Pharmncological Characterization of An Esophago-Cardiovascular Reflex in the Rat

Pharmncological Characterization of An Esophago-Cardiovascular Reflex in the Rat

by Dongyuan Yao

A the sis submittedto the Schoolof GraduateStudiesin partialfulfilmentof therequirements for the degreeof

Master of Science

SchoolofPharm acy Memorial Univ ersity ofNewfoundlan d

1996

51.John's Newfoundland

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ABSTRACT

The distension of hollow viscera is known to evoke a range of autonomic visceromotorandbehavioralresponses subsumed underthe tenn"pseudoaffective"

reflex. Although some physiologicalproperties of"pseudoaffective" reflex responses to esophagealdistension have been previously described in the rat, only limited pharmacologicaldata are currently available. The present investigation focused on an esophageal dlstensicn-evcked preseorrcerdloacceterator response in the urethane-anaesthetized rat. Our primary objectives were:i)to determine whether relevant sensory input from the esophagus to the eNS is conveyedby vagal orspinal afferents;and Ii) to study the effects of antinociceptive agents on this reflex,and their sites of actions.Experiments were carried out on male,Sprague- Dawley rats(300-450g),previously prepared for intrathecal (i.t.) injectionat predetermined spinalsegments,and anaesthetizedwith urethane(0.8 9+20 mglkg/hl.v.).Esophageal distension evoked a reproducibleincr easein arterial blood pressure and heart rate. Both components increased linearly withthe logof inflation pressure(25-150mmHg).lower,beingmore effective than upper esophageal distension (100 mmHg for20sec),was insensitive toi.t.thoracic morphine (4-16 1-19).However,l.v.morphine(1.04.0rngIkg) producedadose-dependent inhibition of the evoked responses.This esophago-cardiovascularreflex was:attenuated by unilateral,and abolished by bilateral cervicalvagotomy;andinh ibited by theLt. thoracic, butrotbyl.tlumbar or tv.injed;onofdexmedetom idine(OX,0.05-0.5I-Ig).

The evoked response was inhibited by neonatalcapsaicin.and by thetopical administration ofOXor morphine to the solitary complex (NlS).The pressor response persisted aftertv.pancuronium,scopolamine, and methsccplarmne.The data indicate that: 1) the sensory information of this esophaqo-cerdioveeculer reflex is conveyed to theeNSvia vagal afferents;2)the efferent limb of this reflexis comprised of sympathetic cardioacceterator- and vasoconstric:tor-preganglionic patlTways in theIMLof the thoracic spinalcord;clod3)the internuncial connections are made up of bulbo-spinal pathways probably originating in thenVLMwhichin tum.receive input from the NTS.

Key words: esophageal distension; morphine;dexmeoetcmldlne:intrathecal ; solitary complex:spinal;vagal;pseucloaffective reflex;esophageal pain;rat

ACKNOWLEDGMENTS

Iwish to expressmy sincereappreciationto myresearch supervisors,Drs, ChristopherW.loomis and DeUef 8ieger,for their expert guidance and encouragementthroughoutmyresearch.I am alsogratefulfortheir thorough review of this thesis.Iam verygratefulto Or.Loomis for providingfinancialsupport.

Iwould alsolike to extendmygratitude to the following indi viduals:

Or.Slave Shermanfor hisgeneroushelp and constantsupportthroughout my graduateprogram,and forhis expertadvicein experimentalandstatisticalmethods.

Mrs.Nagwa Sherman forherkindness, support,and constant encouragement ,nol tomentionherpatient correctionofmyEnglishpronunciation.

Mr.JohnCruzBautistafor expert adviceand kindhelp,bothacademicandnon- academic,and forhis friendship.

Ms. SueEnenMaher,Ms.ElizabethHodge,Ms. Hemalkhencwala fortheirkind help andfriendship.

Dr.WeiyangLu,Dr.KanthaD.ArunachalamandMrs.JanetRobinsonfor skilful technlcatassistance.

Ms. Margaret MHierfor herkindhelpand forprovidingabloodpressurerecording instrument.

Facultymembersandstaff of School of Pharmacy,especiallyDr.Krishnan Tirunellai,Or.HuLiu,Dr.Uli Wang,fortheirexpert advice,encouragement. kind help.

:amgratefulto the Schoel ofPharmacy and SchoolofGraduateStudiesfor providing financialsupportduringmy degreeprogram.

iii

TA B LE OF CONTENTS

. vii

ABSTRACT.. ACKNOWLEDG EMENTS. LIST OFFIGURES.

LIST OFTABL ES

LIST OF ABBREVIATIONS ANDSYMBOLS INTRODUCTION

Statement of theResearch Problem. GeneralFeaturesof VisceralPain. SensoryInnervation oftheEsophagus.,

VagalAfferentFibers Spinal AfferentFibers.. EsophagealReceptorsand Pain PseudoaffectiveReflexesto Pain. AnimalModelsof VisceralPain.

. ii

...iii

... . ...ix

...1 ...1 ..2 ..3 .... .. .. ...S ...7

.13

.15

PharmacologicalPropertiesof Pseudoaffective

Responses toVisceralStimuli. . 18

ImplantationofIntrathecalCatheters .

RationaleandSpecificResearch Objectives.

MATERIALSAND METHODS

Animals .

.24

...2B ...28 .2B

iv

CapsaicinTreatment.

SurgicalPreparationand PhysiologicalReco rding.

... ..29 .30

NoxiousMechanical and ChemicalStimulation 31

DrugAdministration. Immunohistochem istry. OtherDruqs. Data Analysis.

RESULTS....

The relationship between esophagealdistension pressure and cardiovascularresponses.

..32

..32 ..34 .34

... . . .36

.. .. ..36 Theeffect of balloonpositionontheesophago-cardiov ascular

reflex. . 40

The pressorandheartrate responsesevokedbyesophage al distension depend on theintegrity ofvagal innervation 40 Theesophaqo-cerdiov ascula r reflex persists afteresop hage al

paralysis. ..45

Neonatalcapsaicin partia lly allen uatesthe esopna qo-

cardio vascularrefl ex.. . .46

Supraspinal,butnolspinal,morphine inhibitsthe esophago -

cardiovascula rreflex.. . 47

Supraspinal andspinal dexmedelomidineinhibits the

esophag o-car diovascularreflex. . 53

Esophagealinflationinducesperistalsis that modulates

arterialblood pressure. ....55

DISCUSSION ..62 Methodological considerationsand relevanceof the model

for studying esophageal pain. ..62

Is the esophago-cardiovascular reflex suitable for studying

esophageal noclceptlon? .65

Is the sensory informationtriggering the esophago- cardiovascular reflex conveyed to the eNS through

vagal or spinal afferents? .. . 66

PhysiologicalJphannacologicalpropertiesand central pathways of the esophago-cardiovascular reflex. .... . .67 The effect of capsaicin on nociceptive reflexes and the esophago-

cardiovascular reflex., 69

02Adrenoceptorsin the NTS andthe esophago--cardiovascular

reflex. . ...71

Opioid receptors in the NTS and the esophago-cardiovascular

reflex. ..72

NTS efferenls 10the RVLM. 73

The efferent limb of the esophago-eardiovascular reflex. . ..75

CONCLUSIONS.. ... .. .77

REFERENCES. 79

vi

usr

OF FIGURES

Figure 1 Neuralinnervationofthe humanesophagus ...4 Fig ure 2 Distribution ofretrogradelylabelled cells in dorsal

root gangliaafter injectionoffastblue lnlothecervical striated muscle and thoracic smoothmuscle of the

esophagus. ..9

Figure 3 The spinalandvagal afferentinnervationof the

esophagus. . .25

Figure 4 Representative heartrate and meanarterialblood pressureresponses to lower esophagealdistension

in urethene-aneetheflzecrats. . 37

Figure5 Therelationshipbetween esophagealdistension

pressure and evokedcardiovascularresponses. ..38 Figure6 Distension oftheloweresophagus reduces the

frequencyofrespiration 39

Fig ure 7 Esophageal distensionhadno detectable effects

on theEKG exceptfor anincrease in heartrate. ... . . .41 Figure 8 The positionof theballoon intheesophagus affects

thedistension-evokedchange in mean arterialpressure

and heart rate. . ...42

Figu re SA The change inmean arterialpressure(AMAP) evoked byesophagea l distensiondependsonthe integri tyof

vagalinnervatio n. . 43

Figure98 The changeinheartrate (AHR)evoked by esophagea l distension depends onthe integrityof vagalinnervation 44 Figure10A Neonatal capsaicin partially attenuatesthe meanarterial

blood pressure response to esophagealdistensio n .48 Figu re108 Neonatal capsaicinpartiallyattenuatestheheart rale

response toesophageal distension .49

vii

Figure 11 The effect of neonatal capsaicin onpeak tailflick latency

inadult rats ,.. , 50

Figure 12 The effect of neonatal capsaicin on peak paw pressure

withdrawalthreshold in adult rats. . 51

Figure13 Morphine, appliedtopically tothe solitarycomplex(NTS), inhibits the esophago-cardiovascularreflex. ....54 Figure 14 Dexmedetomidine dose-dependentlydecreased basal

mean arterialpressure ....57

Figure 15 Dexmedetomidinedose-dependenlfydecreased basal

heart rate. . ".,58

Figure 16 Distension of thelower esophagusevokesesophageal peristalsis that modulates thereflex pressor response. ...59 Figure 17 Dexmedetomidine,applied topically to the solitary complex

(NTS).inhibits esophageal distension-evokedperistalsis and modulation of the reflex pressorresponse.. ....60 Figure 18 The postulated esophago-carcto....ascular reflex arc.

viii

. ... .78

LIST OF TABLES

Table I Esophageal DistensionThreshold for AMAP and AHR ..36

Table II The Effect of Vagotomy on the CardiovascularResponses to NoxiousMechanicaland ChemicalStimulation

45

Table 1II The Pressor Reflex Persists after Esophageal Paralysis ..46

Table IV Supraspinal,butnot Spinal,Morphine Inhibits the Esophago-CardiovascularReflex

... .. ... ... .. .. .. ... ...52

TableV Supraspinaland Spinal DexmedelomidineInh ibits the Esophago·CardiovascutarReflex

.... .. .... .. ..56

ix

'C

~A 1'9~I

urn

~M pmol nmot + AO ANOVA bpm C Cl CCK C.I.

CGRP CNS Co.

DRG OX e.g.

ED"

EKG eta/.

EMG Fig.

g h

LIST OF ABBREVIATIONSAND SYMBOLS

alpha,a Greek letter beta,a Greekletter gamma,a Greek tatter

mu,a Greek leiterused10indicated microin metricunits, also a subtypeof opioid receptor

delta,aGreekletter usedto lnclcatedmicroin metric units,also a subtypeof opioidreceptor

degreesCelsius equa ls greaterthan lessthan

mlcroernperets),1_0~amperes,unitofelectriccurrent microgram(s),10'"grams,unit ofmass mtcrollterfe),10-$lilr es,unitofvolume micrometer(s),10~meiers,unitofdistance micromolar,10~moles perlitre,unit of concentration micrcmolets),10.6moles,unitofmolecul es nanomole(s) ,10-limoles,unitofmol e cules plus , in combinationwith

A-de lla,a classofprimary afferent neurons analysisof variance

beatsperminute

a classof pr imaryafferent neurons cervicalvertebranumberone cholecystokinin

confidenceofinterval(aboullhemean) calcitoningene-relate dpeptide centra lnervoussystem company,corporation dorsalrootganglion dexmedetc midln e exempli gratia,(for example) effec tivedo se for50percentresponse electroca rdiogram

et alia(and others) electromyography figu re gram hour(s)

Hel Hg H20a HR HTMN Hz I l.e.

IGLE l.p.

Lt.

tv.

II III IV IR IML Inc.

in vitro in vivo kg Kpa L1 LES L-EN K log

m

MAP mg MK-212 ml

mm

mm' M N nmol NTS NTSc NX PAP P.E.

PE-lO

hydrochloricacid elementalsymbolformercury hydrogenperoxide heartrate

high-thresholdmechanonociceptor(s) hertz,5-¹,reciprocal seconds,unit of frequency Romannumeralone,used to denotespinal lamina id esf,that

inlraganglioniclaminar nerve ending inlraperitoneal(ty}

inl rathecal( ly) inlravenous(ly)

Roman numeral two,usedto denote spinallamina Romannumeralthree,used to denotespinal lamina Roman numeralfour,usedto denotespinallamina immunoreactivity

intermediolateralcellcolumn incorporated

in glassware in theliving body kilogram(s), 10³grarns kilopascal(s),unitof pressure lumbarvertebranumberone lower esophagealsphincter leucine enkephalin logarithm (base 10) metene)

mean arterialblood pressure milligram(s),10.³gram 6-chloro-2-(1-piperazinyl)-pyrazinr miltHiter(s),10.³liter millimeter(s),unitof distance millimeter{s)squared,unit ofarea molar(molesfliter) number of determinations

nanomole(s), 10'&moles,numberof molecules nucleus of the solitary tract

centralsubnucleusof the solitarytract naloxone

peroxidase-antiperoxidase polyethylenetubing

size10 polyethylenetubing(diameter..O.61mm) xi

PpH RVLM sec s.c.

SEM SP SST T1 U VIP WDRMN WY27127 wlv V VI X

probabilityoferror

nega tivelog of the hydrogen ion concentration rostrovenlral lateral medulla

second(s) subcutaneous(lY) standard errorofthe mean substanceP somatostatin thorac ic vertebranumberone biologicalunits vasoactiveintestinal peptide wide-dynamicrange mechanonociceplor(s) Wyeth27127 (disulphonamido-benzoquinoline ) weightpervolume

Roman numeralfive,usedto denotesplnallamlna Romannumeralsix,usedto denotespinallamina limes(multipliedby),alsoRoman numeralten

xii

INTRODUCTiON

Statement ofthe ResearchProblem

Recurrentchestpainis an importantand potentiallyseriousclinicalsymptom because of its associationwithcardiacdise ase.Howe ver,not all recurrentchest painis of cardiacorigin.Oftheapproximately600,000new patientswithanginal symptomslNho undergocardiaccatheterizationintheUnitedStales each year,30%

havenormalcoronaryarteries.Of thosepatientswith normalcoronarycirculation who report chest pain.approximately 50%have a demonstrableesophageal abnormality(Richteretal.,1989).

Esophagealmotilitydisorders,characterized byhighamplitude,longlasting, peristaltic contractions,are observedinapproximately 28% of patientswith noncardiacchest pain(Richter

at

81.,1989). Chest painin humans alsoarisesfrom spontane ouscontrac tions of the esophagusagainst anon-compressible objectin thelumen (PayneandPoo'tcn,1927;Nixon andKoch ,1989), and from distension of the esophagusitself(DeV ault and Castell,1992).Dependingonthe degree of distension,patientshave described esophageal chestpainas'a severe dunache' and 'something sharp'10'a continuous burning and grippingpain' (Payneand Poulton1927;PayneandPoulton,1928;Polland andBloomfield,1931a).Prolong ed distension also causes cutaneoushyperaesthesia intheskinabovethe sternum.

Esoph ageal painis oftenreferredfromthe area ofthe suprasternal notchto xiphoid pro ce ssandsometimesto the costarangle, withradiationthrough tothe

angleofthescapula.Asaresult,esophagealpaincan mimicthe painof myocardial ischemiamakingit veryimportantforthe physician tofirst properlydiagnosethe causeofthepain.AJ.the presenttime,it is verydifficultto distinguish cardiacfrom esophagealchest pain until afterextensivetestinghas been completed. This delay in diagnosiscanresult inunnecessary stresstobothpatientand physician,andthe needfor extensivecardiac testsimposessignificantcosts to the healthcare system.

It IIIestimatedthat more than315milliondollarsarespenteachyear intheUnited Statesto diagnosepatientswithesophagealpain(Richteraf af.,1989).

Underlying this clinicalproblemis a lackofinfonnation about visceralpainin general,andmore specifically,the lack of informationabout themechanism(s)of and theneural pathways mediatingesophagealpain. Although the extrinsic innervationofthe esophagusis well known,the roleofspinal and/or vagal afferent fibersin esophagealpain is unclear.Using gradedesophagealdistension asthe visceral stimulus,thepurpose ofthis studywas:1)to determinetherelevant sensorypathway(s) thattriggerreflex "pseudoaffective" cardiovascularresponses inurethane-anesthetized rats;and 2)to investigatethe pharmacologicalproperties ofthis esophago-cardiovascularreflex.

GeneralFeaturesof VisceralPain

Pain is generallyclassifiedas eithersuperficial or deep dependingon its location in thebody.Superticial painarisesfromnoxious stimulationto cutaneous

struct ures and the mucousmembranes of body orifices.Deep pain originatesin muscle,fascia,bone,joints,vascular structures and thevisc era,Visceral painisa special kindof deep pain.It is evokedby stretching ,twisting, orcontractionofan orgooand is (hemostcommon type of pain treatedby physicians,Unlike superficial pain. visceralpai.,is dullin nature, poorly localized,and generallyevokesan intense emotionalresponse.IIis often referred tocutaneous but otherwisenormal siteson thebody,and has a propensityto trigger tonicmuscle contractionsas wellas autonomic responses(collect ively called the pseuooettective responses).Whereas deeppain frommu sculoskeletal andarticular tissuecan generallybe diagnosed usingenertesuch as the rate ofonset of pain,the locationofreferre d sensations andthe presence of aggravatingfactors,visceral pain isvery diffi cultto diagnose on the basisofthequality,loca tion and intens ityof sensationalone.Cardiac pain is particula rlydifficultto distinguishfromth at originatingin the esophagus,liver, appendixand gallbladder(seeNess and Gebhart,1990 for review). Clinical tests andimaging techniques are currentlyrequ iredfor this purpose.

SensaI}'Innervatio nof the Esophagus

The innervationof the esophagus issupp lied bilaterallyby the vagus nerve andby spinal nerves that extend from the cervicalto thoracic segments ofthe spinal cord(Fig, 1). Each ofthe se two general pathw ays are discusse dindetatlbelow.

Figure 1. Neural Innervationof the humanesophagus. (Source:Ellis, 1981)

Vagal Afferent Fibers

Vag al afferentfibers arise fromcell bodiesin the superior(jugular)and inferior(nodose) gangliaofthevagus.Thecentral axonal processes project via the tractus solitarius to the racleusofthetractus solitarius tractus, while the peripheral axons primarilyterminatebetweenthe longitudinaland circularmuscle layers, in and aroundthemyenteric gunglia,withonlyscattered fibers innervating thesubmucosal andmucosal layers(Neuhuber,1987).The ratio of afferentefferenl fibersfor indiv iduc11branchesof the vag u sisnot clear,butabout80-90% ofthe fibers in the abdominal vagi areafferent,unmyelinatedC fibers innon-rumina nt animals (Andrews,1986).The remainderare small myelinated andlarge myelinatedfibers (Wo odbury andWoodbury,1990).

Different types ofvaga l afferent fibers subserve differentsensory functions inthe esophagus.Recent studies(Senguptaat a/.,1989) have shownth attension- sensitive(mecnenoreceptor)afferent fibersinthe opossum areof the C- or AI5-lype.

Mechanoand chemoreceptorsin the mucosa are present onB-fib ers (Harding and Titchen,1975). Thermoreceptorsin the esophagealmucosa of the cat are located onsmall diameter myelinated fibersin thevagus nerve(El OuazzaniandMei,1982).

Mechanoreceptive afferents fromthe esophagus have alowlevel of spontaneous activityand exhibit an increased dischargeto either moderate distensionor muscle contraction (Andrews,1986).In the opossum,the threshold distension pressure(applied 2-7emaboveloweresophageal sphincter) requiredto

evoke activityinthese vagal afferentfibers is<10 mmHg.Ma ximal dischargeis achieved at a pressureof56mmHg for discretedistension(70 mmHgfo rstepw i se distension). Three generalphases of response to esophagealdistensionare exhibitedby these fibe rs.Th e first phase is characterizedbyarapidincrease in firingdurillQ the onsetof the distension.Thisis followedbya lower dischargera te (second phase) that remainsabove thelevelof restingactivityfor aslo ng as the esophageal distensionpressur e is maintained.Beforereturnin g to normalrestin g activity ,thefrequency dischargefallsbelow theresting levelandsome fibers even stop firingfor 2-4 seconds afterthe distensionpressure is discontin ued(thirdphase) (Seng uptaatal.,1989).

The dala indicate that thesevagal afferentfibers ,whichoccur in serieswith thecircular or longitudinalmuscle layers ofthe esophagus:a) areslowlyadapting;

b) have a low thresholdof activation«10mmHg)and a maximalresponse within thephysiologi cal range of esophageal distensionpressure;andc)aremaximall y activatedbynormal peristalticcontractions (Senguptaat al.,1989).Recent studies (e.g.Randichet al.,1990,19 9 1,Msllerat al.,1991,Renet al.,1991)have confi rmedtherole of vagal afferentsinnociception.For exam ple,Randichat a/.

(1991)!howed that the antinociceptiveeffects of low doses ofl.v.morphine(0.1 or 0.5mg/kg) Involveactivationofvag alafferents. Bilateral cervicalvagotomy significantly allenuated the antinocicept ionproduced bytv.morphine and the degreeofattenuationwasinversely related to drug dose.As reviewedbyRandich

and Gebhart(1992),electrical stimulationof right orleft cervical vagalnerve (afferent) either facililatestailflickreflexatlesser Inlenslties (typically2.5-20~, 20Hz, 2 ms) or inhibitsthe tail flick reflex atgreaterintensities(>30~)in an in lensi ty-depe ndentmanner intherat. Theseda ta suggesttha ttheeffects of periphera lstimuli on nociceptiondepends on integrity of vagalnerves. The importance of vagal afferents in esophagealnocicepl\on is not fully understood.

SpinalAfferentFibers

Whileall regions of the esophagus receive both vagal and spinalafferent innervation,the cervical and upper thoracic parts of the esophagus are predominantlysu ppliedbyvagal atterente. The lower part of thoracicesophagus andthe acoominet esophagusare primarilyinnervatedby sympathetic etterents. The abdominalportion of the esophagus is also innervated by thephrenic nerve (Pope al1d Bonica,1990).Inthecat,spinal nerves inneNatingthe10000r esophageal sphincterextendfrom spinal segmentsT1tol3,witha peak distribution inT1-T12.

The innervationof esophageal smooth muscle extends from spinal segments C4 to L3,witha peak distributio ninT1-T1 2.Esophagealstriatedmuscle Is supplied by nerves from spinal segments C1 toT8,with peak distributionsin C4-C6 andT2·T7 (Fig.2) (Collman

et

al.,1992).In thedog, thecervical esophagus isinnervat ed by spina! afferentswhose cell bodies are locatedin C1-T9dorsalroot ganglia;the peak distributionis locatedinC1-C6andT3-T4 (Khurana and Petras,1991).In general,

spinal afferents to esophagus, which arefewin number, arise from lower cervical andupperthoracic dorsatrootganglia (see Cunningham and Sawchenko, 1990 for review).

The sympathetic fibers that supply the esophagus originate in the Intermediolateral (IML) column of the spinal cord.In humans, theCdilbodies of preganglionic sympathetic neurons are located in the spinal segments12-T8(Pope and Bonica,1990).Their axons pass from the spinal cord through the ventral nerve roots and continue via the corresponding spinalnerves to reach the paravertebral sympathetic chain. Primaryafferent fibers from the esophagusprotectto spinal cord mostly in the splanchnic and sympathetic nerves. After passingthrou gh the paravertebral sympathetic chain and the ramicommunicantes,they join the spinal nerves before entering the dorsal root ganglia.

Thecentral terminations of these spinal afferent fibers have not been studied in detail but Euchner-Wamserat al.(1993) showed Ih3i neurons in spina! segments T2.T4,v.-tllchare activated by distensionin the middleregion of the esophagus,are distributed throughout lamina I-VI,with a peak distribution In the deeper laminae.

Esophageal Receptors and Pain

As described above, sensory information is conveyed from the esophagus to the eNS by vagal and spinalafferent fibers.Vagal afferents in the submucosal and mucosal layer may act as mechano-,thermo-, or chemoreceptors (Cunningham

W~

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Fig ure2. Dlstrib utlon ofretrograd elylabelled cells Indorsal root gangli a (ORG)(right) after Injectionoffast blueInt othecervical striatedmuscle(black blocks)andthoraci csmooth muscle (graybloc k) ofthe esophagus. The rectangularportionof eachblockdenotestheORGs thatwerelabelledinfour of four cats;thepointedpartofeachblocksignifiesthatnotallanimalshadlabelling at this level.lES

=

lower esophagealsphincter(FromCollmanet af.,1992 )

1.0

andSawehenko,1990;Harding and Titchen, 1975).These mechanoreceptors and chemoreceptors are ideallysuitedfordetectinggastric contentsthat arerefluxed into theesophagus andwhichnormallyyield distension pressurestoo lowto activate muscle receptors.These mucosal receptorsmayplay an important rolein vagatly mediated reflexessuchas secondaryperistalsisandthe clearing of refJuxedgastric contents (Christensen ,1984, Andrews,1986).

Thennoreceptors are alsofound on the peripherallerminalsof vagalafferents in the esophageal mucosa.Three types of slowly.adaplingthermoreceplors have been detected in the cat: 1) warmreceptors thaidisch arge between 39 to 50°C;2) cold receptors that discharge between10 and35°C;and 3) mixedreceptors, responding tobothwarm and cold temperatures(EI Ouazzaniet al.,1982).These receptors were silent at normal body temperature.The receptordischarge was slow (1-20impulsesls), lasted for1-20seconds,and exhibiteda discharge frequency that was generally related to the stimulustemperature. These receptors areimp ortant in the detectionand transductionof temperature changes in the lumen of the esophagus.

Slowly adapting mechanoreceptors,located inthe muscular and"serosal"

layers of the esophagus, are connected to bothvagal and spinalafferent fibers (Clerc and Mei,1983a,1983b,Satchell,1984). Mechanoreceptors on spinat afferent fibers existmainly in the musclelayers ofthe esophagus.They are less frequent in the "serosal"layer and absent in the mucosa(Lynn,1992).There is also

11

evidence that slowlyadapting esophagealmechanoreceplorscanbe functionally subdivided(Satchell,1984),suggestingthatthe cterecterietceof mechanoreceptors on vagal afferent fibers ere different from those on spinal efterents.

These characteristicshave been studiedin the opossum(Sen g uptaet al., 19B:3, 1990,1992) andin the dog (Satchell, 1964). Dlstenslon-seneitive vagal afferentshave a low threshold of activation(ai9ragethreshold pressure=10 mmHg),a narrow stimulus-responsetonctton inwhich the unitdischarge rete increases with increasing distensionpressure,and reaches a maximumdischarge rate at distensionpressureswithinthe normal physiologicalrange«56 mmHg) (Sen g upta et ai,1989). In humans endexperimentalanimals,esophageal distension pressures greater than 60mmH g are usuallyrequired to elicit a nociceptiveresponse (lynn,1992). The functional import ance of the vagal mechanoreceptors is not fully understood.However, itis likelythat pressure infor m ation on tension in circular and longitUdinalmuscle layers of the smooth

muscle portion of the esophagusis sentto thecentra l nervous systembyvagal nerves during esophageal distensionand esophageal peristalsis (Seng uptaat al., 1989).

In contrast, the mechanoreceptors on spinal afferent fibersin the opossum have been shown 10be primarily nociceptive(Senguptaefal.,1990, Lynn 1992).

They appear to exist"in series"with th e longitUdinalmuscle in the esophagus and canbedivided intotwotypes:wide-dynamic range mecharcnoctceptors (WDRMN)

12

and high-thresholdmechanonociceptors (HTMN).TheWDRMNIscharacterizedby alowthresholdof stimulation and a linearstimulusresponsefunc'ionextendingfrom innocuous to noxious intensities. Itcan distinguish between physiologicaland nociceptive stimuli, and may subserve a role in both physiological reflexes and nociception (Senguptaet al.,1990). The HTMN responds only to high-intensity mechanical stlmuliin the noxiousrange and may servea specificnociceptive role (Sengupta

et

af"1900).Thedischargerate ofmech anorece ptors located

on

spinal afferent fibers, activatedbyesophageal distension,does notreach plateauat pressures up10120mm Hg.

Informationaboutthe structureofesophageal mechanoreceptors is limited.

The spindlesfoundin somaticstriatedmuscle are mechanoreceptive sensors and it has been suggested that they serve a similarfuncti on in the striated muscleof the esophagus(Ch r istensen,1984). In the dog,thesespindles are found in the muscularispropria andintermuscular space inthelower one-third of the esophagus (Asaadet al.,1983).However,they are not present in smooth muscle,suggesting that at least one othertype of mechanoreceptorexistsin the esophagus. Itis possible that nerve cell bodies themselves are mechanoreceptive or that special strudures existwithinth~nervous elements of smooth muscle(Chris t ensen,1984).

StudiesbyNeuhuber (1987) have shcrM1that myentericvagal sensory terminals are identical with the intraganglioniclaminarnerve endings or IGlEs describedby Rodrigoet a/.(1982),whichmaybe these special mechanoreceptive structures.

13

Althoughmanydetailsremain to be determined aboutthe sensoryinne rvation ofthe esophagus,itisclearthatprimaryafferentfibers Inboth the va gusand spinal nerves subservea sensoryroleintheesophagus.Their periphera lterminals containmedlanoreceptorsthatareactivatedbydistension of the esophagus,and are thus theoretically capableofconveyingvisceral nociceptiveinform ation from the esophagustotheeNS.Theactual role playedbyeachofthese nerveroules in esophageal pain isunknown and is amajo r focus ofthepresentstudy,

Ps eu doaffecfiveReflex es

'0

Pain

Besidesevokingthesensat ion of "pain" ,visceral noxiousstimulieliclta characteris ticpatternof reflexre sponse s that were in itiallycharacteriz edby Sherrlnglon (1906).Theyincludegrimacing, vocalization,increasedheartrate and blood pressure, increasedrespiratoryrate,generalized orregionalmuscle contractionswithnon-Ialera lizedvisceralstimuliand f1exionlwithdrawaland head- lumingwithlateralizedsomaticstimuli (Woodworthand Sherr ington,1904).These so-called'p seudoaffective'responses areonly trigg ered bynoxiousstimuli,cease when the noxiousstimulus isremoved ,andarereflexly media tedbyneurons within the brain-stemand spinal cord.Theypersistinthedecerebrated anima l preparat ion (Nessand Gebhart, 1988).

Unlike humans,experimental anim a ls areincapa bleof reporting and describingtheirexperience to painfulstimuli. Consequently, investigatorsmust rely

14

on other indices of noclceotlon such as molor reflexes, autonomic responses and pain-like behaviours (i.e.scratching, biting, vocalization, escape efforts,hyper- responsivenessto touch).The characteristic patternsof pseudoaffecliveresponses to noxious stimulation have made them a useful and commonly employed measure of nodceptlon in experimental animals(Gebhartand Ness, 1990,Nessand Gebhart, 1990)

The useofpseudoaffedive responses to actuallycharacterize a stimulusas

"noxious", as suggested by Cervera (1981),is less reliable.In the cat, blood pressure and single unit electrical adivity in viscero-somatic neurons of the thoracic spinal cord were increasedbynoxious distension of the biliary system,but were unchanged at innocuous distension pressures (Cervera,1981, 1982a, 1982b).

However, pseudoaffective responses can be evoked by visceral stimuli that elicit no behavioral evidence of pain(Nessat al.,1990). Intra-coronary injections of bradykinin (10-300 ng/kg) produced a graded pressor response in unanesthetized dogs without any signs of a painful reaction such as vocalization and agitation (Paganiet af.•1985). The dogs remained clam or somnolent and showed an increase in depth of respiration (Paganiet al.,1985). Thus, pseudoaffective responses are used as an quantitative index of nocicepncn to a stimulus that is knovmtobenoxious. By themselves,pseudoaffeclive responses are not sufficient to characterize a stimulus as being 'noxious'.

15 AnimalModelsof VisceralPain

Ithaslong beenknownthat visceralorganscanbe exposedto cutaneous noxious stimuli(l.e.cullingorburning)without evokingpain.VisceralpainOCCUfS whenthe capsulesurroundinganorganis distendedor theparenchymabecomes inflamed bypathological processes(l.e.obstruction of the commonbileduel triggeringsevere visceral pain). Thus,ananimalmodelofvisceral rccrcepucn requiresa stimulusthatstretches, twists or contractsvisceralstructures.Ideally, theexperimentalstimulusshouldbe:a) similar to a natura! stimulus;b) minimally invasive:c) controllable;d)reproducible;ande)quanlifiable (Ness andGebhart, 1990).To date,four types ofstimulihave beenused inbasicstudies of visceral nociception;electrical,mechanical, ischemicandchemicalstimulation.

Electricalstimulationofa nerve or grwp ofnerves innervating a given organ can be reliable andhighlyreprOducible.Unfortunately,itis not specificfor any sensol'Y modality.In COfllrast,interruplionofthebloodsupply to a visceralorganis notareliablestimulussincetheischemicmyocardium ortheinfarcted bowelIs often clinically"silent" andonly recognizedas abnormalbyanalteration inthe eleclroc:adiogram(ECG)or theonsetof associatedsymptomssuchas peritonitis.

The markedvariabilityin the neuronal responsestoischemia,bothwithin and betweenpreparations,alsomakethistypeofnociceptivestimulusunreliable (as reviewed by NessandGebhart,1990),

11

Algogenic chemicals,administrated topicallytothe exposedSl.riacesof selectedorgansor

ir1eded

into theirbloodsupply,have alsobeen used for visceral nodception.However,topicalapplicationlirritsthesiteofactiontoalocalizedarea oftheorgan, andthe intra-arterialor I.v.administrationmay produce widespread activationofthe neural affer ents inall layersofanorgan , depending on the site of injection.In therodentYKithing test,algogenicsubstancesareinjed edIpand the numberof evoked'stretchin g'or'writhing' respon ses,comprised ofalternating flexionandexten sionof the abdominalmuscles, isthen counted per unit lime.

Although thistestis commonlyusedforscreening antlnocceptlve drugs,the onset, intensity,andduration of'Nlithingare noteasilycontrolled,the actual sourceof the ''pain'' isun!<no'Ml, andnon-analgesic,as well as analgesic,drugs are"efficacious"

Inthismodel(NessandGebhart, 1990,GebhartandNess,1990). Inaddition.the stirmiusis inescapableandlonglastingraisingeth icalconcernsabout thismodel.

Mechanicalstirrutationofthe viscera,sudlas distensionofaholloworgan, stretchingofserosaltissue,compressionofanOfgan(i.e.testes),andtractionofthe mesentery,all~ucepaininhumans.PollandandBloomfield(1931b)studied painarisingfromthedistension of differentregions in thegutofhumans.Gastric, duodenalorcolonicdistensioneach triggeredreport sofpain thatranged from 'severe sharp pain' toa'buming and gnawing pain'.Bentley and Smithwick (1940) also studiedpainevokedbydistendingthejejunumof6healthy individuals.Atlow distensionvolumes, thispain was roughly localized tothe epigastricregionbut

17

Spreadto include the entire abdomenwhen greater distension volumes were used.

These resultswere confirmedby Ray and Neil (1947).Painwas most intense when long continuoussegments of gut were distended simultaneously(Lewis, 1942).

Thus,graded distensionof a hollowvisceral organ (i.e.the gastrointestinaltract.

urinary tract,vagina,uterus,biliarytree and gallbladder) mimicsa natural and painful stimulusin humans.Unlike the natural stimulushowever,the distending pressureis controllable, reproducible and can be easilyquantified.

Based on Ihese clinicalobservations,investigatorshave used mechanical distension10study visceral nociceptionin animals.Such studiesfrequenllyinvolve the gutbecauseit canbeeasily accessedthrough the mouthor the anus.Noxious mechanical distension of the gut triggers reproducible pseudoattecnve cardiovascular(Lembeck and Skofitsch,1982),respiratory (Satpathyand Al-Sallar, 1984) andvisceromotor responses (Ness and Gebhart,1988) in experimental animals.Distension of middle portionof esophagus has been reported to trigger a depressor response followed by an increase in blood pressure in nembutal- anesthetizeddogs(Salpathy and AI-Saltar,1964).An interesting study by Gayheart at al.(1991) showedthat esophagealdistensionin c-chtcretcee-eneethetlzeddogs

causes coronary vasoconstriction;an effectmediated by c-adrenoceptors. Euchner-Wamserata/.(1993) also showed that distension of middle and lower esophagus triggersbradycardiainpentobarbital-anaesthetizedrats and found that neurons in T2·T4 spnel dorsal hom.responsive 10graded esophagealdistension,

1.

exhibiteda linearand accelerating stimulus-reapcnee function.The type and dO'18 of anaestheticsmay affect the direction of cardiovascularresponses tc the distension of gut(Ness and Gebhart, 1988).

Overall,thesedataindicatethaIdistensionof a visceralorgan is an effective noxious stimulus evoking both pain and reflexpseudoaffecliveresponses.In the present study, graded esophageal distension was used as the experimental stimulus,and the evoked pseudoaffectivecardiovascula rresponsesware measured in order to investigate the afterent palhway{s)throughwhich this esophago- cardiovascularreflex is triggeredin the rat.

Pl,annaco/ogical Properties of Pseudoaffective ResponsestoVisceral Stimuli

Pseudoaffeetive responses to noxious stimulation are physiological reflexes havingan afferent and an efferent pathway.Sensory spinal andfor cranial nerves comprise Ihe afferent arm of therefl ex. The efferent armis composed of sympathetic fibersthat mediate the autonomicresponses 10noxiousstimulation.

Intemuncial fibers in the medulla and spinal cord playavital role Inthe activat ion and coordination of these reflexes but their identity and pharmacology are poorly understood. Efforts to characterize these pathways withneuroablativeand pharmacological techniques have been attempted usingdifferent modelsofvisce ra l nociception.

19

In early studies, pseudoaffective visceromotorand cardiovascular reflexes evokedby intestinal manipulation,including pinching thesmall or largefntestlne, pinchingthe pancreasand pulling the mesentery(Millerat al.,1924; 1925 a,b;lewis and Kellgren. 1939; Downman and McSwiney,1946) wereretained in animals transacted below the medulla (Oownman and McSwiney,1946). In a more comprehensive study,the heart rate and pressor responsesevoked by graded colorectaldistensionwere not significantlyaffectedby partialsympathectomy(L6-S3 segments) orby mid-collicular decerebration(Ness and Gebhart, 1988) as compared 10 lntact control rate However, both responses were markedly attenuatedor absent in spinallzed rats,eitheracutely at the C1 spinal segment, or chronically at mid-thoracic (T6) spinal level. Similarresults were obtained using the visceromotorresponse to coloredaldistension (a contraction of the abdominal and hind limb muscctatorerecorded on the EMG and observed as 'hunching' of the rat).

These dala indicate that both the cardiovascular andvisceromotorresponses evoked by colorectal distension are activated by brainstem loops. The loop underlying the pressor and heart rate responses probably involves sympathoexcilatory neurons located in the bramstem. Because surgical demedullationofthe adrenalglands significantlyreduced the heart rate. but not the pressorresponse, as compared to sham operatedcontrols, the heartrate response appears to also involve the adrenalglands.

2.

In the samemodel,Nessand Gebhart{198P\ showed thai I.v.

chlorisondamina(8ganglionic blocker)and phentolamine (anon-setecnve0- adrenoce ptor antagonist),eachinhibitedthe distension-evokedrisein heartrate, and converted the normal pressorrespo nse toa slightdepre ssorresponse.

Propranolol or atropinealso markedly imbited theheartrateresponse,bli.hacllittle effect on thepressor response. These dalaindicate that thecardiovascular responses e'JOkedbynoxiouscolorectal distension arisefromthereflex activation of sympath etic outflow and theremova l of vaga l tone.Comparableresults were reportedbyPittma nat al.(1988)using gastricdistensionin an esthetized rats.

Indeed,a reflex increaseincardiacsympatheticactivityandaconcurrentdecrease invagusnerveactivitywere recordedduring urinarybladder distensionin anesthetizedcats {Taylor,1968} and dogs(Hassan

et

81.,1987a,b).Moreover, urinarybladder distension inanesthetizeddogsinduced a pressure-dependent atlerKJationdvagus nerve activityin response to inaemenlal increasesin carotid sirlJSpr9SSl.18(Ramadan

et

aI.,1989). These physiologicalandpharm acological data are consistentwith a reflex modulationof the autonomicnervous systemby noxious visceralstimulation.

Asa presumptiveresponsetonoxiou s stimulation,pseudoaffectivereflexes should be sen siti ve to antinocic eptive drugs. Ingeneral, all classesof entooccepuve agents,Inc luding oz-adrenoceptor agonists,serotoneralcagonlsts andcapsaicin,significantlyattenuatethe pseudoaffeetiverespon ses to noxious

21

visceral stimuli.ThJs, intrathecal (Lt)andi.v.morphine dose-dependentlyinhibited thecardiovascularandvisceromotorresponses to cclcrectaldistension;aneffect reversedbynaloxone (NessandGebhart,1988).Raldorsa!hom neuronsinthe L3-l5segments,activatedbynoxiouscoloreclaldislensioo,exhibitedacomparable sensitivity10morphineandnaloxone(Nessand Gebhart, 1969). Antinociceptive synergybetweenintrathecal morphine andlidocainehas alsobeenshownin the colorectaldistension model(MavesandGebhart.•1992).

However,sensitivityto cplotd analgesicdrugs is nol restrictedto the cclorecta!model.The cardiovascular responses evokedbydistension of the proximaljejunum inurethane-anesthetized rats wareinhibitedbymorphine and reversedby naloxone (Clarkatal.,1968;ClarkandSmith,1985;Lembeck and Skofltsch,1982).Interestingly,thethresholdintraluminalpressure required to evoke a reflex responsewasincreasedby codeine, butnotbythe quaternary agonist,N- methylmorphine.The effectof codeinewassignificantlydecreased afterbilateral vagotomy(Clark and Smith,1985).Collectively,these resultssuggestthat the opioideffectonthettveshold forthe reflexis centrallymediatedand requiresbtact vagal inneNation.Incontrast, theinhibitory effectofopioids on the cardiovascular responses waslargely independent ofvagalinnervationand appearedto involvea peripheral mechanism(Ctal"k andSmith,1985).

Thereis eblJ'ldant evidence thaibulbospinalnoradrenergicandserotonergic muons modulatetheprocessingofnociceptiveinformationinthe spinalcord(e.g.

22

Zemlanatai,1988,Yakshand Wilson.1979,Yaksh.1965.Proudfit 1988)..

Consistentwith these resu!ts, andtheroleofspinaloz-adrenoceptorsin noradrenergicantinociception,theoz-agonistsdoni dineand ST-91 inhibitedthe cardiovascularand visceromotor responses to c.:olOfectal distension inoose- dependent,yohimbinereversible fashionfollowingl.t.injection(Oanzebrinkand Gebhart,1990;1991b).Theseinhibitory effectswere similarto theiractionsin animal models of cutaneousnocicepl ion (hoi plate, tail flick.pawpressure withdrawa llesl).Singleunitrecordings of ratdorsalhom neurons,activatedby colorectaldistension,were alsoinhibitedbyintra-arterial clonidine in adose- dependent.yohimbine-reversiblemanner(Nessand Gebhart,1989). Unlike clon idineandST-91,1.1.tiZanidine(anotherQ2-adrenoceptoragonisl)faile(4to elevate the visceromotor ttTeshold,despitehavingasignificantinhibitoryeffecton thepressorresponse to coloredal distension(DanzebrinkandGebhart, 1990).

Taanidinealso increasedrestingmean arterialpressure,suggestive of aneffect on cardjoregulatorynetIOI"lSin the spinalcord.oradirecteffect on peripheralvascular smoothmusclefollowingsystemicredistribut ionfromthe spinalsubarachnoidspace.

The abilityof02-agonists10 inhibit bothafferentnociceptiveinputand efferent s~theticoutflowatthe spinalcord levelmake itimportantto distinguish between thesetwo effects when assessing anlinocicepliveactivityon thebasisof pseudoaffectivecardiovascular'iuponses

23

In the same model,Danzebrinkand Gebhart(1991a)showed thatl.t.5-HT11.,

5-HT1B>5-HT2and5-HT,agonistsinhibitedIhe pressorresponseand elevatedthe

threshold pressure required to evoke the visceromotorresponse in a dose- dependent memer,The antooclcepnve effects of5·HT,RU~2496915-melhyoxy-3­

(1,2,3,~etrahydr0-4-pyrindinyl)-1H·indole;a 5-HT1E15-HT1/\agonist], alpha-methyl- 5-HTand001 (5-HT2agonists)were antagonizedby pretreatment with intrathecal methysergide(a 5-HT

is-HT

,c antagonist).Pretreatmentwithintrathecal ketanserin (5-HTI5-HTICantagonist)blockedlheeffects ofMK-212(6-chIOfo-2-(1-piperazinyl)- pyrazine;a5-HT₂agonisl);MDL-72222 (a 5-HT,antagonist)blocked the effects of 2-melhyl·5-HT(8 5-HTJagonist). Whereas spinal 5-HT₂receptors appear to mediate serotonergic antino ciceptionin a cutaneous painmodel (Solomon and Gebhart,1988),spinal 5·HT1,5·HT2and 5-HT3receptors inhibitthevisceromotor and pressor responses evokedby cclcrectat distension in the rat.

Finally,primary afferent C-fibers have long been implicatedin the transmissionof pain (Ganong,1993).Theirselective destruction by the neurotoxin, capsaicin, has made capsaicina useful pharmacological tool for determining the fibertypes mediating noxioussensory transmission (Fitzgerald,1983,Holzer,1988) as well as an antinociceptive and clinicalanalgesic drug (Lynn,1990).Neonatal treatment with capsai cinreducedthe behavioralresponses to chemically induced visceral pain and inhibitedsomata-visceral reflexes in adult rats (Cervero and McRitchie,1982). Using a similar treatment protocol, capsaicin inhibited the

..

cardiovascularresponse evokedby distensionofthe proximaljejunumin pentobarbital-aneslhelizedrats(lembeckand Skofitsch,1982).Neonatalcapsaicin has also been reportedto selectivelyaffect thevisceromotorresponse to colcrectal distension(Gebhartand Ness,1990).Thus,incapsaicin-treatedrats, the threshold colorectatpressureevokingthevisceromotor responsewas doubled,whereasthe cardiovascular responses remainedunchanged.Non-steroidalanti-inflammatory agents,such as indomethacin and dipyron e,thatare effective inrodent writhing tests,hadno significanteffect on the cardiovascularresponses evokedby noxious distensionof the jejunumin urethane-anesthetizedrats(Clarketal.,1988).

W'hllenot a comprehensivereview,data from these representativestudies confirmthe sensitivity of pseudoaffectiveresponses,triggeredby noxiousvisceral stimuli,to kOO'M1 anlinociceptiveagentsatdoses that inhibitcutaneous nociception.

Theyalsoimplicatethe roleof C-fibers invisceralpain that triggersthesereflexes.

Overall,these resultssupport the hypothesis that: a) pseudoattective responses representnccrcepuco-evckecrenexes;and b)that theseresponsescanbe used as indices of visceral ncclcepticn inexperimentalanimals.

Ration ale and Speclf/ cResearchObj ecti ves

Abnormalitiesinthe esophagus are known to cause chestpainin humans thatis difficult to distinguishfromthe pain of myocardialischemia.Atthe present time, thediagnosisofchest painoftenrequiresa completecardiacworkup before

as

Esophagus

Figure 3.The spinal and vagal afferentinnerv ati on of the esophagus.NTS

=

Nucleusof the solitarytract;RVLM

=

rostroventrar lateral medulla

26

an esophageal etiology is identified. Underlying this clinical problem is a lack of infonnationabout the sensorypathwaysthat conveypain fromtheesophagus to the eNS (Fig.3)Thus,spinal and/or vagal afferent fibers from the esophagealmaybe important in esophageal chest pain.

Graded esophageal distension, using a balloon catheter inserted into the lumen of the esophagus, not only evokespain in humans, but has been used to evaluate patients with suspected esophageal chest pain(Barish at al.,1986, Patersonet a/.,1991).The results of these studies showed that patients with esophageal chest pain are not only sensitive to distension,but have a lower threshold for esophageal pain as compared to control subjects.

Esophagealdistension in the rat (MellerandGebhart,1991) has been shown to evoke a pattern of reflex responses, subsumed under the term of

"pseudoaffective" reflexes (Sherrington, 1906), that are suggestive of a painful event. This maybea useful model for investigatingthe mechanisms underlying clinical esophageal pain. In the present study,we used urethaneanesthetized rats to characterize the physiological and pharmacologicalproperties of esophageal distension-evoked cardiovascular (pressor and canffoaoceterator) responses.

The purposes of this research were to: a) determine whether the relevant sensory input evoking these pseudoaffeClive responses is conveyed to the central nervous system (eNS) by vagal andlor spinal afferent fibers; and b) study the effects and the sites of action of antinociceptive agents on this reflex. The specific

27

objectiveswere:

1. To developan esopheqo-cerdiovascular reflex modelin urethane- anesthetizedrats.

2. To determinethe mostsensitiveportionof the esophagusfordistension- evokedcardiovascularresponses.

3. To determinethe relationshipbetween esophagea ldistensionpressu reand evokedcardiovascular responses

4. To determinethe effect ofvago~myon the esophago-cardiovascularreflex, and10compareits effecton the responsesevokedbycutaneous mechanical and chemicalnociceptivestimuli.

5. To determine the effectof neonatal capsaicin on the esophago- cardiovascularreflex and 10 compareilseffect on the respon sesevokedby cutaneousthermal and mechanical nociceptivestimuli.

6. Tocompare lheeffectof morphine,givenl.t.i.v.Dod topicall y10 the surface of the solitarycomp lex,onthe esophag o-card iovascularreflex.

7. To compare the effect of dexmedetomidine ,given i.t.,i.v.and topically tothe surface ofthe solitarycomp lex, on the esophago-cardiovascurarreflex.

8. To determinetheeffect of esophageal paralysis, induced withi.V.N₂-and M- receptor antagonists,on the esophago-cardiovasc ularreflex.

28

MATERIALS AND METHODS

All proceduresin this studywere approvedby the AnimalCare Committee of MemorialUniversity ofNewfoundlandin accordance with theGuidelinesof the CanadianCouncilon Animal Care.

Animals

MaleSprague -Dawley rats(350-450 9 atthe time oftheexperiment),and femalepregnantrats(15·16daysof gestation on arrival)wereobtainedfrom CharlesRiverInc.(St.Constant,Canada).Animalswerehoused in groups of2per cage ata roomtemp erature of22°C witha12-hourlight-dark cycle (lights on at 0800 h).Ratswithintralhecalcatheters,and female rats werehoused individuall y.

Purina^tlrodent laboratorychow and lap waterwere providedadfibitum.

ImplantationofIntrathec al Catheters

Intrathecalcatheterswere constructed from polyethylene{PE-10} tubingand implantedunder halothane (Halocarbon Laboratories,North Augusta,USA) anaesthesia.The catheterwasinserted througha slit inthecisternal membrane terminating al the mid-thoraciclevel(T4·T5)orthe rostralendof thelumbar enlargement (L1-L2)ofthespinal cord.Afixedloopnear therostraltip ofthe catheterwassutured to the overlyingmuscle andthe incisionclosed.Therostral end of the catheter wasexternalized on thetopof thehead andsealed witha

2'

stainless steel plug. Animals with Intrathecal catheters were then housed individually and allowed to recover for a minimum of 5 days.Only those animals without overt signs of neurological impairmentwere used for experimentation. At the end of the experiment, methylene blue was injected through the intrathecal catheterandthe position of the catheter tip was visually oetermlned in the spinal cord. Data from individual animalswere excludedifthe catheter was not in the subarachnoid space with the tipat the appropriate spinal segment.

Capsaicin Treatment

Capsaicin(SigmaChemical Co.,St. Louis,USA) was dissolvedin a mixture of absolute ethanol(Aldrich Chemical, Milwaukee,USA), TWEEN 80 (Willer Fine Chemicals,london,Canada) and 0.9%sallne (2:2:16volume)and injected s.c.on thebad<.of the neck under halothane anaesthesia. Capsaicin was administeredat a dose of 25 mglkg on post-natal day 2,and at 50 mglkg on post-natal days 3, 4,11, 25, 55 and 85.Rat pups were returned to their motbe-Jafter capsaicin or vehicle treatment. At 6 weeks of age, rats were housed in separate cages.

To confirm the neurotoxic effect of capsaicin onC~fibers,behavioral responses to thermal and mechanicalnociceptive stimuli were determined at 3- months of age (before the experiment).Tail flick latency was measured With a tail flickanalgesy meter (Model MK-330, Muromachi Kikai

cc.,

Tokyo, Japan) (D'Amour and Smith, 1941).A cutoff time of 10 seconds was used to avoid tissue damage.

3.

Paw pressure withdrawalwas measured with a Ugo Basile Analgesy-meter (Biological ResearchApparatus,Comerio-Vaese,Italy)(Randall and Selitta,1957).

Pressure was appliedtothe dorsalsurface ofthe non-inflamedhindpawuntil a completewithdrawal or wilhdr9'N81attempt wasobserved.The maximumpressure C:lpplied to the hindpaw was750grams.

Surgical Preparation and PhysiologicalRecording

Under halothaneanaesthesia,cemuta e were surgically placed inthefemoral artery, the externaljugularvein andtracheafor recordingarterialbloodpressureand heartrate(HR),l.v.druginjection,and mechanicalventilation(vagotomized rats), respectively.The incisionwas suturedandcoated with2%lidocaine gel(Astra Pharma,Mississauga.Canada).After surgery,halothane was discontinuedand anaesthesia wasmaintainedwithLv.urethane (0.8g/kg+20mglkg/h).Urethane (Sigma ChemicalCo.)wasdissolved insterilesalineandadministered asa 10%w/v solution.Bodytemperaturewas maintained at 37_38°C throughoutthe experiment usinga thermostatically controlled heatingblanket (Harvard Instruments,51.

Laurent. Canada). Arterial bloodpressure and HRwerecontinuously monitored using aStathampressure transducer coupled to a Gould80005Recorder which was equippedwith an ECG/Biotech amplifier. The EKG was recorded on a polygraph (model 7P1,GrassInstruments,Quincy.USA) with inputleads onthe right and leftarmsand the isolated groundelectrode ontherigh·leg. Respiration

31

was monitoredon the polygraphusinga temperature sensitivetransducer (mod e l TCR-1R,Grass Instruments)insertedin the upper thirdofthe tracheal catheter.

A small balloon made from PE-50 tubing wasfilled withwate rand placedin the lower esophagus(10.5-11.0emfromthe incisors).In someexperiments,similar balloonswereinserted in iheupper andmiddleesophagus(5.0-5.5 emand 8.0

em

fromincisors,respectively).Animalswere allowedto stabilize for 30 minutes before experimentation.Theesophagus wasdistended byinflatingthe balloonwith water usinga hand held syringe.The resulting esophageal distension pressure was calibratedwith a manometer or measuredusinga pressuretransducer coupledto the polygraph.

In thoseexperime ntswhere cervi calvagot omy was perf ormed, ratswere mechanically ven tilated through a tracheal catheterwitha smallanimal respirator (Harvard Instruments).The ventitatlonratewas 100 strokesper minutewith a tidal volumeof 2 ml per 100 grams of body weight (maximumvolumeof8 ml).The cervical vagi were separatedfrom thecarotid arteries and sympatheticnerves,and severedet the level of the larynx.

Noxious Mechanical and Chemical StimufaUon

Two typesofnoxiousstimuliwere used;calibrated paw pinch(Sherman and loomis,1994)and the topical applicationof xylene (Olsen and lund, 1991). To applythe pawpinch, the left paw was gripped with a hemostat at the post axial

aa

border (on the medial surface,distal to the tarsal joint), such that the region of skin coveredby the hemostat was about 20 mm²•The forceof thepinchwas produced by 500gram-weightsattached to the handlesofthehemostat. This resulted in final pressureon eachpawofapproximately0.7kPa,Reflex withdrawalof the hind paw was prevented until the end of the 20 s stimulus period. Noxiousrb emlcal stimulalion was produced by the application of 10 IJI of mixed xylene (Sigma Chemical Co.)on the plantarsurface ofone front paw (before or after unilateral vagotomy) followedbyone hind paw(after bilateral vagotomy).

Drug Administration

All drugs,except capsaicin, were dissolvedin 0.9%salineand doses are expressedas theirsalts. Drugs were injected tv.in a volume:!OO.1 ml followedby a O.1-m1 saline flush.Intrathecaldrugs were injected thn:.ughthe spinalcatheter in a volume of 6 jJl and flushed with 10 jJt of sterile saline. For medullary drug administration,the rat wasplaced in a stereotaxicapparatus andthe dorsal surface of the solitarius comptex and area postrema was exposed. Drugs were applied manuallyin vokmes of 0.02-Q.05jJlfrom a Hamilton microlitersyringe (ljJl) under microscopic control.

Immunohistochemistry

At the endof the experiments,substanceP (SP)- and calcitonin gene related

33

peptide (CGRP)-likeimmun oreacti vity(IR)were determined in the esophagiof vehicle-and capsaicilHreatedanimalsto confirmthe neurotoxiceffect of capsaicin.

Ratswereinjected with an overdoseofi.p.urethaneand the esophaguswasrapidly removed.The esophaguswas flushedwithphosphatebufferedsaline(PBS),tied witha ligatureattherostral end,andfilledwith0.4%parafonnaldehyde (0.8-1.0ml).

Th e caudal end of the esophaguswas closed withanothe r ligatureand fixation allowedtocontinue for30-45 minutes.The esophaguswas cullongiludinallyand thestriatedandsmooth musclelayerswereseparated.

The esophaguswas then:1}washedwith PBS for20 minutes;2)immersed in 50%alcoholfor 5 minutes,70%alcoholfor 5 minutes,100%alcoholfor 10 minutesand xylene for 30-40 seconds;3)immersed in100%alcoholfor 10 minutes, 70%alcoh olfor10 minutes,50%alcoholfor10 minutes,and PBS;4)washed four limeswilh PBS (3Q-40 ml for30min perwash) using a mechanicalshaker (modified fromCosta andFurness,1983).

Wholeesophageal mountswereprocessed for$P-or CGRP-IikeIR employingtheperoxlcase-antiperondase(PAP) method (Stember qer.1979).

Tissues were incubatedin 10%normal goatserum (ICN Biomedicals ,Toronto, Canada) containing0.3%H₂0:and0.4% TritonX-100for1 h,followedbyrabbit antiserumto SP(1:2000 dilution,lnestarccrp., Stillwater,USA ) or CGRP(1:10,000 dilution;Amersham CanadaLtd.,Oakville,Canada)for24 hat roomtemperature and at 4°Cfora further24 h.They were then:wa shed fourtimes with PBS (as

"

describedabove);dried;Incubatedwith1:150 dilutionof goat-anti-rabbil-lgGserum (Boehringer Mannheim Biochemicals,Indianapolis, USA) for 3 h atroom temperature;and storedovernightal 4 "C. AfterwashingwithPBS,thetissueswere incubated with a 1:300dilution of rabblt PAP (Sternberger Monoe/onals Inc., Baltimore,USA)for 3 h at room temperature and at 4"C ovemigi1t,washedagain with PBSand incubatedin a stainingmedium containing3',3'diamino-benzidineHel (0.5 mglml,SigmaChemicalCo.),glucoseoxidase(3.8Ulml,Aspergillus nigertype V, Sigma Chemical Co.) and D-glucose (2 mg/ml,Sigma ChemicalCo.) in 0.1M phosphatebuffer (pH 7.2).

Other Drugs

Methylene blue,scopolaminebromide ,and methscopolamine bromide (SigmaChemical Co"SI. Louis,USA).morphinesulphate(SOHChemicals, Toronto, Canada),and naloxone hydrochloride(Research BiochemicalsInc.,Natick, USA) were purchased from commercial suppliers. Wyelh 27127 (Wyeth Ltd., Philadelphia, USA), de:..nedetomidinehydrochloride(Orion Corporation Ferrnos, Turku, Finland),and pancuronium bromide(Organon Canada ltd.,Toronto, Canada) were generously supplied by theirmanufacturers.

Data Analysis

All cardiovascular data are presented as the maximumchangein mean

3!

arteria lpressu re(.IiMAP)andheartrate(~HR)duringesoph ageal distension relativetotheimmediate pre-stimulus(co ntrol)period.Mean arterialblood pressure (MAP)wascalculated fromthe followingequation:

MAP

=

Diastolicbloodpressure+1/3pulse pressure where pulsepressure is thedifference betwee nsystolic anddiastolic pressure.

Variabilityassociated with single measurements isexpressedasthe stand arderror ofthemean(SEM).Variabilityassociatedwithblocksof dataisindicatedby pooled 95%confidenceinlElfVals.TheED5(land95%confidenceinleNalofmorphineorOX was calculatedfrom their respe ctive dose-responsecurveusingthemethodof Tal1ar id a and Murray (1987). Significantdiffer encesamongmultiple treatment groups weredet erminedusingone -wayanalysisof variance(ANO VA) followedby theNewmea-K eels test. A Student's t-lest wasusedtocom paredifferences betwee ntwogroups.Tw o-tailed . unpairedMann·Whitneyor Wilcoxontests were used whentherewas hetorogeneity ofvaria ncebetw een twogroup s. The relationshipbetweeneso phageal distensionpressureandcardiovascul arresponses wasde terminedbylinearregressionanalysis.

..

RESULTS

Therelationshipbetweenesophageal dIstens ionpf8SSureandurdiovS$cular responses

Gradedesophagealdistension (20secondduration)inuremene-eneethetlzed rats evokeda pressor and tachycard icresponse,hereafterreferred10 as the esophago-cardiovascular reflex,thatwas lime-linked10thestimulus(Fig.4). MAP andHRincreasedlinearly with thelogofthe distensionpressureover the range 25- 150 mmHg(Fig.5).Asindicated bytheSEMand95% CI,themonotonicstimulus- resp onse re lationship s forMAPandHR were highlyreproducible (Fig.5).The preparallonwasalso sensitive toesophageal distension asindicated bythe relativelylow thresholdpressuresyieldingdetectable and quantilalivecardiovascular cha n ge s(Ta b !GI).

TableI.EsophagealDistensionThreshold (mmHgl for.o.MAP and.o.HR DistensionThreshold" 95%ConfidenceInterval AMAP

AHR

10.2 11.4

8.63-12.2 8.73-14.9 ..Es t imatedfrom th emean stimulus -responsecurves (n=7)usingthe linear transf ormatio n:

AMAP= (log distensio npressure)(slope )+logthreshold pressure

Respira tion wasreduced duringesophagealdistension(Fig.6).Respir atory frequency,averagedover 20sec beforeandduring esophageal distension ineach

[500

HR ^{bp m}

350 200

""'2 ^{1& J. [ mmHg}

AP

50

~ n ^COO

EDP ommHg

20 sec

Flgur.... Representa tiveheart rate (HRJ and mean arterialblood pressure (AP)responses10lower esophageal distensionIn urethane-enesthetJzedrats.

Inflationwas applied tothedisla!esophagus,10.5--1 1.0an fromincisors,ata pressureof100mmHg for 20 seconds.EOP

=

esophagealdistensionpressure

31

40

35

30

I

²⁵

e:

"

e ²⁰

. _'"

_c

0 15

u~

10

o Cha ng e in MAP

o

Chan ge inHR

40

35

30

'"

"

25 E ,§.

c, 20 ;]

10

20 30 40 50 60 80 100 200

Esophageal DistensionPress u re (mmHg)

Figure5. The relationship between esophagealdistensionpressure and ev o ked card iovascularresponse s. Themaximumchangeinmean arterial pressure(MAP;0)andheartrate(HR; 0)followinggraded esophagealdistension isst1cM'n.The distensionballoonwas locatedintheloweresophagus,10.5-11.0em fromthe incisors. Eachpointrepr esents themean:t.:SEMof 6-7 urethane- anaesthetizedrats. Theleast squaresregressionline andcorresponding95%

confidenceintervals (dottedlines)are shown.

Jt

before esophageal distension

Innate _{dellate 5 S}

Figure6. Distensionof the lower esophagus reduces the frequency of respirati on.Esophagealdistensionwasapplie datapressure of100mmHg fo r 20 sec.Respir ationwasmostaff ected duringthe first 5 sec of distension.

••

of6 rats,was significantly lowerduringesophag£'laldistension(p<O.OOO1; datanot sho'Nn). Exceptfortheincreaseinheartrate,esophageal distensi ondidnotcause anyEKGwave change oratrio ventricu lar01'sin usnode block (Fi g.7).

Theeffectof balloonposit/ononthe esophago-camio llsscufa,reflex

The positionof the ball o onalon g the lengthoftheesoph agusaffected the magnitu de ofthecardiovascula rresponsesevoked by esophagealdistension.

Usinga fIXedpressure stimulus(100mmHg for205),the cardiovascular responses to tower(10.5-11.0cmfromincisors)and middle (8.0 emfromincisors) esophageal distensionweresignificantlygreat erthanthoseevoked by distens ionofthe upper esophagus(5.5-6.0em from incisors )(Fig. 8).Ther efore,lo wer esophageal distensi on wasusedintheremainderoftheexperiments.While not significant, there wasatrendtowardsincrea sedrefle xresponsesinthelowerascomparedto themid dleesophagus.

The pressorand haalt rale responsesevo k edbyesophage al dis tension depend on theIntegrity ofvag alInnerva tion

AsshowninFig. 9, un ilateralvagotomyatlenuatedthecardiovascular responses 10esophagealdistension byabout 50%of control.Bilateral cervical vagotomycompletelyabolishedthis reflex.Incontrast,vagotomyhad no significan t effectonthechangeinMAPevokedbycutaneous mechanicalorchemical noxious

..

before esophage al distension

0.5 S during eso phag e al distensio n

FigUf81. Esophagealdistensionhad no detectableeffect on the EKG except for an Increase In heartrate.

12

35 35

30

o

^{Change in HR} ₃₀

~Cha n geinMAP

E

^{25 25}

en

I

o,

E

fe- ^E

~

^{20 20~[L}<!

L

"

.

0'^{C 15 15}

.

^c0'

2

^c

u _{10 10} ⁰

6

lO,S-II-O em a.Oem 5.S-6.0 e m

(rom ln ebors frominc isor, CromInci sors

Figure8. The position of the ballooninthe esophagus affects the distension-evokedchange In mean arterialpressure(il.MAP) and heart rate (a HR). Theballoonswereinserted intheesophagus 5.5-6.0an,6.0em,and 10.5- 11.0emfromthe incisors.Esophagealdistensionwas appliedat a pressureof 100 mmHgfor 20 seconds.Eachpointrepresentsthemean ± SEMof 6-8 rate. The change in MAP and HRevokedbylower and middleesophageal distensionis significantly greaterthan that evokedby distension in the upper esophagus (" P<O.05:r iP<0.01).

45 40

35 (j; ₃₀ I

£ E

²⁵

D-

<t 20

'"

^c

"

¹⁵

'"

c

0 10

.c U

"

o

Con t ro l

• Unil a te ralvagotom y 'VBilater a lVa go t om y

Esophag ea lDistensi onPressure (mmHg)

Figure 9A. The change Inmean arterialpressure (.6.MAP)evoked by esophageal distension dep ends onthe Integrityof vagal Innervation.The maximum changein meanarterialpressure(MAP) fol101Ning esophageal distension wasrecorded ineachanimalbef0l'9(opencircles), afterunilateral(solidcircles)and after bilateralvagotomy (triangles).Each point represents the mean:tSEMof 7 rats.least squares regressionlineand 95%confide~intervals(dottedlines)are shown. Infl ation was applied to the distal esophagus,10.5 to 11 em from the incisors.

..

45

40 a Con lrol

• Unilateralvagotomy

35 ^'VBilate r al vagot omy

E

_a. ³⁰

~ 25

cc

I

c 20

e

'"

¹⁵

c .c0

u 10

EsophagealDist ension Pressure (mmHg)

Figure99.Thechange Inheart rate(.o.HR)evoke dbyesop ha geal distension depends on theIntegri ty of vagalInnervation.The maximum changeinheart rale(HR) following esophagealdistension was recordedin each animalbefore (opencircles),after unilateral (solidcircles) andafter bilateral vagotomy(triangles).

Eachpointrepresentsthemean±SEMof 7rats. Least squaresregressionline and95%confidenceintervals (dotted' lines)areshown.Inflation was appliedtothe distal esophagus,10.5to11emfromtheincisors.