1、Vol.36,No.2ANTIMICROBIALAGENTSANDCHEMOTHERAPY,Feb.1992,p.473-476 0066-4804/92/020473-04$02.O/O Copyright1992,AmericanSocietyforMicrobiology UtilizationofPulseOximetryfortheStudyoftheInhibitoryEffects ofAntiviralAgentsonInfluenzaVirusinMice ROBERTW.SIDWELL,*JOHNH.HUFFMAN,JOHNGILBERT,BRETMOSCON,GORDON

2、PEDERSEN, ROGERBURGER,ANDREEDP.WARREN InstituteforAntiviralResearch,UtahStateUniversity,Logan,Utah84322-5600 Received9July1991/Accepted22November1991 Pulmonarydiseaseinmiceinducedbyinfluenzaviruswasmonitoredbymeasurementofoxygensaturation (SaO2)inbloodwithapulseoximeter.TheSaO2declinedininversepropo

3、rtiontotheviralinoculum.The knownantiviralagentribavirininhibitedtheSaO2decline,preventeddeath,loweredlungconsolidation,and reducedthelevelofrecoverablevirus.Pulseoximetryisaneffectivemeansofmonitoringmurineinfluenzal diseaseandcanbeusedinthestudyofpotentialantiviraldrugs. Ithasbecomestandardpractic

4、einhospitalsthatcarefor patientsinpotentialrespiratorydistresstocontinuously monitortheirrespiratoryconditionbyattachingapulse oximeterprobetoafinger,toe,foot,orearlobe.Theoxygen saturation(SaO2)ofthearterialhemoglobinismeasuredand isbasedonthepulsatileabsorbanceoflight(1). Therehasexistedaneedforim

5、provedmeansofmonitor- ingtherespiratoryconditionofmiceinfectedwithagents suchasinfluenzaviruswithouthavingtosacrificetheanimal atvariousstagesofthedisease.Inhibitionofrales(5)and weightloss(4,7)havebeenusedinfrequentlyinattemptsto satisfythisneed.Inthisreportwedescribetheuseofpulse oximetryforthestu

6、dyofmurineinfluenzaldiseaseandthe applicationofthismethodologytosupplementthecom- monlyusedparametersofdeath,lungconsolidation,and virustitersinlungstoevaluatetheinhibitoryeffectsofan antiviralcompound,ribavirin(1-P-D-ribofuranosyl-1,2,4-tri- azole-3-carboxamide)(6,9),oninfluenzavirusdisease. Younga

7、dultmaleandfemaleBALB/cmiceandC57BL/6 mice(SimonsenLaboratories,Gilroy,Calif.),whichwere maintainedondrinkingwatercontaining0.006%oxytetracy- cline(Pfizer,NewYork,N.Y.)tocontrolpossiblesecondary bacterialinfections,wereusedinthisstudy.Themicewere lightlyanesthetizedwithether,andindesignatedstudies t

8、heywereinfectedintranasally(i.n.)withoneofthefollow- ingviruses:influenzaA/NWS/33(HlNl),obtainedfrom K.W.Cochran(UniversityofMichigan,AnnArbor);influ- enzaA/Japan/305/57(H2N2),providedbyF.M.Schabel,Jr. (SouthernResearchInstitute,Birmingham,Ala.);andinflu- enzaA/PortChalmers/1/73(H3N2)andB/HongKong/5

9、/72, bothobtainedfromtheAmericanTypeCultureCollection (Rockville,Md.).Allviruseswerepassagedmultipletimes throughspecific-pathogen-freemicetodeveloplethalityfor theseanimals.Eachwaspassagedseparatelywithnoother virusesintheareatoavoidrecombination.Viruspoolswere preparedandtitratedinconfluentmonolay

10、ersofMadin- Darbycaninekidney(MDCK)cells. TheviralinfectionwasmonitoredbyusingaBiox3740 pulseoximeter(Ohmeda,Louisville,Ohio).Boththefinger andearprobesaccompanyingtheinstrumentwereusedin separatestudies.Thenumeric-dominantinstrumentdisplay wasusedwiththeinstrumentintheslowmode.Initial experimentses

11、tablishedthattheSaO2valuesdetermined * Correspondingauthor. withapulseoximeterwereessentiallyduplicatedbydirect measurementsofSaO2inheparinizedbloodperformedwith aOSM-3hemoximeterandanABL-2bloodgasanalyzer (Radiometer,Inc.,Copenhagen,Denmark). Comparisonofmethodsfordeterminingpulseoximeter readingsi

12、nmice.Thefollowingtwomethodswerefoundtobe usefulinobtainingSaO2readingsbyusingthepulseoximeter: (i)insertingtheentiremouse,tailfirst,intothefingerprobein amannerthatthelight-emittingdiodesandthephotodiode wereoppositeeachotheracrossthemidsectionoftheanimal, and(ii)placingtheearprobeontheinnerthighmu

13、scleofthe animalsothatthelight-emittingdiodesandthephotodiode wereonoppositesidesofthethighmusclemass.Thefinger probemethodyieldedsomewhatlower(2to4%)readings; however,thehealthyanimalstendedtobecomeoverexcited whentheywereplacedinthefingerprobe,andmorestrenu- ousmeasureswererequiredtoholdtheminthep

14、robefora sufficienttimetoobtainanaccurateSaO2measurement. Intraperitonealinjectionof a1:80dilutioninsterilesalineof 94mgofAvertin(1.5goftribromethanolin1.0mlofisoamyl alcohol;AldrichChemicalCo.,Milwaukee,Wis.)perkgof bodyweightadministered5minpriortoSaO2determination successfullyeliminatedtheanimals

15、struggleswithoutaf- fectingtheSaO2valuesthatweredetermined. Suchtranquilizationwasnotnecessarywhenthemeasure- mentsweretakenbyusingtheearprobeonthethigh.Itwas found,however,thatcarehadtobetakeninplacingtheear probeapproximatelyanequaldistancebetweenthehipandthe knee.Determinationstakennearthehipofte

16、nvariedby+10%, apparentlybecauseofobstructionbythepelvisboneofthe lighttransmissionthroughthevascularbed.Whentheprobe wasmovedtooneartheknee,thevascularbedwasapparently toosmall,andexternallightoccasionallyinterferedwiththe determinations,againresultinginsignificantvariations. Todeterminewhetherhold

17、ingtheanimalstootightlymay tendtoconstrictthechestandhinderappropriaterespiration, fivemicewereheldbythescruffoftheneckverytightly,with SaO2measurementstakenonthethighfiveseparatetimesfor eachanimal.Theywerethenheldverylooselyandthe measurementswererepeated.ThemeanpercentSaO2forthe animalsheldtightl

18、ywas86.3+1.0;foranimalsheldloosely itwas86.5+1.1.Thesedataindicatethatthechestwasnot beingconstrictedsufficientlytoaffectthereadings. LiteratureaccompanyingtheOhmedainstrumentusedin theseexperimentsrecommendedmassagingtheskinregion onwhichtheprobeistobeattachedwithanisopropylalcohol 473 ANTIMICROB.A

19、GENTSCHEMOTHER. uXU tV_- _U-DI 8-I 60-*S/T:0/10;MST:3.9days *S/T:0/10;MST:7.2days AS/T:0/10;MST:6.8days 50 A S/T:1/10,MST:7.1davs 01234567 123456 DaysPost-VirusInoculation 7v 80- 60-*S/T:0/10;MST:4.3days *S/T:0/10;MST:4.9days AS/T:6/10;MST:5.8days BS/T:10/10;MST:21daVS so .I . 123456 0123456 DaysPos

20、t-VirusInoculation FIG.1.EffectofpulmonaryinfluenzavirusinfectionsonmurinearterialSaO2determinedbypulseoximetry.Thetitersofeachundiluted virus,expressedas50%tissuecultureinfectiousdosespermilliliter,wereasfollows:influenzavirustypeA(HlNl),107-5(A);influenzavirus typeA(H2N2),108.5(B);influenzavirusty

21、peA(H3N2),107-7(C);influenzavirustypeB,108-5(D).*,10-1virusdilution;0,10i-virus dilution;A,10-2virusdilution;*,10-2.5virusdilution;O,normalcontrols.S/T,Numberofsurvivorsbyday21/totalnumberofmicetested. MST,meansurvivaltimeofmicedyingonorbeforeday21. (70%)padorwitharubefacientcreamfor20to30stocause l

22、ocalvasodilationand,hence,toincreaseperfusion.Several experimentswithmiceinwhichtheskinwasinitiallydamp- enedwithisopropylalcoholindicatedanincreaseofapprox- imately4%inSaO2readingscomparedwiththereadingsin thesameanimalsnotexposedtoalcohol.Wefeltthatrelative readingsweremostimportantinantiviralexpe

23、riments,sothe alcoholpretreatmentwasnotdoneinthesestudies. SaO2determinationsininfluenzavirus-infectedmice.Young adultBALB/cmicewereinfectedi.n.withvarious0.5log1o dilutionsofeachinfluenzavirus.Thearterialoxygensatura- tionwasdeterminedintheseanimalsdailyfor7to10daysby usingtheearprobeplacedonthethi

24、gh;deathswerealso recordeddaily.Topreventskewingofdata,micethatdiedof obviousrespiratorydistresswerearbitrarilyassignedanSaO2 valueof65%untilallanimalsdied,atwhichtimenofurther valueswererecorded.AsshowninFig.1,thethreeinfluenza Avirusesathigherinoculawerelethaltothemice.In contrast,only40%oftheinfl

25、uenzavirustypeB-exposedmice died,andthesedeathsoccurredsomewhatlate(meansurvival time,6to7days).Inmicewithlethalinfections,theSaO2 valuesdeclinedinadose-responsefashion.Inthecaseofthe influenzavirustypeBinfection,inwhichtheviralinoculum didnotcausedeathinthemajorityoftheanimals,theSaO2 12345 DaysPos

26、t-VirusInoculation 67 FIG.2.ComparisonofoccurrenceofdeathandSaO2declinein influenza(H3N2)virus-infectedBALB/cmice.A,occurrenceof death;0,SaO2.Thebarsindicatestandarderrors. 474NOTES VOL.36,1992 01234 5 DaysPost-VirusInoculation 6 FIG.3.Effectofintraperitonealribavirintherapyoninfluenza virustypeA(Hl

27、Nl)infectionsinBALB/cmice.*,normalcon- trols;A,infected,saline-treatedmice;0,infected,ribavirin-treated mice.(A)ArterialSaO2;(B)lungconsolidation;(C)lungvirustiter. Thebarsindicatestandarderrors. declinewaslesspronounced.AsshowninFig.2,themajority ofthedeathsoccurredwhentheSaO2valuesdroppedbelow 75%

28、.Thelinearcorrelationcoefficient(r)forthesedatawas 0.908,indicatingastrongcorrelationbetweenSaO2anddeath intheanimals. Anti-influenzaviruseffectsofribavirin.BALB/cmicewere infectedi.n.withanapproximately90%lethaldoseof influenzavirustypeA(HlNl)(10550%cellcultureinfec- tiousdosesperml).Ribavirin(ICNP

29、harmaceuticals,Inc., CostaMesa,Calif.),atadoseof75mg/kg/day(n=30),or saline(n=40)wasadministeredintraperitoneallytwice dailyfor5daysbeginning4haftervirusexposure.Ten infected,ribavirin-treatedmiceand18virus-infectedplace- bo-treatedcontrolanimalswereobserveddailyfor21 days fordeath;pulseoximeterread

30、ingsweredeterminedonthese micedailyfor7daysbyusingtheearprobe,asdescribed above.Frompoolsoftheremaininginfected,treatedmice, fivemicewererandomlyselectedandkilledondays3,5,and 7aftervirusexposure.Visualevidenceoflungconsolidation wasscoredonablindbasis,with0beingnormaland4being indicativeof100%conso

31、lidation.Thelungswerethen homogenizedandassayedforvirustiterinMDCKcellsby testingvariouslogarithmicdilutionsintriplicate,asde- scribedpreviously(8).AsshowninFig. 3,theribavirin treatmentpreventeddeath,reducedthedepressionofSaO2, inhibitedlungconsolidation,andtoalesserextent,reduced virustitersinthel

32、ungs.Visiblelungconsolidationhada moderatecorrelation(r=0.792)withSaO2values;theSaO2 valuesdeclinedmorerapidlythanconsolidationincreased. Thisdifferencebetweenthetimeofloweringof SaO2 valuesanddevelopmentofvisiblelungconsolidation sug- geststhatthepulmonarytissueswereexperiencingsufficient damagetol

33、owertheirgastransferencecapacitypriortoa significantchangeinlungcolor.Lungconsolidationin influenzavirusinfectionsinanimalsisaresultofacombi- nationofevents.Thevirusinfectiondamagesthealveolar cellsandcausesnecrosisofthecapillarywalls,leadingto hemorrhageinthelungs.Thealveolarexudate usually consist

34、sofneutrophilsandmononuclearcells(2).Thein- creasingcolorseeninthelungslaterintheinfectionis probablyamanifestationofvascularphenomenaresulting fromalaterimmuneresponsetotheinfection(3).Thedata fromtheantiviralexperiment,whichshowedanearlier indicationofseriouslungdamagebySaO2 measurement, illustrat

35、eanadditionalvalueoftheuseofthepulseoximeter forstudyingmurineinfluenzavirusinfections. Thesedataindicateadefiniteapplicationforpulseoxim- etryinlaboratoryanimals,thatis,asameansofmonitoring moderatetosevererespiratorydisease.Themethodappears tobeofparticularvalueasanadditionalparameterfor studyingd

36、rugsthatarepotentiallyinhibitorytoinfluenza virus. ThisstudywassupportedbycontractN01-AI-15097,Antiviral ResearchBranch,DivisionofMicrobiologyandInfectiousDis- eases,NationalInstituteofAllergyandInfectiousDiseases,Na- tionalInstitutesofHealth. REFERENCES 1.Bowes,W.A.,III,B.C.Corke,andJ.Hulka.1989.Pulse oximetry:areviewofthetheory,accuracy,andclinicalapplica- tions.Obstet.Gynecol.74:541-546. 2.Douglas,R.G.1975.Influenzainman,p.315-320.InE.D. Kilbourne(ed.),Theinfluenzavirusesandinfluenza.Academic Press,Inc.,NewYork. 3.Hers,J.F.P.,J.Mul