Braz. j. . vol.80 número6

Brazilian

Journal

of

OTORHINOLARYNGOLOGY

www.bjorl.org

ORIGINAL

ARTICLE

Evaluation

of

the

ability

of

an

experimental

model

to

induce

bacterial

rhinosinusitis

in

rabbits

夽

,

夽

Eduardo

Landini

Lutaif

Dolci

,

Carlos

Augusto

Correia

de

Campos

,

Leonardo

da

Silva

_,

_Ricardo

_Landini

_Lutaif

_Dolci

,

José

Eduardo

Lutaif

Dolci

a_{FaculdadedeCiênciasMédicas,SantaCasadeSãoPaulo(FCMSCSP),SãoPaulo,SP,Brazil} b_{DepartmentofOtorhinolaryngology,SantaCasadeSãoPaulo,SãoPaulo,SP,Brazil}

Received14March2013;accepted22July2014 Availableonline16September2014

KEYWORDS

Sinusitis; Animalmodels; Rabbits

Abstract

Introduction:For decades, animals havebeen used in sinonasal experimentalmodels, and thepracticehasincreasedsubstantiallyinthelastfewyears.Thisstudyaimedtoassessthe pathogenesisofinfectiousprocessandmedicationefficiencytotreatrhinosinusitis.

Objective:Toevaluatetheefficiencyoftheproposedexperimentalmodeltoinduceanacute bacterialsinonasalinfectiousprocessthroughhistologicalanalysisandsinussecretioncultures. Methods:Thiswasanexperimentalstudywith22NewZealandrabbits,dividedinto:groupA (sixrabbits),groupB(sevenrabbits),groupC(sevenrabbits),andgroupD(controlgroupwith tworabbits).Rhinosinusitiswasinducedbytheinsertionofasyntheticspongeintotheright nasalcavityof20animals(studygroups),followedbytheinstillationofbacterialstrains(50% Staphylococcussp. and50%Streptococcus sp.).The groupswere euthanizedwithin10 days (groupA),17days(groupB),and30days(groupsCandD).

Results:Alltherabbitsofthestudygroupdevelopedacutebacterialrhinosinusitis,whichwas diagnosedthroughmacroscopicevaluation,histologicalanalysis,andsinussecretionculture. Conclusion:The proposedmodelistechnically simpletoperform,itissimilar tothe rhino-genicmodelinhumanbeings,anditishighlyefficienttoreproduceanacutebacterialsinus infection.

© 2014Associac¸ãoBrasileira de Otorrinolaringologiae CirurgiaCérvico-Facial. Publishedby ElsevierEditoraLtda.Allrightsreserved.

夽 _{Pleasecitethisarticleas:DolciEL,deCamposCA,daSilvaL,DolciRL,DolciJE.Evaluationoftheabilityofanexperimentalmodelto}

inducebacterialrhinosinusitisinrabbits.BrazJOtorhinolaryngol.2014;80:480---9.

夽 _{Institution:IrmandadedaSantaCasadeSãoPaulo,SãoPaulo,SP,Brazil.}

∗_{Correspondingauthor.}

E-mail:[email protected](E.L.L.Dolci).

http://dx.doi.org/10.1016/j.bjorl.2014.09.001

PALAVRASCHAVE

Sinusite;

Modelosanimais; Coelhos

Avaliac¸ãodacapacidadedeummodeloexperimentalparainduc¸ãoderinossinusite bacterianaemcoelhos

Resumo

Introduc¸ão: Arealizac¸ãodemodelosexperimentaisnasossinusaisemanimaisvemsendo real-izadahádécadas,comsubstancialaumentonosúltimosanos.Temcomoobjetivosidentificar asalterac¸õesfisiopatológicasocasionadaspeloprocessoinfecciososinusaleavaliaraeficácia demedicamentosnotratamentodarinossinusite.

Objetivo: Avaliaraeficáciadomodeloexperimentalpropostoparaainduc¸ãodeumprocesso infecciosonasossinusalagudobacteriano,utilizandoparâmetroshistopatológicoseculturada secrec¸ãosinusal.

Método: Estudoexperimentalcom22coelhosdarac¸aNovaZelândia,divididosem:grupoA(6 coelhos),grupoB(7coelhos),grupoC(7coelhos)egrupoD(controlecom2coelhos).Induzido quadroderinossinusiteatravésdainserc¸ãodeesponjasintéticanasfossasnasaisdireitados20 coelhos(gruposdeestudo),seguidoporinstilac¸ãodetoxoidebacteriano(50%estreptocócico, 50%estafilocócico).Osgruposforamsacrificadoscom10dias(grupoA),17dias(grupoB)e30 dias(gruposCeD).

Resultados: Todososcoelhosdogrupodeestudoapresentaramquadroderinossinusiteaguda bacteriana,atravésdaidentificac¸ãomacroscópica,análisehistológicaeculturadassecrec¸ões. Conclusão:Omodelopropostoapresentasimplicidadetécnicaparasuaexecuc¸ão,similaridade aoquadrorinogênicoqueacometeoshumanoseéaltamenteeficaznaproduc¸ãodeumquadro infecciosobacterianoagudosinusal.

©2014Associac¸ãoBrasileiradeOtorrinolaringologiaeCirurgiaCérvico-Facial.Publicado por ElsevierEditoraLtda.Todososdireitosreservados.

Introduction

Rhinosinusitis is currently one of the most prevalent dis-eases; it is the fifth most common disease that requires antibiotics.1_{Approximately25millionpeoplearediagnosed}

withsinusitiseachyearintheUnitedStates,makingitone of the main diseases that require medical attention with otorhinolaryngologistsandgeneralpractitioners.Thedirect and indirect costs associated withrhinosinusitis arehigh, andincludediagnosticmethods,therapy,procedures, med-ications,anddecreasedproductivity.2

Theprevalenceofacuterhinosinusitis(ARS)andchronic rhinosinusitis(CRS)inthegeneralpopulationisquitehigh, but it is difficult to accurately estimate this prevalence, primarily because many of the episodes are self-limited andmildlysymptomatic,anddonotcompelthepatientto seekhealthcare.Rhinosinusitisisresponsiblefor 9%ofall antibioticsprescribedtothepediatricpopulationand21%of thetotalprescribedfor theadultpopulationintheUnited States,costingapproximatelyUS$5.8billion;US$150 mil-lionarespentonantibioticsalone.3,4

Several attemptstomodel thedisease inanimalshave been made; studies with rabbits are the most frequent. Theseanimalshavesinonasalanatomyandphysiologyvery similartohumans.Theyarewellsuitedforstudiesinvolving surgical procedures,but theyexperiencea high mortality wheninprolongedstress.Otheranimalsusedforresearch aretheWistarand Sprague---Dawleyrats,guineapigs, and sheep.5---7

Experimentalmodelsofrhinosinusitisdiscussedinthe lit-eratureaimtoinduceinflammationintheparanasalsinuses

similartothatexperiencedbyhumans.Experimentalmodels havebeenusedtostudythephysiopathogenesisof inflam-mationandtoevaluatetreatmentoutcomes.8---10

Studies have utilized obliteration of nasal passage maneuvers,sinusdrainageostiumobstruction,and instilla-tionofinflammatoryprocessmediatorsandevenmaterials thatactasaculturemediuminthenasalpassages.11---14

Theliteraturesearchretrievednodetailedexperimental modelofacutebacterialrhinosinusitisthatincludeda thor-oughhistologicalanalysisofbothmaxillarysinuses(induced sideandcontralateralside)andamicrobiologicalanalyses ofbothmaxillarysinusesinthepresenceofabacterial infec-tion.

Nostudiesthathadanalyzed thenasalpackingwhenit wasusedasthemethodfor bacterialrhinosinusitis induc-tionwere retrieved. Suchan analysiscould correlate the microbiological findings of the nasal cavity with those of themaxillarysinus.Thefewstudiesthathaveassessedthis correlationdidnotfindsignificantresults.15

It was also observed that many studies aimed to pro-ducean infectious picture or toevaluate the therapeutic efficacy of drugs. Therefore, few studies have evaluated thehistologicalandmicrobiologicalalterationsintheperiod followingthediagnosisofacutesinusinfectionprocess with-outtheuseofany medication,afterremovalofthenasal packing,i.e.,theydidnotanalyzetherecoveryperiod.

Table1 Descriptionofthegroupsofanimals,numberof rabbitsandeuthanizationdaysandremovalofnasalpacking.

Group Rabbits(n) Euthanization (day)

Nasalpacking removal(day)

A 6 10 10

B 7 17 10

C 7 30 10

D 2 30

theassociation betweenthebacteria foundin thesinuses andinthenasalpassages.

Therearestillsomedoubtsintheliteratureregardingthe physiopathologyandtreatmentofacuterhinosinusitis,such astheexpectedperiodafterdiagnosis tostartthe antibi-otictherapy,theevaluationofpatientswithrecurrenceright aftertheendoftreatment,andwhysomepatientsdevelop recurrentand/orchronicconditionsafteranacute rhinosi-nusitisepisode.

Methods

The studywas approvedby theEthics Committeeon Ani-malExperimentation(EAEC)andwasperformedinaprivate institute,underapprovalnumber008-12.Theanimalswere kept in individual cages, appropriate for the breed and weight.Theywereofferedfreefoodandwaterthroughout theperiodtheywereconfined.Allanimalswerekeptunder standardconditionsforaperiodofeightdayspriortostudy initiation. All surgical and experimental procedures were performedinthesameinstitute,inaccordancewiththe eth-icalprinciplesinanimalexperimentation,postulatedbythe Braziliancodeforanimalexperimentation(CódigoBrasileiro deExperimentac¸ãoemAnimais[COBEA]).Preparationsand histologicalanalyseswereperformedinaprivatelaboratory. Microbiologicalstudieswereconductedinthemicrobiology laboratoryofatertiaryhospital.

Atotalof 22NewZealandwhiteadultrabbits,of both sexes,weighingbetween2500and3000gwereusedatthe beginningoftheexperiment.Therabbitsweredividedinto fourgroups:groupA(sixrabbits),groupB(sevenrabbits), groupC(sevenrabbits),andgroupDascontrol(tworabbits). Therabbitsin groupAwereeuthanizedonthe dayofthe nasalpackingremoval,10daysaftertheexperiment induc-tion.TherabbitsingroupBwereeuthanized17daysafter induction,and therabbits ingroup Cwere euthanized30 daysafter induction. The tworabbits fromgroup D were keptin aseparate environment,in adifferentroom from theanimalsinthestudygroupsfor30days,andeuthanized togetherwiththegroupCrabbits(Table1).Thenasal pack-ingandbacterialtoxoidwerenotintroducedintothesetwo animals.

An experiment was performed in order to obtain a sinonasalinflammatoryprocessthroughthenasalcavityof theanimals, simulating an acuteinfectious rhinosinusitis. Therabbitsweresubmittedtogeneralanesthesiaanda ster-ileMerocel® _{nasalpackmeasuring0.3cm}

×0.5cm×2.5cm wasintroducedintotherightnasalcavityusingsterile bayo-netforceps,followedbyinstillationof1mLofstreptococcal andstaphylococcal toxoid (Toxoidepot®_{) in the ipsilateral}

nostrilusingasterilesyringeandinsulinneedle.Thetoxoid instillationwasperformed inthenasal cavityafter place-mentofthenasalpacking,inordertostandardizethetoxoid amountinallanimals.Thepackingwasremovedonthe10th dayafterthestartoftheexperimentfromallanimalsinthe threestudygroups.

Openingandexposureof theanteriorwallof the max-illary sinuses were performed bilaterally in each animal, initially on the leftside to prevent contamination of the contralateralsidetothatoftheinductionexperiment,using swabstocollectsecretionfromthem(Cuturet®_).The

mate-rialswere keptat roomtemperature without exposureto sunlightandsenttothelaboratory24---36haftercollection. Allsecretionsampleswerepreparedonslidesandstained by the Gram technique for bacterioscopic analysis. Thus, the slides were stained with methyl violet, fixed with Lugol’s solution, discolored withethyl alcohol, and again stainedwithsafranin.Slidereadingwasperformedbylight microscopywithoilimmersionobjective(1000×).

After the bacterioscopic analysis, the materials were seeded inbloodagar, chocolateagar,andSabouraudagar. Platesofbloodagarandchocolateagarwereincubatedat 35±2◦_{C.Daily readings of theplateswere performed up}

to48hforbloodagarandchocolateagarmedia,andup15 daysforSabouraudagar.

Immediately after euthanization, the structures lining thefaceoftherabbitsweredissected,andtheanteriorwall ofthemaxillarysinuswasopened,aswellastheouterwall ofthenasalcavities.Then,theinnermucosaofthemaxillary sinuswasremoved(Fig.1).

Mucosal samples wereobtained fromthe rabbits after removaloftheinnermucosaofthemaxillarysinuses,and immediately fixed in 10% buffered formalin; the samples remained in this solution for a minimum of 24h for ade-quatefixation. Theyremained24---48hunder theauthors’ responsibility,andwerethensenttothepathology labora-tory.Subsequently,thecleavageofsampleswasperformed and the histological processing itself was started (dehy-drationin successivebathswithincreasingconcentrations of ethyl alcohol, diaphanization in xylene; samples were then paraffin-embedded at 60◦_{C). Then, the microtomy}

wasperformed,witheach histologicalslice havinga max-imum thickness of 4␮m. Finally, the slides were stained

withhematoxylin-eosin(HE)andmountedoncoverslipsfor microscopicexaminationbythepathologist.

Allslideswereanalyzedbyasinglepathologist,whowas unaware of thegroup towhich the specimenbelonged.A semi-quantitativeassessmentofthealterationsfound dur-ingtheanalyseswasperformed.

Subsequently, the samples were classified using the adoptedcriteriaforinflammationaccordingtoMarks16,17_:0,

Figure1 Outerwalloftheopensinonasalcavities,exposing themaxillarysinusesandnasalcavity.Evidentinfectious pro-cessontheright,characterizedbymucosaledemaandpurulent discharge.Anatomicalsitesrepresentedbythemaxillarysinus (M),nasalseptum(S),inferiorturbinate(T),andnasal cavity (N).

wasevaluatedaccordingtoitsintensity,classifiedasabsent, present,orsevere(Figs.2and3).

The nasal packing removed from the rabbits’ nasal passages wasanalyzed for microorganism culture through

Figure2 Sinusmucosaontherightsideofthestudygroup euthanized after 10 days,with moderateinflammation char-acterized by diffuse inflammatory infiltrate in the lamina propria,withoutformationofinflammatoryaggregates(grade 2),delimitedbyarrows---opticalmicroscope,HEstaining,100× magnification.

Figure3 Sinusmucosa ontherightsideofthestudygroup euthanizedafter17days,withsevereinflammation(grade3), demonstratingtheepitheliumpermeatedbyneutrophils(filled arrows)andfibroblasts(hollowarrows)inthesuperficialcorium. Opticalmicroscope,HEstaining,200×magnification.

Hemobac® _{in the same microbiology laboratory. The}

HemobacTriphasic® _{System is a product used in cultures}

of blood and blood components, stem cells, body fluids, andparenteralnutrition;inthepresentstudy,thePediatric HemobacTriphasic®_{systemwasused.Thesystemconsistsof}

twoelements:aplasticcontainercontaining30mLofbroth supplemented withyeast extract and sodiumpolyanethol sulfonate(SPS)and adipslide(Fig.4).The latterhas two faces:a broad face,consisting ofchocolate agarand CO2

indicatortodetect bacterial and/orfungal growth, anda dividedface,consistingofSabouraudagarandMacConkey agar.

Figure5 Systemaftertheendoftheincubationperiod.Nasal packing(T)inside the containerofsupplemented broth, CO2

indicator(i) withastrong pink colordueto thepresence of micro-organismsandchocolateagarmedium(H)withthe pres-enceofbacterialcolonies.

Thenasalpackingwasplacedinside thecontainer with thebroth,soonafter itsremovalfromthenasal cavityof therabbit,followedbyocclusionofthecontainer.Foreach nasalpackingremoved, onePediatricHemobac Triphasic®

systemwasused.Thiswasgentlyshakenforhomogenization ofthesamplewiththebroth.Thematerialwaskeptfor72h atroomtemperaturewithnosunlightexposureor manipu-lationofthematerial.Thesystemwasrepositionedatthe originalpositionsothatalltheliquidmediumwouldreturn tothe bottom of the container. All materials were again incubatedat 35±2◦_{C andobserved twicedailyforcolony}

identificationand/orchangeintheCO2indicator(Fig.5).

The incubation period was seven days and then the separationof thetwocontainers andtheidentification of colonies present on solid medium were performed. The chocolateagar and MacConkey agar mediawere used for bacterialidentification.Thematerialswerekeptincubated foranothereightdays,totaling15daysatroom tempera-ture,fortheinvestigationoffungionSabouraudagar.

Statistical analysiswasperformed toassess thedegree of mucosal inflammation, connective-fibrous proliferation andthepresence or absenceofbacteria accordingtothe

dayofanimals’euthanization.Allgroupsofbacteriafound inthesampleonbothsidesweredescribedusingabsolute and relative frequencies. The degrees of mucosal inflam-mationandconnective-fibrousproliferationwerecompared betweenthedaysofeuthanizationusingtheKruskal---Wallis test,18_{followedbyDunn’snonparametricmultiple}

compar-isons test19 _{tocomparedaystwobytwo, whennecessary.}

Forthepresenceorabsenceofbacteria,theassociationwas verifiedusingthelikelihoodratiotest.18

The concordance between the swab and culture per-formed in thebuffer wasdescribedaccording tothedays ofeuthanizationandtheexistenceofanassociationofthe concordance with the days of euthanization was verified using the likelihood ratiotest. The tests wereperformed withasignificancelevelof5%(p<0.05).

Results

Atotalof22rabbitswereusedinthestudy,with20rabbitsin thestudygroupandtworabbitsinthecontrolgroup.Noneof theanimalsdiedduringthestudyperiod.Afternasal pack-ingremoval,onthe10thdayoftheexperimentinduction, allrabbitshadunilateralpurulentrhinorrhea.Thus,allthe rabbits euthanized in group A had purulent rhinorrhea at thatmoment.FewrabbitsfromgroupBshowedevident rhi-norrheabeforeeuthanization.NorabbitfromgroupChad evidentrhinorrheainthenasalcavityatthetimeof euth-anization. However,many rabbits fromgroup Band some rabbitsfromgroup Chadpurulent secretionin the maxil-larysinusaftereuthanizationoftheanimalsandexposure oftheseanatomicalstructures.

Secretioncultureandbacterioscopicanalysis

Secretions from the right and left maxillary sinuses of all rabbits were collected and analyzed. The bacteria foundduringtheanalyseswereclassifiedintothefollowing groups:non-fermentingGram-negativebacilli( Acinetobac-ter baumannii, Acinetobacter Iwoffii,Achromobacter sp., andPseudomonasaeruginosa),Gram-negative enterobacte-riabacilli(Escherichiacoli),Gram-positivebacillui(Bacillus

sp.),andGram-positivecocci(Micrococcussp., coagulase-negativeStaphylococcus,andStaphylococcusaureus).The resultsaredescribedinTable1.

Table2shows thatthemostcommongroupofbacteria foundonthesidesubmittedtorhinosinusitisinduction(right side)wasthegroupofGram-positivebacilli(44.4%),while onthecontralateralsideitwasthegroupofnon-fermenting Gram-negativebacilli(40.9%).Ahigherincidenceof Gram-negative bacteria was identified in the total number of assessedrabbits.Thesewereidentifiedin15animals(75%); andGram-positivebacteriawerefoundinonlynineanimals (45%).Themicroorganismmostoftenfoundintheculturesof therightmaxillarysinusoftherabbitswasBacillussp., iden-tifiedin11rabbits.Sevenrabbitshadtwomicroorganisms inculturetestsfromtherightside.

Swabpositivitywasevaluatedbilaterallyinthecollected samples.TheresultsareshowninTable3.

Table2 Descriptionofgroupsofbacteriafoundinthe sam-ples according to the sideof the 20 rabbits inthe study groups.

Variable Frequency %

Bacterialgrouprightside

Negative 2 7.4

Non-fermentingGram-negativebacilli 5 18.5 Enterobacter,Gram-negativebacilli 4 14.8

Gram-positiveBacilli 12 44.4

Gram-positiveCoccus 4 14.8

Total 27 100

Bacterialgrouprightside

Negative 6 27.3

Non-fermentingGram-negativebacilli 9 40.9 Enterobacter,Gram-negativebacilli 1 4.5

Gram-positiveBacilli 5 22.7

Gram-positiveCoccus 1 4.5

Total 22 100

Absoluteandrelativefrequencies.

associatedwiththedayofeuthanizationandthatpositivity decreaseswitheachpassingday(p=0.039).

The culturesofthemaxillarysinusesofthetworabbits usedascontrolsshowednobacterialgrowth.

Histologicalanalysisofsinusmucosa

Histological analysis of the right and left maxillary sinus mucosa showed some degree of inflammation in most samples. The results of the semi-quantitative histological evaluationofmucosalsamplesareshowninTable4.

Therewasastatisticallysignificantdifferencebetween groupsregardingthedegreeofinflammationintheinduced side (p=0.009), with no statistically significant differ-enceinconnective-fibrousproliferationbetweenthegroups (p=0.420;Table4).Ontheleftsidetherewasno statisti-cally significant differencein either parameter. Regarding the degree of mucosal inflammation in the left maxillary sinus,onlygrades0and1wereobserved.

Table 5 presents the comparison of the inflammation gradeontherightsideinrelationtothethreestudygroups,

showingthatthedegreeofinflammationingroupA(day10) wasstatisticallyhigherthaningroupC(day30),p=0.002.

Histologicalanalysisoftherightandleftsinusesofthe tworabbitsinthecontrolgroupshowedgrade0 inflamma-tionandabsentfibrous-connectiveproliferation.

Nasalpackinganalysis

Theassociationbetweenthebacteriafoundintheswabfrom theright side andthe bacteria identifiedin the Merocel®

nasalpackingwasanalyzed.

No statistically significant association was observed betweenconcordanceof bacteriafoundin thenasalswab and nasal packing according to the groups (p=0.171;

Table6).

TheMerocel® _{nasalpackingusedascontrolthroughthe}

Hemobac® _{systemshowednobacterialgrowth.}

Discussion

Severalexperimentalmodelshavebeen utilizedtoinduce rhinosinusitisandaredescribedintheliterature.20 _Rabbits

arethemostcommonlyusedanimalsinthistypeofstudy, followedbyratsandsheep.Itwasdecidedtouserabbits, since they show more anatomical and physiological simi-laritieswiththe human sinonasalcavities, asreportedby Casteleynetal.21

These experimental models have assessed several aspectsof sinus infection. Anatomical, physiological, and pathological alterations of the paranasal sinuses have beenevaluated,13,16,22,23 _{andcomparisonsofthe}

effective-ness of different treatments for rhinosinusitis have been conducted.8,9,11,24---26 _{The study and identification of the}

presenceofbacterialbiofilmshavealsobeenperformed.27

Different methods have been used toinduce bacterial sinusinfectioninrabbits.Earlierstudiesadvocated defini-tivemaxillaryostiaobstructionthroughsurgicalprocedures oruseofglue.6,22---24_{Althoughthismodelofrhinosinusitishas}

beenshown tobeextremelyeffective intheformation of purulentrhinosinusitis,inflammationwasgenerallylimited tothemaxillarysinus,asasinusabscess.Furthermore,the manipulationusedtogeneratetheostiumobstruction was performedthroughthesinuscavityintothenasalcavity.

Table3 Descriptionofswabpositivityaccordingtothedayofeuthanizationofthegroupsandstatisticaltestresults.

Variable Groups Total p

A B C

n % n % n % n %

Rightswab 0.468

Negative 0 0.0 1 14.3 1 14.3 2 10.0

Positive 6 100.0 6 85.7 6 85.7 18 90.0

Leftswab 0.039

Negative 0 0.0 2 28.6 4 57.1 6 30.0

Positive 6 100.0 5 71.4 3 42.9 14 70.0

Total 6 100 7 100 7 100 20 100

Table4 Descriptionofthedegreeofinflammationandfibrous-connectiveproliferationaccordingtothedayofeuthanization ofthegroupsandstatisticaltestresults.

Variable Groups Total p

A B C

n % n % n % n %

Rightfibrous-connectiveproliferation 0.420

Absent 3 50.0 4 57.1 2 28.6 9 45.0

Present 3 50.0 3 42.9 4 57.1 10 50.0

Verypresent 0 0.0 0 0.0 1 14.3 1 5.0

Leftfibrous-connectiveproliferation 0.786

Absent 5 83.3 5 71.4 6 85.7 16 80.0

Present 1 16.7 2 28.6 1 14.3 4 20.0

Degreeofinflammationrightside 0.009

0 0 0.0 0 0.0 0 0.0 0 0.0

1 0 0.0 1 14.3 2 28.6 3 15.0

2 1 16.7 3 42.9 5 71.4 9 45.0

3 2 33.3 3 42.9 0 0.0 5 25.0

4 3 50.0 0 0.0 0 0.0 3 15.0

Degreeofinflammationleftside 0.267

0 1 16.7 4 57.1 4 57.1 9 45.0

1 5 83.3 3 42.9 3 42.9 11 55.0

Total 6 100 7 100 7 100 20 100

Kruskal---Wallistestresults.

Table5 Resultofmultiplecomparisonsofthedegreeof inflammationintheinducedsidebetweenthegroups.

Comparison Z-value p

A−B 1.82 0.069

A−C 3.05 0.002

B−C 1.28 0.200

In the present study, rhinosinusitis induction was per-formed by inserting a synthetic sponge in the right nasal cavityoftheanimals,whichwasremovedafter10daysin allgroups.This methodis technicallysimple,causes little damage tothe nasal mucosa, and the packing is easy to removeintherabbitsthatcontinuedinthestudy. Further-more,thismethoddoesnotcausepermanentalterationsin

the nasal mucosaof the animalsand is reversible, allow-ingfortheassessmentof histologicalrecoveryafteracute rhinosinusitisinduction.

Some authors performed the inoculation of the infec-tiousagentinsidethemaxillarysinus7,22,25,26,28,29_andothers,

insidethenasalcavity.15,20,30 _{Theauthorschosenottouse}

thefirsttechnique,asitismoreinvasiveandcauses iatro-genic alterations in the sinus mucosa. Some studies have demonstratedthatthesimpleobliterationofnasalpassages wouldbeenoughforthedevelopmentofabacterial infec-tioncondition.7,13,31

The inoculation of streptococcal and staphylococcal toxoid wasusedin conjunction withthesynthetic sponge packinginthesamenasalcavity.Itwasdecidedtousethe toxoid inoculation aimingat standardizing thepathogenic agents present in thesamples withthe agentsthat cause

Table6 Descriptionoftheagreementofthenasalpackbacteriaandmicrobiologyintheinducedsideaccordingtothegroups andstatisticaltestresults.

Variable Groups Total p

A B C

n % n % n % n %

Hemobac®_{concordancewithrightswab 0.171}

Differentbacteria 4 66.7 7 100.0 6 85.7 17 85.0

Samebacteria 2 33.3 0 0.0 1 14.3 3 15.0

Total 6 100 7 100 7 100 20 100

sinusinfectioninhumans,aswellastoacceleratethe rhi-nosinusitisinductiontime.Thisfindingwasdemonstratedby Karaetal.,15_{whousedcomputedtomographytodisclosethe}

presenceofinfectioninthemaxillarysinusofrabbitsfrom daysix afterinduction ofrhinosinusitiswithtoxoid inocu-lationandontheeighthdaywithouttoxoidinoculation.

After the spongeremoval onthe 10th day, all animals hadunilateralpurulentrhinorrheainthenasalcavitywhere theywereinserted.This resultisconsistent withthoseof other studies15,26,32 _{applying similar methods through the}

rhinogenic route, amodel originally proposed byMarks,20

usingonlytheinsertionoftoxoidand/orbufferthroughthe nasalcavity.Marks20_{had83%successwiththismethod;Liang}

et al.31 _{obtained 91.7% success in the induction of acute}

infection.

The presence of purulent rhinorrhea in the nasal cav-ityofthestudyanimalswaschosenasthemaindiagnostic criterionofacuterhinosinusitis.Microbiological and histo-logical evaluations of the animals from group A allowed fortheassessmentoftheinfectiousprocessatthetimeof diagnosis, i.e.,10daysafter thestart ofthe experiment. After that, the acute rhinosinusitis recovery period with-outanytreatment wasobserved.Therefore,some rabbits fromgroupBstillhadsecretionsinthenasalcavityandthe maxillarysinusesafterbeingeuthanized,whilesomerabbits fromgroupCstillhadsecretioninthemaxillarysinusesat thetimeofeuthanization.

In the microbiological and histological analyses of the rabbits in group A, all animals had positive swab of the maxillary sinus for microorganisms and signs of mucosal inflammation bilaterally. The rabbits in groups B and C showedsomenegativeresultsofthemaxillarysinusswabs, whilethehistologicalevaluationdemonstratedthatall ani-mals still had some degree of inflammation in the right maxillarysinus.Consideringtheseresults,it canbenoted that,duringtheevolutionofanacutebacterial rhinosinusi-tispicture,inflammatoryfindingsinthesinusmucosapersist longerthanthepresenceofthemicroorganismscausingthe infectiousprocess.

Most of the studies that used strains of Streptococcus pneumoniaetoaidintheinductionofrhinosinusitis demon-strateditsreplacement byother opportunisticpathogenic agents.Westrinetal.33_{usedthisbacteriumfortheinduction}

andobserveditsreplacementafteranaverageoffivedays. Chengetal.26_{didnotisolatethisagentinanyrabbit10days}

afterthestartofinduction.Inthepresentstudy,S. pneu-moniaewasnotidentifiedinanyoftheassessedrabbits.

Mostmodelsdescribedintheliteraturedidnotevaluate thecontralateralsinustothatoftheexperimentinduction. Fewstudiesreportedcultureofthematerialfromthe con-tralateral sinus, and when performed, the studies had a small sample size. Liang etal.31 _{reported little bacterial}

growth in nasal cavities without the presence of rhinosi-nusitis. In the present study, bacteria were found in the contralateralsinusof14rabbits(70%oftotal).Theauthors believethattheprogressionoftheinflammatorycondition inthenasalmucosaalsofavorsthegrowthofopportunistic bacteriaonthecontralateralsideoftheinducedexperiment side.

Histological analysis of the sinus mucosa in rabbits submitted to experiments for rhinosinusitis induction was performed in most studies, with several goals. Its

assessment was used as a diagnostic criterion for the presence of sinus infection,15,20,29,31 _{in the assessment of}

infectionseverity,16,34 _{inthe comparisonof the}

effective-ness of different treatments for rhinosinusitis,12,14,25 _and

inthephysiopathologicalchangesaftertheinductionofan acutesinonasalinfection.13,24,30

The semiquantitative analysis was usedin the present studyinordertoassessinflammationintensityandcompare thethreestudygroups(euthanizationafter10,17,and30 days).Astatisticallysignificantdifferencewasobservedin thedegree of inflammation between rabbits ofthe group euthanizedat10daysandthegroupeuthanizedat30days. Thus,it wasdemonstrated that theinflammatory process willregress after removalof the nasal packing.But even after30daysofthestartoftheexperiment(20daysafter packingremoval),manyrabbitsstillshowedinflammationin thesinusmucosa.Thisfindingindicatesthatacute rhinosi-nusitiscausesmoreprolongedhistologicalchangesthanthe macroscopicfindingsandthattheypersistforafewweeks untilcompleteregression.

Fewstudieshave performedthehistologicalanalysisof themaxillarysinusmucosainthecontralateralsideofthe affectedsinus. Jyonouchi etal.23 _{observedan increase in}

glandularand goblet cells in thecontralateral sinuses, as well asmild stromal thickening. However,they used per-manentobliterationofthesinusostiumwithcyanoacrylate as the induction method. Gentian et al.30 _{observed no}

alterationsin thecontralateralnasalcavity. Liangetal.31

evaluatedonlysomecontrolsidesandfoundnohistological alterations.

Theauthorsbelievethatthisis aquitesignificant find-ingandthataprogressionofinflammationasaresponsein theentirerespiratoryepitheliumtoalocalinfectiousfocus canoccur,anditcantriggeragreaterresponseeveninareas withoutdirectcontinuity.Thisalsooccursinasthmapatients withallergicrhinitisand/orrhinosinusitis,whosepulmonary conditiondeteriorates asaresultof the worseningofthe nasal condition, an association known as unified airway. Therearesomeother hypothesesfor itsoccurrence,such asnasobronchialneuralreflex,contaminationofthelower airwayswithinflammatorycellsandmediatorsthrough pos-teriornasalsecretion,or absorptionofinflammatory cells ofthenasalepitheliumbythesystemiccirculationand con-sequently,intothebronchialmucosa.35,36_{Futurestudiesare}

neededtoassesstheassociationofthisresponseinthe pres-enceofrhinosinusitis.

The assessment of the connective-fibrous proliferation showednostatisticallysignificant differencebetween the groups. Therefore, this parameter could not be used as a markerof infectious process chronicity. The number of rabbitsmaynothavebeensufficienttoreproducestatistical alterationinthisassessedparameter.

Inrecent decades,most oftheexperimental studiesin rabbitsusedtheinsertionofanasalpackingfor rhinosinusi-tisinduction.ThematerialusedinthestudieswasMerocel®

syntheticsponge,15,20,26,31 _{gelatinoussponge,}32 _orpolyvinyl

sponge.7,29 _{Theobjectivewastoestablishaninflammatory}

Nonetheless,the literaturesearch retrievednostudies thatperformed the analysis ofnasal packing for microor-ganismculture,orwithsomeotherobjective.

Karaetal.15_{culturedtherightmaxillarysinus(infection}

inductionside)andrightnasalcavityofallrabbits,through swabcollection.This wasperformed inonlyoneregionof thenasalcavity.Theyexpectedthedisseminationof bacte-riafromthenasalcavityintothesinus,butdidnotobserve thatfinding.

The assessment ofnasalpacking wasperformed sothe microorganismspresent in the nasal passages, where the induction ofthe infection wasperformed, couldbe accu-rately analyzed. This method was used to correlate the bacteriapresent inside the nasalcavity withthebacteria foundinthemaxillarysinus.Nosignificantassociationwas observedbetweenthebacteriapresentinthesetwo differ-entanatomicsites.Thisassociationwasnotobservedeven inthefirstgroupofeuthanizedrabbits,inwhomtheremoval ofthenasalpackingwasperformedonthesameday,10days afterthestartofthestudy.

Conclusion

The experimental model, conducted and assessed by histopathologicalparametersofthesinusmucosa,through cultureofsinussecretionandnasalpackingandbyassessing thepresenceofnasalsecretion,wasshowntobecapableof inducingacutebacterialrhinosinusitisin100%oftheanimals usedinthestudy.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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