www.bjorl.org
Brazilian
Journal
of
OTORHINOLARYNGOLOGY
ORIGINAL
ARTICLE
The
effect
of
melatonin
and
vitamin
C
treatment
on
the
experimentally
induced
tympanosclerosis:
study
in
rats
夽
Sema
Koc
a,∗,
Halil
Kıyıcı
b,
Aysun
Toker
c,
Harun
Soyalıc
¸
d,
Huseyin
Aslan
e,
Hakan
Kesici
e,
Zafer
I.
Karaca
eaAntalyaEducationandResearchHospital,DepartmentofENTHeadandNeckSurgery,Antalya,Turkey bMevlanaUniversity,SchoolofMedicine,DepartmentofPathology,Konya,Turkey
cNecmettinErbakanUniversity,SchoolofMedicine,DepartmentofBiochemistryandClinicalBiochemistry,Konya,Turkey dGaziosmanpasaUniversity,SchoolofMedicine,DepartmentofOtorhinolaryngology,Tokat,Turkey
eGaziosmanpasaUniversity,SchoolofMedicine,DepartmentofHistologyandEmbryology,Tokat,Turkey
Received1June2016;accepted24June2016 Availableonline20July2016
KEYWORDS Tympanosclerosis; Melatonin; VitaminC; Totalantioxidant status
Abstract
Introduction:Theethiopathogenesisoftympanosclerosishasnotbeencompletelyunder-stood yet.Recentstudieshaveshownthatfree oxygenradicalsareimportantintheformationof tympanosclerosis.MelatoninandVitaminCareknowntobeapowerfulantioxidant,interacts directlywithReactiveOxygenSpeciesandcontrolsfreeradical-mediatedtissuedamage. Objective: Todemonstrate thepossiblepreventativeeffectsofmelatoninandVitaminCon tympanosclerosisinratsbyusinghistopathologyanddeterminationoftotalantioxidantstatus totalantioxidantstatus.
Methods:Standardmyringotomyandstandardinjurywereperformedinthemiddleearof24 rats.Theanimalsweredividedintothreegroups:Group1receivedmelatonin,Group2received vitaminC,andGroup3receivedsalinesolution.
Results:Themeanvaluesoftotalantioxidantstatusweresimilarintheallstudygroupsbefore thetreatmentperiod.Themeanvaluesoftotalantioxidantstatusweresignificantlyhigherin themelatoninandvitaminCgroupscomparedtocontrolgroupbutvitaminCwithmelatonin groupsweresimilarafterthetreatmentperiod(p<0.001).Minimumandmaximumwall thick-nesseswerelowerinthemelatoninandvitaminCgroupscomparedtothecontrolgroupbut thedifferenceswereinsignificant.
夽 Pleasecitethisarticleas:KocS,KıyıcıH,TokerA,Soyalıc¸H,AslanH,KesiciH,etal.TheeffectofmelatoninandvitaminCtreatment
ontheexperimentallyinducedtympanosclerosis:studyinrats.BrazJOtorhinolaryngol.2017;83:541---5.
∗Correspondingauthor.
E-mail:drsemakoc@gmail.com(S.Koc).
PeerReviewundertheresponsibilityofAssociac¸ãoBrasileiradeOtorrinolaringologiaeCirurgiaCérvico-Facial.
http://dx.doi.org/10.1016/j.bjorl.2016.06.008
Conclusion:Melatoninincreasestotalantioxidantstatuslevelandmighthavesomeeffecton tympanosclerosisthatdevelopsaftermyringotomy.
© 2016 Associac¸˜ao Brasileira de Otorrinolaringologia e Cirurgia C´ervico-Facial. Published by Elsevier Editora Ltda. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/).
PALAVRAS-CHAVE Timpanoesclerose; Melatonina; VitaminaC; Capacidade antioxidantetotal
EfeitodotratamentocommelatoninaevitaminaCnatimpanoescleroseinduzida
experimentalmente:estudoemratos
Resumo
Introduc¸ão:Aetiopatogênesedatimpanoesclerose(TE)nãofoiaindatotalmenteesclarecida. Estudos recentes têm demonstrado que os radicais livres de oxigênio são importantes na formac¸ãodeTE.MelatoninaevitaminaCsãoconhecidasporserempoderososantioxidantes, interagiremdiretamentecomespéciesreativasdeoxigênio(ROS)econtrolardanosemtecidos mediadosporradicaislivres.
Objetivo:DemonstrarospossíveisefeitospreventivosdamelatoninaedavitaminaCnaTEem ratosusandohistopatologiaedeterminac¸ãodacapacidadeantioxidantetotal(CAT).
Método: Miringotomiaspadronizadasforamrealizadasnaorelhamédiade24ratos.Osanimais foramdivididosemtrêsgrupos:oGrupo1recebeumelatonina,oGrupo2recebeuvitaminaC, eogrupo3recebeusoluc¸ãosalina.
Resultados: OsvaloresmédiosdeCATforamsemelhantesemtodososgruposdeestudoantes doperíodo de tratamento. Osvalores médios deCAT foramsignificativamente maioresnos gruposquereceberammelatoninaevitaminaCemcomparac¸ãocomogrupodecontrole,mas osgruposvitaminaCemelatoninaforamsemelhantesapósoperíododetratamento(p<0,001). Asespessurasmínimasemáximasdeparedeforammenoresnosgruposmelatoninaevitamina C,emcomparac¸ãocomogrupocontrole,masasdiferenc¸asnãoforamsignificativas.
Conclusão:AmelatoninaaumentaosníveisdeCATepodeteralgumefeitosobreaTEquese desenvolveapósamiringotomia.
© 2016 Associac¸˜ao Brasileira de Otorrinolaringologia e Cirurgia C´ervico-Facial. Publicado por Elsevier Editora Ltda. Este ´e um artigo Open Access sob uma licenc¸a CC BY (http:// creativecommons.org/licenses/by/4.0/).
Introduction
Tympanosclerosis(TS)isaconditionthatmayaffectboththe tympanicmembraneandthemiddleearmucosa.Itis char-acterizedhistologicallybyanincreaseincollagenousfibers, decreasedvascularization andcellformation hyalinization calcificationanddegenerationofthecollagenlayer.1,2TSis
usuallyidentifiedaswhitechalkypatcheseitherinthe mid-dleearmucosaorinthetympanicmembrane(TM).Atthe beginningofthepathologyitconsistsofcheese-likemasses oftympanoscleroticmaterialandbythetimesimilartobone itgets harder.3 The ethiopathogenesis ofTS hasnot been
completelyunderstoodyet.Recentstudieshaveshownthat freeoxygenradicalsareimportantintheformationofTSin traumatizedTMandantioxidantagentsdecreaseorprevent myringosclerosisandTSafterexperimentalmyringotomy.4---7
Melatonin(N-acetyl-methoxytryptamine),anindolamine derived from tryptophan, is mainly produced in the mammalian pineal gland but it is also produced in the other organs.8 It has been shown to act as a potent
anti-inflammatory,antioxidantandfree radical scavenger, protectingagainstanumberofradicalspecies.Various stud-ieshaveestablished thatduetothesefeatures melatonin decreasessclerosis.9---11VitaminCisknowntobeapowerful
antioxidant,interactsdirectlywithReactiveOxygenSpecies (ROS)andcontrolsfreeradical-mediatedtissuedamage.12
Theaimofthisstudywastoinvestigateandcomparethe possiblepreventativeeffectsofmelatoninandvitaminCon TSinrats,whichhadTMmyringotomyandastandardinjury inthemiddleear,byusinghistopathologyanddetermination oftotalantioxidantstatus(TAS).
Methods
International Standardsfor thecare oflaboratory animals werefollowedandtheprotocolofthestudywasapproved bytheresponsiblelocalethicalcommittee.
Animalmaintenance
Operatingproceduresandexperimentaldesign
Allinterventionswereperformed understerile conditions. After anesthesia with intramuscular ketamine hydrochlo-ride30mg/kg, undertheotomicroscope,throughan aural speculum,themyringotomywasperformedsimilarlyonboth earsof rats, witha radialorientation inthe upper poste-rior quadrant of the TMs, length approximately 3---4mm. Subsequently, a standard injury was formed in the mid-dle ear. They were divided into three groups (Groups 1, 2, and 3). Eight rats (Group 1) received 5mg/kg mela-tonin via orogastricgavage, eight rats(Group 2) received 75mg/kgvitaminCviaorogastricgavage,eightrats(Group 3)receivedsaline(0.9%NaCl)solutionviaorogastricgavage; thiswascontinuedfor10days.Theamountofthemelatonin andvitaminCsupplementweredeterminedwithreference totheliterature.9,13
Biochemicalanalysis
Forbiochemicalanalysesblood sampleswerecollectedat thefirstdayfromthetailveinsoftheratswhentheywere anesthetizedpriortomyringotomyandatthe28thdayfrom intracardiacrouteagainwhentheywereanesthetizedprior toscarification.
Totalantioxidantstatus(TAS)
Totalantioxidant status levelswere measured using com-mercially available kits (RelAssay). The novel automated methodisbasedonthebleachingofcharacteristiccolorof amorestableABTS(2.2′ -azino-bis[3-ethylbenzothiazoline-6-sulfonic acid]) radical cation by antioxidants. The commercialkitworkcolorimetricmethodisbasedon660nm absorbance. The results were expressed as mmol Trolox equivalent/L.
Tissuecollectionandhistopathologicalanalysis
On the 28th day, all animals were euthanized painlessly followingadministrationofhighdose (80mg/kg) intraperi-toneal pentobarbital. The temporal bones of the animals were removed, enumerated, and the middle ear cav-ity, tympanic membranes, and external ear canals were isolated.The specimenswere fixatedwithin4% formalde-hyde,andthendecalcifiedusing0.1moL/LEthlenediamine Tetra-acetic Acid (EDTA). Following tissue processing and blocking,specimenswerecuttoobtain3mthicksamples. Hematoxylin---Eosin (H&E) and Masson’s trichrome stains wereusedforhistopathologicevaluation.Thicknessof lam-ina propria at the middle ear was measured by a graded ocular,underOlympusBX53lightmicroscope.Exudateswere notincludedinthicknessoflaminapropria.Whenavailable, thicknessoftympanicmembranewasmeasuredbythesame technique.
Statisticalanalysis
All statistical analyses were performed using SPSS for Windows version 15 (SPSS, Chicago, IL, USA). The
Figure1 Thereisnosignificanttympanosclerosis(arrow)in thissampleofcontrolgroup;withmoderateotitismedia(left sideofarrow).H&Estain,100×magnification.
Kolmogorov---Smirnov test was used to evaluate whether the distribution of variables was normal. The Student t or Mann---Whitney Utests were used tocompare continu-ousvariablesbetweenthetwogroups.Continuousvariables were presented as mean (standard deviation [SD]) or as median(interquartilerange [IQR]). Ap-value ofless than 0.05wasconsideredtobestatisticallysignificant.
Results
ThemeanvaluesofTASweresimilarintheallstudygroups beforethetreatmentperiod.ThemeanvaluesofTASwere significantlyhigherinthe melatoninandvitaminC groups comparedto control group but vitamin C with melatonin groupsweresimilarafterthe treatmentperiod(p<0.001) (Table 1). Minimum and maximum wall thicknesses were lowerin themelatoninandvitaminCgroups comparedto controlgroup but the differences wereinsignificant. Min-imumand maximum wall thicknesses were similar in the melatoninandvitaminCgroups(Table1)(Figs.1and2).
Table1 Totalantioxidantstatusandwallthicknessinthestudygroups.
Control(n=8) VitaminC(n=8) Melatonin(n=8) p
TASbeforethetreatment(mmoLTroloxEq/L)a 2.97±0.86 2.51±0.39 3.20±0.91 0.199
TASafterthetreatment(mmoLTroloxEq/L)a 2.51±0.64 5.68±0.28 5.46±0.23 <0.001c
Minimumwallthicknessb 2.35(1.25---4.5) 2.0(1.0---6.9) 2.0(1.25---3.6) 0.946
Maximumwallthicknessa 38±27 33±26 32±17 0.847
TAS,totalantioxidantstatus. aValuesarepresentedasmean
±standarddeviation.
b Valuesarepresentedasmedianandinterquartilerange(Q1---Q3).
c Therewerestatisticallysignificantdifferencebetweencontrolwithmelatoningroups(p<0.001)andcontrolwithvitaminCgroups
(p<0.001)butvitaminCwithmelatoningroupswassimilar.
Discussion
Ourstudy revealed that systemicadministration of mela-toninreducedorinhibitedtheformationofTSbyactingas afreeradicalscavengerinratswhichhadmyringotomyand formationofastandardinjuryin themiddle ear.As faras weknow,thisisthefirststudyintheliteraturetoevaluate theeffectivenessofmelatonininpreventionofTS.
Reactive Oxygen Species are oxygen-containing molecules that are produced during normal metabolism under aerobic conditions. ROS include superoxide anion radical(O2•−),hydrogen peroxide(H2O2),hydroxylradical (•OH)andsingleoxygen(1O
2).14 Theseoxygenradicalsare considerably reactive and posses the capacity of causing irreversible cellular destruction. The antioxidant systems containenzymaticandnon-enzymaticmechanismsagainst the harmful effects of the endogenous ROS products.15
Enzymatic system involves Superoxide Dismutase (SOD), Catalase(CAT)andGlutathionePeroxidase(GSH-Px). Ascor-bic acid, glutathione, ˇ-carotene, tocopherols, and uric acidcanbeincluded asnon-enzymaticdefensesystems.16
Formation and elimination of free oxygen radicals are in a balance known as oxidative balance. As long as the oxidative balance is maintained, free oxygen radicals do not harm the organism. When the oxidative balance is corrupted,freeoxygenradicalselevateandtissuedamage occurs,thisfinalsituationiscalledasoxidativestress.17Itis
possibletomeasureonebyonetheantioxidantparameters in serum. Antioxidant parameters have additive effects, thusindividualvaluesmaynotcompletelyexhibitthetotal antioxidant status. Therefore TAS measurement is more appropriateproceduretoassessantioxidativestatus.18
Tympanosclerosis is caused by recurrent acute otitis media, treatment of serous otitis media with ventilation tubeinsertionintotheTM,chronicotitismedia, immunolog-icalhypersensitivityreaction,genetictendency,ortrauma. Recentstudieshaveinvestigatedformationof myringoscle-rosis after myringotomy and pointed out a relationship between thedevelopment of TS andROS.1,4,5 When
com-pared to 10% in ambient air, approximately 5% oxygen concentrationinthemiddleearcavityismuchlowerthan that in ambient air. Myringotomy leads to an increase of oxygen concentration in the middle ear cavity, result-inginarelativelyhyperoxic condition.19 Hyperoxiacauses
formation of ROS thereby might provoke inflammatory process.TheincreaseofROSandtheimpairmentof antiox-idant defense mechanisms cause tissue damage involving
fibrosis,hyalindegeneration,accumulationandaggregation ofcalciumandphosphorusformingscleroticdeposits.ROS scavengershavebeenshowntodecreasetheinflammatory reaction which also decrease the adhesion formation.4---7
Therefore,antioxidanttreatment maybe hypothesizedto reduceTSformationandreformation.
Melatonin is a potent antioxidant and free radicals scavenger.9 Bothdirectandindirectantioxidanteffectsof
melatonin have been reported.Melatonin has an indirect antioxidant effect by inducing SOD and GSH-Px activi-ties. By scavenging oxygen-derived free radicals, such as hydroxylandperoxylradicals,melatonindisclosesitsdirect antioxidanteffect.14 Inaddition,melatoninpreventsfrom
sclerosisbyinhibitingaggregationandsecretionofplatelets, prostaglandinsynthesis,andfibroblastproliferation.20 Rosa
etal.21 havereported thatmelatoninreducesdestruction
andfibrosiscausedbyoxidativestressbymeansof elevat-ingantioxidantenzymessuchasSODandGSH-Pxincirrhotic rats. Koc etal.11 have examined effects of melatoninon
oxidative stressandadhesioninratsafterlaparotomyand standardinjuryonrightuterinehorn.Inthemelatoningiven group they have found adhesion to be less, SODand CAT activitytobehighandMalondialdehite(MDA)leveltobelow. Inanotherstudymelatoninhasshowntopreventperitoneal adhesionbyreducingoxidativestress.9
Acurativetreatmentformyringosclerosis(MS)andTShas notbeenidentifiedyet.Variousantioxidantshavebeenused intreatmentandprovedtoreduceMS.21Emiretal.22have
statedthatginkgobilobaextracts,whichhaveantioxidant and anti-inflammatoryeffects,reduced or inhibitedMS in ratswhohadmyringotomy.Dundaretal.12usedascorbicacid
inmyringotomized ratears andshowedthatascorbicacid reducedthe occurrenceof MSboth inotomicroscopic and histopathologicexaminations.Karlidagetal.23 have
evalu-atedoxidativestressandTSin65patientswhounderwent tympanoplastyor tympanoplastytogetherwith mastoidec-tomy.InthepatientswithTSlevelswerefound tobehigh andCAT activity lowascomparedtothepatientswithout TS, whereas, no differencewas found between groups in SODactivity.Kazikdasetal.5havedemonstratedthatalpha
tocopherol reduces oxidative stress and MS by means of biochemical analysis, otomicroscopic, tympanometric and histopathologicassessments.
itwasshown that themeanvaluesof TASwere similarin theallstudygroupsbeforethetreatmentperiod.Themean valuesofTASweresignificantlyhigherinthemelatoninand vitamin C groups compared to control group but vitamin Cwithmelatoningroups weresimilarafterthetreatment period.Minimumandmaximumwallthicknesseswerelower inthemelatoninandvitaminCgroupscomparedtothe con-trolgroupbutthedifferenceswereinsignificant.Minimum andmaximumwallthicknessesweresimilarinthemelatonin andvitaminCgroups.
Conclusion
This study suggests that the administration of melatonin increasesTASlevelandmighthavesomeeffectonTSthat developsfollowingmyringotomy,buttheseobservationsare not statistically significant. Nevertheless, further experi-mentalstudiesonlargenumberofsubjectsusingmelatonin and/orvitaminCinhighdosesandforvariousperiodsshould bedesignedtoaffirmtheeffectsofmelatoninand/or vita-minConTSinducedbymyringotomy.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
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