thoracic segments45minutesafterdrugadministrati on.Thesedata clearlyindicatethatc- adrenergic agonistscan impairblOOdflow through cerebralandspina lvesse ls.
A direct compa risonofthe hemodynamic effect of neu rotox ic peptideswith thatofa-agonistsiscomplicatedbytheuse ofthe different methodologies,species and spinat routes of drug administration.Nevert he le ss,both classes ofdrugshave
28
beenshowntodeaease spinal cordblood flow to a compara bledegreewithinthe first30min ofl.t,drugadministration.The 25-35%decrease inbloodflowrecorded 45 min afterepidxaIdonidine (Gord hetal.,1986a)suggeststhata-agonistshave a sustainedeffect.and that theymay induceasubstantial disturba nce in spinal perf usion. Unfortunately. thereare no nme-cocrse data bywhichto judge the actual durationofthis disturbance.
Theadrenergic nerve supplytomost blood vessels origi natesin the pre-or para-vertebra lgangliaof thesympatheticnervous system. In the brain.the inne rv ati onofsomebloodvessel s may originate from centralcatecholaminergic neurons (Edvinssonat aI., 1973).Thereis greatvariationinthepattern and den sity ofthesympatheticimervation of
vascne-
smoothmuscle (Bumstock,1975 ).Large elasticarteriesgenerallyshow asparse patternofinnerv ation while capillaries.venutesandsmallveinshave virtuallyno adrenergic nerv e supply.In contrast, smallerarteries displayanncreased densityofimervation.TheyareSlI'TOUnd ed byahair~ikenet'Nork of sympathetic postganglioni cnervefibersinfl uenci ng smooth muscle cells.Ind eed,eachterm ina l branchof a postganglionic fiber coursesacr o ss thesurfaceofoneor more vaSOJlarsmoothmusdecell s.releas ing norepineptrine fromthe ve sic le s withinits multiplevancoeftes.
Vasoconstrictionis mediatedbypost-junctiona t01·ando,-adrenoceptors.
Whereaslarger aterio lesand venutesare regulatedbyboth
°
1•and oz·adrenoceptors,vasoconstriction inthetenn inal vessels ismedi a tedprim ar ily by
0,·
29
adrenoceptors(Faber,1988).a,-Adrenoceptormediated contraction ofsmooth muscle relies on the releaseofintrace llular, rather than on extracellu lar,storesof Ca'". Thus. agonist occupation of vascular or-adrenoceptors activates phosphol ipaseCwhichactsonphosphotidyl inosito l bisphosphate,a membrane phospholipid,to produce two second messengers.Inositol triphosphate(IPJ is refeasedinto the cytosol.anddiac:ytglycerol (OAG)remains in the membrane and activatesprotein kinase C(PKC).IP~evokes releaseofCa-fromintem al stores withinthesaaopIasmicreticuIun of themuscle cell;PKCsensitizesthecontractile apparatus to Ca".Thenet effect of ar-adrenoceptoractivation is contractionof vescner smooth musclewithout themovementofionsacrossthecellmembrane and witha minimal cha nge inthemembranepotentia l.
The contractionofvascularsmooth musclemedi ated by02-adrenoceptors depen ds almost totally on extrace llu larea- andanincreaseinthe membrane permeability to thision.It has beenproposedthata2.-adrenoceptoractivationmay becoupled.via a pertussistoxin-sensitiveG-protein(GJ•to phospholipid hydrolysisinvolvingphospholipase0(PLO) andthefonnation of phosphatidic acid (Abu1 0etat.1995).Onceformed.phosphatidicacidmay giverise toOAG.via the action ofphosphatidate phosphohydrolase,leading toincrease of activity of PKC act iv ityand sUbseque ntcontraction.Basalinfluxof extracellu larCa-.andan as yetunidentifiedtyrosinekinase,are critical regulatorsofa2-adrenocept orcoupling to phospholip aseO.
30
Clearty,thesmooth muscleatcentralbfood vessels containsboth
0,-
and a~ptors.Eachofthesereceptor subtypes is coupledto a dist incteffector mechanismthat mediatesvasoco n stri cti on.The spina ladmi nistration ofeither0,·
or02-agonistscouldbe neurotoxic ifprolongedvasoconstrictionandthus a sustainedischemic stat e wereto arise.
AsSU'TUllaIiZedinTableIII,a.JITentexperimentalevidencesuggeststhat the spinal administrat ion of individua l a-adrenergic agonists is without neurotoxicolog icaleffects.However.the safetyofa spinala, - +a)-a gonist combination,effecting independentvasoconstrictormechanisms,hasnot been in vestigated.
Table )
StudiesofSpinalNeurotoxicityFollowing Intrathecal or EpiduralAdministrationofa-AdrenoceptorAgonists.
Drug!RootelReference Species Dose Tes' Histology Spinal
Neurotoxicity
clonidincJepiduraV sheep daily dose onweekly SEP,HD ught No
Eisenachetal.(1984) schedulefrom0.1 10 0.6 mglkglday
c10nidincJepidurall sheep total dose 0£2.010 2.8 AN,HO, Light No
Eisenach et al.(1984) mglkg BO
c10nidincJepiduraU sheep dailydose on weekly SEP.HD Light No
Coombset al.(19841) schedule(rom0.110 0.6
mglkglday
c10nidinelintrathecall dog u.s or 2SIlglkg once Behaviour light No
Gordhetal.(1984) dailyfor14days Body
Weight c10nidinelintrathecaV
'"
I,63 or 16.31lg once Behaviour Light+EM NoGordh 1:1al.(1986b) daily(or 14 days Locomotion
ST-91/intrathecall cal 2000ugoncedailyfor7 • light No
Yaksh and Reddy (1981) days
ST-9lJintrl theclll monkey 4000ll g AN
Yaksh andReddy (1981) Behaviour"
Tendonreflexes,locomotion,musclestrength,foodandwaterconsumption
!:1
Abbreviations:AN,entinociception;BO,bloodgases;lID,hemodynamics;SEP,somatosensoryevoked potentials
, ." ', , , ' "'-VII'U
Drug! RoutelRefen:nce Species Dose Test Histology Spinal
Neurotoxicity guanfacine/intrathecal! rat 16.3 or75Iigoncedaily - Light+EM No
Gordhetal.(1986b) for 14days
oxymerezotine/ rat 100nmol twicedailyfor AN Light+ No
intrathecal!Loomiser al. three days or200 or300 Immunehistc-
(1992c) nmol oncedailyfor chemistry
three days
- -
-
~
33 1.7 Combination Drug Therapy
While spinalopioidsandloca l anestheticscan achieve clinically effective acutepain control,none do so without attendant adverse effects. Spinal opioids can produce respiratory depression (immediate or delayed), neurological abnormalities (i.e. confusion). urinary retention and pruritis. Spinal local
anesthetics cause postural hypotension,bradycardia and urinary retention.Elderly patients or patientswithchronic obstructive pulmonary disease are also at risk of local anesthetic-induced respiratory depression (Cousinsand Bridenbaugh,1988).
To minimizethe problem of adverse effects and toxicity,alternate strategies have evo lvedin acute pain management. One example is the conceptofbalancedspinal anesthesia. This is achievedbyadministeringlow dosecombinat ionsofthe analgesicdrugs,each with separateand potentiallysynerg isticmechanismsof action. In appropriate doses, these combinations produce a level of anesthesia/analges iacomparableto that of a large dose of a single drug while minimizingtheir individual adverse effects.
c-Adrenerqtcagonists represent a novel drugclass of spinal analgesics.
Used alone,analgesic doses produce pronounced hypotension and bradycardia.
Additionally,the variabilityin theanalgesicefficacyof a-agonists in humans makes the irindepende nt use unreliabl e.However,whenusedin combinationwithlocal anesth et ics and opioids,the spina ladministra tion of a-agoni sts has been demonstra tedtohavepositivepharmacokineticand pharmacod ynam iceffects.For
34
example,Lt. clonidine, at doses wtIich were otherwise inactive,prolonged the duration of action of tetracainein laboratory animals (Sadder at aI.,1986;Mensink at al.,1987)and ofbupivacaine in humans (Raele at al.,1987;Racle at al.,1988).
This effect, which was dose-related (Bonnet at al.,1992),is suggestive of a pharmacokinetic interaction. In contrast, i.t. clonidine or norepinephrine significantlyreduced the E050of i.t.morphine or DPDPEin mice (Roorig at at, 1992). Intrathecal metomidine and oxymetazcline produceda similarinteraction with i.t. morphine in rats (Sherman at al.,1988;Ossipov at al.,1990a).In humans, the combination of epiduralclonidine and fentanyl produced additive analgesia in a study of cesarean section (Eisenach at at 1994). Clinical studies have also shown a synergism between opioids and a2-agonistsin the treatment of opioid refractory cancer pain (Pclati et at, 1996). These results suggest a pharmacodynamic interadion between a-agonists and opioids,consistent with their antinociceptive/analgesic activity. Interestingly, clonidinehas been shown to prolong the duration of analgesiaof epidural morphine and fentanyl in humans (Motsch et aI.,1990;Rostaing et aI.,1991). These data suggest that both pharmacodynamic and pharmacokinetic factors underly the interaction of a- agonists with spinal cpioids.
The combination of low dose local anesthestics and opioids in spinal anesthesia/analgesia has alsobecomepopular(Miller,1994).For example,Cullen et al. (1985) studied a mixture of morphine (0.01%)and bupivacaine (0.1%)in the
35
control of
ma;or
post-operative pain.In this double-blindstudy.painscoresinthe morphine alone and the morphine-bupivaca ine combination groups were signlficanttyreduced cxrnparedtothoseof bupivacainealone.Mixturesof epidural sufe nta nil(O.1.Q.2IJglml)or fenta nyl(1 IJglm l) with0.125%bupivaca inearenow commonly usedduring labour (youngstrom ataI.,1984;Vert om man at al.,199 1).Interestingly,the adcfrtioodepineptrine(diluted1:800.000toafina l ooncenuation of 1.45IJgl ml) prolonged the duration of actionofabupivaca ine-fentanyl combi nati on,there byreducing the needforsupplementary doses to sustai n pain control(Grice et at,1990).
1.8Rationa le and SpecificObjec tives
LOW"dose combinationsof analgesicand/or anestheticdru g s arebeingused incre asinglyto optimizepain controlwhile minimizingthe problemofadverse effects.a-Agonistssuchas epinephrineand phenylephrineare routinelyused in combinat ionwith spinalloca l anesthetics(Cousins and Br idenbau gh,1988)and a2--agonistsarebeingused adjlSldivelywithspinalopioids.Thus,itis ina eas ingly likelythata-agonistswithvaryingselecti vitytora1-anda2-adrenoceptorswillbe administeredperisp ina lly(either concurrently or serially) ascomponentsofa multiple drug approachto paincontrol.
Currenteviden cesuggeststhat01-andq -adrenergic agonists possess discrete anlinociceplive mechanisms (see section 1.3). Thus. their co-
36
admin istrat ion inthe spinal cord raises several importantpharmacological quest ions.Does thecombinationof l.t,01-anda2-adren ergicagonis tseffectan additiveCX'"~itiveantinociceptive interaction?Cana spinal antinociceptive combinat ionofQl-anda~agonistsbeusedwithoutriskof ischemic toxici ty given theirpotent vasoconstrictive effects andtheovertneurotoxicityind ucedby Lt, peptidesthatcause localized vasocontriction inthespinal cord? Coukt an0,·and Q2-agonist combination exacerb at e the adverse effect of aknown spinal l'lelXOtoxi'l?Thepc.rposeofthisresean::hwastoinvest igatethese questions in an animal speci es (rat)whose spinal bloodsupplyresemb les thatofhumans. andwith aknown vulnerability to ischemicnetxOtoxicity.The specific objectives ofthis researchwere:
1) To determinethe dose-responserelationship ofi.t.OX, alone and in combinationwith afixeddose of MX(10 1-19),in the tailflickand paw pressuretests.
2) To detennine if a combination ofi.tOX and MX,inrectec:ltwice a day forfourconsecutivedays,induces spinal neur otoxicity.
3) Todetermine the effect of a combinationofi.t.OX andMX onLt.
dynorphin-indueedneurotoxici ty.
2.0 METHODS AND MATERIALS 2.1 Animals
Allexperimentswereconduded using virus antibodyfree (VAF),male, Sprague-Dawl eyratsweighing between300to400gramsatthe timeof experimentation (Char1es RiverInc.,St Constant, Que.,Canada).Animal swere housed in a climate controlled room (22-C)witha12 hour(h) Iight-darkcycle (Itghts onat07:00h).Puinalmlaboratoryratchow andtapwaterwereprovidedad fibffum.Ratswerehousedingroupcagespriortosurgery foraminimumofone day. All procedures wereapprov edbytheAnimalCareCommitteeofMelll()(ia l Universityinac:cofdancewith theGuidelinesoftheCanadianCouncil on Animal Care.
Theratwas chosenastheexperimentalmodel for this thesis asthe rat spinalcordarterialsuppfybears an almostexactresemblance tothatofhumans (Scremin,1995). Giventhefocusofthe thesis and the experimentaluseof dexmedetomidine in clinical trials, extrapolation of the results of the neurotoxicologicalevaluationofthisdrugtotheuseinhumanswouldbebest achieved using ananimal modelthatclosely resembledthehumancond ition.
Add itiona lly,in thechoiceofapositivecontrol for thisthesis,itwasimporta nt to selectbothamodelandadrug,thathadbeenwelldocumentedinthe litera ture, agai nstwhichtogaugetheneurotoxici tyof thedrug(s}beingevalua ted.As,the majorityofspinalneurotoxicity studies hasbeendonein therat,itwas chosenas themodel fortheexperimen talwork.
38 2.2 Surgical Procedures
2.2.1 ImplantationofIntrathecal Catheters
Rats wereimplantedwithintrathecalcatheters usingthe methodof Yak$h andRudy(1976)asmodifiedby Sherman atal(1988). Intrathecalcatheterswere preparedfrompolyethy1ene tubing (PE10)stretchedtoapproximately1.5Xits original length.Asmallloop wasmade approximate ly 4 an fromoneendofthe catheter and fixed with adhesive.Thesection of the catheterbetcwtheloop was trirrrnedtoalengthof7.5em.Immediatelypriortoimplantation.the catheter was flushed withsterile (0.9%)saline (Astr aPharma Inc.).
Anaesthesia wasinducecl with4%halothane(Halocarbon Laboratories, Rive rEdg e,N.J.)inoxygen(100%) andtherat wasplaced in astereotaxic apparatus(NarishigeType SN-2,TOkyo,Japan).Anaesthesiawas maintainedwith 2.0-2.5%halothane.A transversecutwasmadeatthebaseofthe head and the atl anto-occi pital membrane wasexposed.Thecatheterwas insertedthrou gh a small slitin thecisternal membrane,andguidedcarefullyttvougtlthe spinal subarachnoidspace so that the tipwas positionednear thelumbarenlarge ment The catheter loop wassuturedtotheoverlyingIT'KJscle andtherostralendofthe cathe terwasthenpassedundertheskinand externalizedon thetop oftheskull.
Thecatheter wasflushed with10 IJIofsterilesalineto clear the catheterofany bloodor debris,and sealedwithastainlesssteelplug.Theinci sion wasthen closed with suturesand halothanewasdiscontinued.
39
Animalswerehoused individuallyaftersurgeryand allowedto recover forat leastfourdays before experimentation.Thet.Lcatheter wereflushedthe next day with sterile salineandthesurgical incisionwas deansedwith10%hydrogen peroxide(H"Oozl andtrealedwithananti-infla mmatorycream (Viaderm KC.Cream (Tewo».On lyan imals with normalpostureandgait,and exhibitingnormal eating, drin k ing and grooming behaviourwereused for experiments.
2.2.2 Implantation of Intravenous Catheters
Jugular vein catheters-.wremadefromtwosizesof$ilasticf)tubing.0.020"
X 0.037"X 0.0085"and O.04IT' X0.085"X 0.0225", Two 7millimeter (mm) segnentsofthelargertubingwerea.rtand ead'l was threaded with a3centimeter (em) length of nonabsorbablesurgical suture(8- 186 Ethicon,Peterborough, Ontario,Canada).A 12-anlength otthe smaller tubingwas then threadedthrough thetwolarger tubingsegments.The first segment was positioned4-an fromthe endofthesmaller tubi ng. Animalswereana e sthet iz edwithsodiumpentoba rbita l (50 mg/k gi.p.)and the left jugularveinwasexposed.Theproximalendofthe smaller tubingwas inserted intothejugularveinin the directionof thehe art.The position of the catheter,inthevein, was then securedbytyingthesuturesofthe over1yingtubing segnent tothesurroundingmusculature.As the tubing exitedthe jugu lar, a ha lf.loop wa s formed so tha t the catheter could be passed subcutaneously to the backof theneckand externalized on thebackof the skull.
The loop was secured by positioning and tyingthesecond overlyingtubing segment
40
to the nearby muscu lature.Theexterna l izedportionof the catheter wastrimmed to a finallength of3-anand sealedwithastainless steel plug.Animalswere a110Nedto recover for48 hpriortotheexperiment and patency ofthecatheterwas maintainedbytwice daily injed ions of 50 IJIofsterile saline containing heparin (100 1.Ulml).
2.3 AntJnociceptive Testing
Atltinociceptiowasassessed usingthetail flick (TF)and pawpreSSt68(PP) tests.For bothofthese tests, ratswere lightly restrained by hand using a cloth to coverthe head.ThisminimiZed exploratory behaviour andvisual stimu li duri ng te sting. Ratswereacclimatized to theTF andPP ecpereuthedaybefore expe rimentationbegan.The mean of threeconsecuti vemeasurements intheTF and PPtestswasdeterminedateachtimepoint
In the TF test {D'Amourand Smith ,1941),theventr al portion of the tail was pla cedove r thet-enopeningofthetailflickana lgesiameter(Muromad'liKikai Company ,Ltd.Tokyo ,Japan ).Radiantlightfrom an
ev
SOWfight bulb.focussed usinga reflect or throug htheopening,wasusedastheheat source.Thetime between the onsetofthe heat source andreflexwithdrawalofthetail(termed the"response latency" )was automatically recorded.Thelight intensitywasadjustedto yie ldcontrolresponses of 3-5 seconds(sec ).In theabsence ofa response, the noxiou sstimu luswas terminatedafter 10sec and the cutoff latencyrecorded.
41
Pawpressurethreshold (PP)wasmeasured usingaUgoBasile Analgesy Meter(UgoBasile.Italy) according tothemethodofRanda ll andSe litto (1957).
The non-inf\amedhind pawwasplaced on a Teflon plinth havinga very low friction coefficientA cone-shapedplungerwas placed direct lyabove thedorsal surface fAtherat'spaw.Theforce exertedon thepawwas gradually naeased'atafixed rate until a complete withdrawal or withdrawalattemptwas made. The force elicitinga reflex withdrawalwasthenrecorded.Aartoffpressureof1250 grams wasusedin allexperimentsto preventrnju'y andinfl ammation.
