www.bjorl.org
Brazilian
Journal
of
OTORHINOLARYNGOLOGY
ORIGINAL
ARTICLE
Use
of
scanning
electron
microscopy
in
the
cochlea
of
guinea
pigs
夽
Luiz
César
Nakao
Iha
∗,
Oswaldo
Laércio
Mendonc
¸a
Cruz
UniversidadeFederaldeSãoPaulo(Unifesp),EscolaPaulistadeMedicina(EPM),SãoPaulo,SP,Brazil Received23August2018;accepted18November2018
Availableonline7January2019
KEYWORDS Electronmicroscopy; Innerear; Cisplatin; EDTA Abstract
Introduction:Theuseofelectronmicroscopyinthestudyoftheinnerearhasallowedusto observeminutedetailsofthehaircells,especiallyinototoxicitystudies;however,the prepa-rationofthismaterialisadifficultanddelicatetask.Inanattempttosimplifythehandlingof thesematerials,twoagents,toluidineblueandethylenediaminetetra-aceticacidweretested, inadditiontotheeliminationofosmiumtetroxideduringthepreparationofalbinoguineapig cochleae.Wealsotestedtheapplicabilityofthesemethodologiesinanototoxicityprotocol.
Objective:Toverifythequalityoftheimagesobtainedwithandwithouttheuseof ethylene-diaminetetra-aceticacid,toluidineblueandosmiumtetroxideinthepreparationofcochleae ofalbinoguineapigsforthescanningelectronmicroscopy.
Methods:Threegroupsofcochleaewereused.InGroup1,10cochleaewerepreparedwiththe usual methodology, dissectingthe opticalcapsule withoutdecalcification andusingosmium tetroxide as a post-fixative agent. In Group 2, we prepared 10 cochleae decalcified with ethylenediaminetetra-aceticacid,injectingtoluidineblueintheendolymphaticspaceto facil-itatetheidentificationoftheorganofCorti.InGroup3,weused4cochleaeofguineapigsthat received3dosesofcisplatin(7.5mg/kg,D1---D5---D6),twopreparedaccordingtothe method-ologyusedinGroup1andtwowiththatusedinGroup2.Scanningelectronmicroscopyimages wereobtainedfromtheorganofCortiregionofthebasalturnofeachcochlea.
Results:The organ ofCorti was more easily identifiedwith the use oftoluidine blue. The dissectionofthecochleawasmoreaccurateinthedecalcifiedcochleae.Thequality ofthe imagesandthepreservationoftheorganofCortiobtainedwiththetwomethodologieswere similar.
Conclusion:Theproposedmodificationsresultedinimagesofsimilarqualityasthoseobserved usingthetraditionalmethodology.
© 2018 Publishedby Elsevier EditoraLtda. on behalf ofAssociac¸˜ao Brasileira de Otorrino-laringologiaeCirurgiaC´ervico-Facial.ThisisanopenaccessarticleundertheCCBYlicense (http://creativecommons.org/licenses/by/4.0/).
夽 Pleasecitethisarticleas:IhaLC,CruzOL.Useofscanningelectronmicroscopyinthecochleaofguineapigs.BrazJOtorhinolaryngol.
2020;86:222---7.
∗Correspondingauthor.
E-mail:lciha@yahoo.com.br(L.C.Iha).
PeerReviewundertheresponsibilityofAssociac¸ãoBrasileiradeOtorrinolaringologiaeCirurgiaCérvico-Facial. https://doi.org/10.1016/j.bjorl.2018.11.008
1808-8694/©2018PublishedbyElsevierEditoraLtda.onbehalfofAssociac¸˜aoBrasileiradeOtorrinolaringologiaeCirurgiaC´ervico-Facial. ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).
PALAVRAS-CHAVE Microscopia eletrônica; Ouvidointerno; Cisplatina; EDTA
Microscopiaeletrônicadevarreduraemcócleasdecobaias
Resumo
Introduc¸ão: Oempregodamicroscopiaeletrônicanoestudodaorelhainternapermitiuobservar detalhes minuciososdascélulasciliadasespecialmenteem estudosdeototoxicidade. Entre-tanto,opreparodessematerialétrabalhosoedelicado.Parasimplificaramanipulac¸ãodesses materiais, testou-seouso dedois agentes,azul de toluidina eácido etilenodiamino tetra-acético,alémdaretiradadotetróxidodeósmionapreparac¸ãodecócleasdecobaiasalbinas. Testamostambémaaplicabilidadedessasmetodologiasemumprotocolodeototoxicidade.
Objetivo: Verificaraqualidadedasimagensobtidascomesemousodeácidoetilenodiamino tetra-acético, azul detoluidina etetróxido deósmio napreparac¸ão de cócleasde cobaias albinasparaamicroscopiaeletrônicadevarredura.
Método: Foramutilizadostrêsgrupos decócleas.NoGrupo 1preparou-se10cócleascoma metodologia usual,dissecandoacápsulaótica semdescalcificac¸ãoeutilizando tetróxidode ósmiocomopós-fixador.NoGrupo2preparamos10cócleasdescalcificadascomácido etilen-odiamino tetra-acético,injetandoazulde toluidina noespac¸o endolinfáticopara facilitar a identificac¸ãodoórgãodeCorti.NoGrupo3utilizamos4cócleasdecobaiasquereceberam3 dosesdecisplatina(7,5mg/kg,D1---D5---D6),duaspreparadascomametodologiadoGrupo 1 eduascomadoGrupo2.Foramobtidasimagensdamicroscopiaeletrônicadevarredurada regiãodoórgãodeCortidogirobasaldecadacóclea.
Resultados: O órgão de Corti foi mais facilmente identificado com o azul de touidina. A dissecc¸ão dacócleafoi maisprecisanascócleas descalcificadasA qualidadedas imagense apreservac¸ãodoórgãodeCortiobtidascomasduasmetodologiasfoisimilar.
Conclusão:Asmodificac¸õespropostasresultaramemimagensdequalidadesimilaras obser-vadascomousodametodologiatradicional.
© 2018Publicadopor ElsevierEditora Ltda.em nomede Associac¸˜ao Brasileira de Otorrino-laringologia eCirurgiaC´ervico-Facial.Este ´eumartigo Open Accesssob umalicenc¸a CCBY (http://creativecommons.org/licenses/by/4.0/).
Introduction
The use of optical microscopy to study biological tissues is ofutmost importanceandwasusedbyAlfonso Cortito performthe first detailedhistologicalobservations of the labyrinthorgans,describingthespiralganglion,thebasilar membrane, the inner and outer hair cells and the tecto-rial membrane.1 But our knowledge about the inner ear increasedevenfurtherwhencochlearstructureswere eval-uatedbyelectronmicroscopy.
Theelectron microscopewasinventedin 1931byErnst Ruskaandusesanelectronbeamtogeneratetheimages.As thewavelengthofthisbeamisapproximately100,000times smaller than that of the visible light spectrum, electron microscopyhasamuchhigherresolutionpowerthanoptical microscopy, allowing a closerobservation of the cochlear microstructure.2
OneofthemodalitiesofelectronmicroscopyisScanning ElectronMicroscopy(SEM),inwhichtheelectronbeam trav-elsalongthesurfaceofthestudiedmaterial,generatinga three-dimensionalimageofthismaterial.Thischaracteristic makesSEMparticularlyinterestinginassessingtheintegrity ofhaircellsinsituationsofinternaleardamage,aswellas instudyingototoxicity.
Ototoxicityistheresultofasubstancecausing damage totheinnerear,withthecapabilityofcausingafunctional
alteration.3 The most often studied experimental proto-colsfor ototoxicity arethosemediated byaminoglycoside antibioticsandcisplatin4---8inwhichthecochleaundergoes atypeofprocessingthatincludesfixationwith glutaralde-hyde,removaloftheoticcapsule,post-fixationwithosmium tetroxide,dehydrationinethanolandCO2criticalpointand
finally receiving a thin coating of metal (usually gold or platinum).However,inprevious studies carriedoutinour service9,10 weobserved thatthepreservationof theorgan ofCortiwasnotalwaysideal,withoccasional mechanical focallesionsin theregionof the hair cellsresulting from theremovaloftheoticcapsule.
InanattempttobetterpreservetheorganofCortiand tosimplifythe dissectionand processingof thismaterial, wedesignedamodelinwhichwemodifiedthepreparation ofalbinoguineapigcochleaefortheSEMusing Ethylenedi-amineTetra-Aceticacid(EDTA)andToluidine Blue (TB) to decalcifytheoticcapsule,facilitatingtheidentificationof the hair cell region and allowing a more delicate dissec-tionofthecapsule.Wecomparedthequalityoftheimages obtainedwitha group ofcochleaeprocessed inthe usual manner.Inthisnewmodel,wealsotestedtheeliminationof osmiumtetroxidetosimplifytheprocessandavoidtheneed touselaminarflowwhenhandlingthematerial.Inasecond stage,weappliedthetwomethodologies(theusualandthe newtechnique)tothecochleaeofguineapigssubmittedto
anototoxicprotocoltoassessimagequalityandapplicability ofthisnewmethodinthistypeofexperimentalprotocols.
Theaimofthisstudyistoassessthequalityoftheimages obtainedwithandwithouttheuseofEDTA,toluidineblue and osmium tetroxide when preparingcochleae of albino guineapigsforscanningelectronmicroscopyevaluation.
Material
and
methods
Twelve healthy female albino guinea pigs (Cavia porcel-lus), withpresent Preyerreflexes, weighing between 350 and450g andapproximately 3 months of age were stud-ied.Theanimalswerekeptincages,atstabletemperature between21and22◦C,with12-hlight---darkcyclesandfree access tofood and water. The project and all its proce-dureswerefinancedbytheresearchersandapprovedbythe ResearchEthicsCommitteeofourinstitution,undernumber CEP1625/04.
Theanimalsweredividedintothreegroups.
Group1---customarypreparation
Five guinea pigs (10 cochleae), with no previous oto-toxicmedications and withpresent Preyer reflexes, were anesthetized with intramuscular injections of xylazine (10mg/kg) and ketamine hydrochloride (40mg/kg), and subsequently euthanizedwithan intracardiac injectionof potassium. The animals were rapidly decapitated, and a medialsagittalcraniotomywasperformed, separatingthe right and left hemicranias. Subsequently, the temporal boneswereisolatedby removing theremainingbony por-tionsofthecranialstructure.
The mastoid bulla was opened, thereby exposing the cochlea.Undermicroscopicview,afenestrationwasmade atthecochlear apexwithamicrolancet,whichwasused asanopening forinfiltrationwith2.5%glutaraldehyde fix-ingsolutionin0.1Mcacodylatebuffer,pH7.2,throughthe roundwindowand,afterwards,thematerialwasimmersed inthesamefixativesolutionfor24h,storedina refrigera-torat4◦C.Afterthistime,two1-hwasheswereperformed, followedbyanovernightrestinthebuffersolutionfor com-pleteremovalofthefixativeagent.
Subsequently,theremainderoftheoticcapsulewas care-fullyremovedandthemodioluswasextractedtogetherwith theorgan of Corti,with this materialbeing submitted to dehydrationinincreasingconcentrationofethanolsolutions (50%,70%and90%)for30mineach,followedbythree 30-min immersions in 100% ethanol and then submerged in 2%osmiumtetroxide,waiting15mintocompletethe post-fixationstep.Thenthedryingwascarriedoutusingcritical pointdryingequipment(Balzers-CPD030),inwhichthe sam-plesweresubmittedtoseveral bathsinliquidCO2,which
removesallthewaterandethanolstillpresentinthe sam-ples.
This final specimen was fixed and mounted on an adequate support using conductive paste coated with a 25---50mm thick gold layer (Balzers SCD 050 --- Sputter Coater) and visualized on a JEOL scanning microscope, model5300.
Aphotographicfieldofthebasalcochlearturnwas ana-lyzedinordertoverifythesharpnessoftheobtainedimage
forcomparisonwiththeothergroups,aswellasthe preser-vationoftheorganofCorti.
Group2---modifiedtechnique
Fiveguineapigs(10cochleae)weresubmittedtothesame preparationasinGroup1;however,afterthe12-hperiodin thebuffersolutiontoremovethefixativeagent,the mate-rialwassubmergedina10%EDTAsolution,alsofor a12-h period,butthepost-fixationstepwithosmiumtetroxidewas omitted.Toluidinebluedyewasusedforbetter identifica-tionandpreservationoftheorganofCorti,utilizinga0.1% solution,whichwasinjectedintotheendolymphaticspace ofthecochlearbasalturnthroughthedecalcifiedotic cap-sule utilizinga syringeandneedlekit(8.0×0.3mm)used forinsulininjections.
UsingaN.11scalpelbladeandmicro-scissorsforotologic surgery,theoticcapsulewasremoved,andthemodioluswas thenremovedtogetherwiththeorganofCorti.
Afterthematerialwassubmittedtoalcoholand critical-point dehydration, mounted in an adequate support and coated witha gold layer, a lowercochlear turn fieldwas photographed.
Group3---useofthetwotechniquesina cisplatin-mediatedototoxicityprotocol
Twoguineapigsweresubmittedtothreedosesof7.5mg/kg ofcisplatinfollowingtheD1---D5---D6schedule(applications on the first day, and on the fourth and fifth subsequent days),aprotocolstandardizedatourservicethatproduces aprovenalterationinfunctionalhearingtestsanddamage tothe outer hair cells. On the day following thelast cis-platin application, the guinea pigs were euthanized, and oneofthecochleaeweresubmittedtotheGroup1 prepa-rationprotocol(Subgroup3a)andtheothercochleatothe Group2protocol(Subgroup3b).Duringthefollow-upperiod, otoscopic examinations were carried out to rule out an inflammatorystateofthemiddleandouterear.
Results
Group1
The imagesobtained inthephotosof thecochleaeinthis groupshowedexcellentimagequality,allowingustoeasily verifythepresenceofboththeinnerandouterhaircells,as showninFig.1.Thelevelofdetailwasadequatenotonlyto verifythepresenceofhaircells,butalsotheintegrityand positionoftheirstereocilia.Somecochleaeshowedlesions in the regionnear thehair cells (Fig. 2).We did notfind anyevidenceofhaircelllesionduetotheproceduresofthe preparationofthismaterialfortheSEM.
Group2
There was noperceptible difference regarding the image qualityofthephotosobtainedinthisgrouprelativetothose obtainedinGroup1,anditwaspossibletoclearlyobserve thepresenceandintegrityofthehaircellsandtheir
stere-Figure1 PhotographsoftheorganofCortiregionofguineapigsfromGroup1(useofOsmiumtetroxide,withoutEDTA).
Figure2 PhotographoftheorganofCortiregionofaguinea pig from Group 1 (use ofOsmium tetroxide, without EDTA), showingthepresenceofafracturenexttotheouterhaircells.
ocilia(Fig.3).Similartotheprevious group,weobserved some lesionsinthe regionclosetothehair cells (Fig.4). Inthisgroup,we alsodidnotobserveanyhaircelllesions that could indicatethat the methodology interfered with haircellpreservation.
Groups3aand3b
Thetwogroupsshowedimageswithsimilarresolution qual-ity,whichallowedtheadequateevaluationoftheorganof Cortitodemonstratehaircelllesions(Fig.5).
Discussion
Ouranatomicalknowledgewasimprovedwiththeadventof opticalmicroscopyandelectronmicroscopy.
Optical microscopyisstillthemostfrequentlyusedfor thestudyofbiologicalmaterialsduetoitslowercostandthe useofsimplerequipmentforitsperformance.Nevertheless, itisundeniablethatEMattainsalevelofdetailthatmakes itidealforthestudyofsomematerials.
OneofthecriticalpointsoftheEMstudyisthe prepara-tionofthespecimensfortheanalysis.
The preparationof the biologicalmaterials tobe eval-uated by EM starts with fixation (using formaldehyde, glutaraldehydeorevenboth11,12),followedbypost-fixation anddehydration.Thepost-fixationstepwithosmium tetrox-ideisroutinelyperformedinanEMlaboratory,becausethe electron beam used for image capture has a small pene-trationpowerandpost-fixationelements containingheavy metalatoms suchasuranium (uranylacetate),lead(lead citrate)andosmium(osmiumtetroxide)arerequiredto cre-atecontrast,anextremelyrelevantfactorfortransmission
Figure3 PhotographsoftheorganofCortiregionofguineapigsfromGroup2(useofEDTAandtoluidineblue,withoutosmium tetroxide).
Figure4 PhotographoftheorganofCortiregionofaguinea pig from Group 2 (use of EDTA and toluidine blue, without osmiumtetroxide),withthepresenceofafracturenexttothe outerhaircells.
Figure5 PhotographsoftheorganofCortiregionofguinea pigsfromGroups3aand3b.
electronmicroscopy,inwhichtheelectron beamtraverses theimagingmaterial.
However,whenpreparingmaterialfortheSEM,the spec-imens must be dehydrated, and their surface receives a metalcoating,alsoknownassputtering,usuallywithgoldor agold/palladiumalloy.TheimagecaptationinSEMismainly basedontheelectrondeflectionpropertyofthismetal sur-face, which even makes possible to analyze the external structure of insects that have rigid exoeskeleton with no otherpreparationthanthesputtering.13,14This factledus tobelievethattheuseofosmiumtetroxidewouldbe unnec-essarytoobtainimagesintheSEM,whichwasthecasewith thecochleaepreparedwithournewmethodology.
Theobjectiveoftryingtoeliminatethe useofosmium tetroxidewas toavoid the high toxicityof heavy metals. Osmium tetroxide is highly toxic and extremely volatile, requiring theuse of a laminarflow hood, protective gog-gles,apronandglovesforitshandling,sinceexposuretothis substancemaycausecoughing,headache,skinburns, con-junctivaledemaandcornealdestruction,pulmonaryedema, aswellasitshavingapotentialforcarcinogenesis. During thehandlingofosmiumtetroxideinthepreparationofthe Groups1and3cochleaeinourstudy,wehadtouselaminar flowandprotectiveglovesallthetime,andyetweobserved
therapidosmiumtetroxideimpregnationinthegloveused whenweopenedthesolutionbottle.
TheuseofEDTAandtoluidineblueaimedtoallowamore delicate and precise dissection,in addition tofacilitating theidentificationandpreservationoftheorganofCorti.
Toluidine blue was injected into the endolymphatic labyrinthof Group2cochleaeanditssimilarspecimensin Group 3b. What we observed was an immediate impreg-nation of the dye intothe structures of the membranous endolymphaticlabyrinth.Afterthesestructureswere ade-quatelyidentified,itwaspossibletobeextracarefulwhen dissectingtheouterhaircellarea.
EDTAiscommonlyusedinthepreparationofbone mate-rialswhereextremelythincutsareessential,butitsuseis notcommoninSEM.15---17TheuseofEDTAinourstudyaimed todetermine whetheritwouldalsofacilitatethe prepara-tionofthismaterialforSEM,andwefoundbothpositiveand negativeaspects.
Ontheonehand,thenaturalrigidityofthecapsule facil-itatesthecreationofsmallfracturelinesthatexpediteits removal,buttheforcenecessarytocarryoutthesefractures hasthepotentialtodamagethestructuretobestudied.
ThedecalcifiedopticalcapsuleofGroups2and3bshowed greatflaccidityandmalleability,requiringtheuseofmicro scissorsfortheircompleteremoval.Theuseofcuttingtools broughtgreaterprecisiontotheremovalofunwantedparts from the material but made the dissection a little more time-consuming.
Macroscopically, the groups in which EDTA was used, structurally seemed to be better preserved, but at the microscopic level we observed fractures similar to those found in cochleaewithout decalcification. This leadusto concludethatthedecalcificationstepshouldbeincludedat thediscretionof thepersonresponsiblefor thedissection of the cochleae,depending onthepreference or need to performamoredelicatedissection.
However,theaimofthisstudywastoverifywhetherthe qualityoftheimagesobtainedwiththemethodologic mod-ificationswereequivalenttotheusualprocessingmethod, inadditiontoassessingtheirapplicabilityin experimental ototoxicitymodels.
Inthenormalcochleargroups(Groups1and2)theimages disclosed identical image definition. By using a protocol standardized in our institution, comprising threedoses of 7.5mg/kg ofcisplatin, we wereable tocreateouter hair celllesionsthatwereeasilyidentifiedbytheSEM,making it possible tofind total disarray or absence of the stere-ocilia inthe lower cochlear turns. Consideringthat these alterationswereobservedwithoutanydifficultiesinallour cochleargroups,weconcludethatthemodificationsapplied tothepreparationmethodologyusedinthesesamplesfor SEM areapplicable in thismodel of ototoxicity caused by cisplatin.
Conclusions
The quality of the images obtained with the proposed modifications wassimilartothatobservedwhenusingthe traditionaltechnique.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
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