Blanca 1 JesseBlanca CD652Audiology MarciaRaggio,Ph.D SanFranciscoStateUniversity Friday,November25,2011 PreventingHairCellDeathwithHsp70 Aminoglycosidesaretypesofantibioticsusedtotreat(Gramnegative) aggressiveviral,bacterialinfectionsanddiseases.TheFoodandDrug Administrationapprovesofnineaminoglycosides(gentamicin,tobramycin, amikacin,streptomycin,neomycin,paromomycin,netlimicin,spectinomycinand Kanamycin)forclinicaluseintheUnitedStates.Aminoglycosidesareinexpensive toproduceandhaveahighefficacyrate,whichmakesthistypeofantibioticvery populararoundtheworld.

.Unfortunatelythesideeffectsaredrastic, aminoglycosidesareototoxic(toxictohearingandbalance)andnephrotoxic(toxic tothekidney).Significanthearinglossandvestibulardisturbancesoccursfor nearly1520%ofpatientswhoreceiveaminoglycosides(varianceoccurson dosagesandlengthoftreatment). Mechanosensoryhaircellsfoundintheauditoryandvestibularareasoftheinner earareaffectedbyaminoglycosidestoxicitybyinducingakindofapoptopiccell death(PCD)atamolecularlevel.Mammalianhaircellsarenonregenerativemaking damageterminal.Aminoglycosideinducedapoptoticcelldeathhavebeenisolatedto twospecificapoptoticproteins,cJunNterminal(JNK)andcaspases(cystein asparticproteases).JNKisactivatedbytheaminoglycosidesabilitytogenerate withintheinnerearreactiveoxygenspecies(ROS),whicharechemicallyreactive moleculesthatplayaroleincellsignalingandhomeostasis.(Tabuchi,Nishimura, Nakamagoe,Hayashi,Nakayama,Hara)Thetwotypesofhaircells(IHC,OHC)inthe cochleahavedifferentsensitivitytoaminoglycosideasdemonstratedbythetypesof damagesthatoccurinhearing.Themostsuciptabletoaminoglycosidesarethe outerhaircells(OHC)andeveninthemostseverecasesinnerhaircells(IHC) survivabilityremainshigh.ItisalsoknownthataminoglycosidesaffectstheOHC spatialsensitivity,moreinthebasalturnthantheapicalturn.Suggestingthathigh frequencytomidfrequenciesarethemosteffectedinhearingloss. Reserchersfromtwouniversities(MedicalUniversityofSouthCarolinaandUC SanDiego)publishedapaper(CellStressSocietyInternationalJanuary152009)on alleviatingtheototoxicsideeffectsofaminoglycosidesbyusingheatshocked proteins. MonaTaleb,CarleneS.Brandon,LisaL.Cunningham(DepartmentofPathology andLaboratoryMedicine,MedicalUniversityofSouthCarolina)FuShingLee,Kelly C.Harris,(DepartmentofOtolaryngologyHeadandNeckSurgery,Medical UniversityofSouthCarolina)andWolfgangH.Dillmann,(Departmentof Endocrinology,SchoolofMedicineUCSD)conductedlabexperimentsthatinhibited JNKandCaspasesfrominducinghaircelldeaths.Theinductionofheatshock

Blanca 2 proteins(Hsp)duringlabexperimentshadsignificantresultsinlabrats.Hsp70 proteinbeingthemostpromisinginthestudyandwouldbetheprincipleinthe experiment. Heatshockedproteinsareaclassofproteinsfoundinvirtuallyallliving organism,inhumansandinbacteria.HSParecommonlynamedaftertheir molecularweight,Hsp60,Hsp70,etc.The70measuredinkilodaltonsoramu (unifiedatomicmassunit),1unitbeing1.660538921(73)1027kg.Thefunction ofHspinnormalconditionsaregenerallytomediatestresswithinthehuman biochemicalenvironment,infections,inflammation,rigorousphysicalactivity, hunger,hypoxia,waterdeprivationamongothers,consequintlyHspisalsocalled thestressprotein.WhatthestudydidisuseHsp70proteininreinforcing(Hsp70 Overexpression)thehaircellsinboththeauditoryandvestibularsystem.Thereby creatingabufferforthetoxiceffectoftheaminoglycosideinvivo. MethodoftheExperiment Theexperimentaldesigninvolvedcarefullybreedmice,twentytwotobeexact, 10withoverexpressed(OE)Hsp70(moremRNAthannormalonaspecificgene,a biotechtechnique)proteinandtwelvewildtypemice,usingbothgender. Distributedrandomly,atonly4weeksoldthemiceunderwentauditorybrainstem response(ABR)thresholdtest.Thetestwasusedtoestablishthresholdlevelsfor boththecontrolsubstanceandtheactualaminoglycosidetobeused.Ototoxicity oftencauseathresholdshiftinhearingashaircellsofaparticularfrequencydies andhaircellsofotherfrequencyencroachthedeadarea. ThemicewereplacedonawarmingpadduringABR,usingplatinum subcutaneousneedleelectrodestheresponseswasrecordedontheTDTSystem3,a NeurophysiologyWorkstation.Insertingthenoninvertingelectrodeonthevertex midlineandoverthemastoidoftherightearthenacommonelectrodewasplaced subcutaneouslyontheleftupperleg.Atoneburstof3msdurationonasampling rateof50kHzwitha1msrise/falltimesdeliveredviaafreefieldloudspeakerat4, 8,11.3,16,22.4and32kHzwasdoneatarateof31/s.Themiceearwas2.5cm fromtheloudspeakerandat0azimuth.AMedusa4channel(TDTRA4PA) preamplifierobtainedtheresponses.Filteringwasdoneat60Hzforthenotch,3kHz forthelowpassand300Hzforthehighpass.1strecordingwasat95105dBSPL witha10dBstepgraduationuntilnoresponsewasrecordable.Againfrom0dBtoa gradualincreaseof5dBtillnorecordableresponsewasseen.Afterwhichthe thresholdresponseswasgiventoacertifiedaudiologistforevaluation. ThentheaminoglycosideantibioticKanamycinorsalinewasadministered regimentallyinvivofor14days.Kanamycinsulfatepowderwasdissolvedina sterilesalinewithafinalconcentrationof45mg/mlwithanexperimentaldosage targetof700mg/kg.Administeredat0.02ml/gperbodyweightofthemicetwice perday.AllHsp70overexpressing(n=5)miceandwildtypemice(n=6)received thesameKanamycintargetdoseof700mg/kg.Thecontrolgroup,(n=5)Hsp70 overexpressingmiceandwildtypemicereceivedsubcutaneousinjectionsofsaline.

Blanca 3 Afterwhichthemicewereeuthanizedandtissuesampleswerecollectedfor immunochemistryexamination. Preparationforexaminationrequiredthedisassemblyofthevestibularand auditorysystemofeachmice.Includingtheremovaloftheutriclesandthestapes fromtheovalwindow,thecochleawasperfusedinicecoldparaformaldehydeat about4%concentrationthroughtheapicalopening.Thedissectedcochleaand exposedhaircellswerelabeledimmunohistochemically.AZeissAxioplan2(high resolutionmicroscopewithadigitalmonochromecamera)wasusedinthe fluorescentmicroscopyaswellashaircellcounts.Statisticalanalysiswasdone underonewayortwowayanalysisofvariance(ANOVA)viatheSYSTAT8.0 software. Results ThemiceinjectedwithsalineboththeHsp70OEandWildTypehadnosignificant hearingloss.Salinemicewheretestedat4kHz:F1,17=0.25andp>0.05;8kHz:F1,17 =0.01andp>0.05;11.3kHz:F1,17=0.07andp>0.05;16kHz:F1,17=0.07andp> 0.05;22.4kHz:F1,17=0.36andP>0.05;32kHz:F1,17=0.00andP>0.05.Whilethe WildTypemicewithKanamycinhadseverehearinglossasthechart(figure1) indicates,testedat4kHz:F1,17= 8.39andp<0.05;8kHz:F1,17= 11.83andp<0.05;11.3kHz: F1,17=9.41andp<0.05;16kHz: F1,17=19.89andp<0.05;22.4 kHz:F1,17=4.75andP<0.05;32 kHz:F1,17=5.11andP<0.05;. Hsp70OEwithKanamycinhad somethresholdshiftbuta significantamountofthehair Figure1 cellssurvived,testingat;4kHz:F 1,17=8.39andp<0.05;8kHz:F1,17=11.84andp<0.05;11.3kHz:F1,17=9.41andp <0.05;16kHz:F1,17=19.89andp<0.05;22.4kHz:F1,17=4.75andP<0.05;32kHz:F 1,17=5.11andP<0.05;. WildtypemicewithKanamycinhaddrasticthresholdshiftsignificantlyathigher frequencies.At22.4kHzrange,threeWildTypeKamamycinmicewerehearing impairedandallfivehadhearinglossat32kHz.ContrastedtoHsp70over expressedmicethathadKanamycinresultedinadramaticallylowerlossin thresholdshift(Hsp70OEKanamycin).AminoglycosidesspecificeffectonOHC canbeseenonfigure2,slideBaseKKanamycinWildTypeshowsnoOHCthat survivedtheexperiment.SlideMiddleGKanamycinWildTypealsoshows significantOHCdeathonthemiddlefrequencyrange.

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Figure2

ThefutureofHsp70 Hspinductionshowspromiseinthesurvivalofhaircellsduringthepresenceof aminoglycosideinthesystem.TheutriclesofmicewithoverexpressedHsp70were alsoprotectedfromapoptotichaircelldeath.Thepaperpostulatesthat,The protectiveeffectofHsp70againstaminoglycosideinducedhaircelldeathwas observedatarangeofneomycinconcentrations,suggestingthatinductionofHsp70 mayholdthepotentialtobedevelopedintoarelevantclinicalcotherapyforpatients receivingaminoglycosideantibiotics(M.Talebetal)suggestingthatfurtherstudy couldleadtoaviableagentagainstototoxicity. Theuseofaminoglycosidetypeofantibioticisoftenusedasalastresort,inthe caseofTobramycinitseitherdeathortheototoxicsideeffect.Gentamicinisoften administeredinconjunctionwithAmpacilintonewbornswithaseverefeverforthe durationoftwomonths.Furtherdicussionontheefficacyofaminoglycosidewithin themedicalindustrypersisitantlyleansonthebasicequationofhearinglossvs. death.Fortunatelyamultifacinitedapproachtomitigatingotoxicityandhaircell deathisbeingstudiesandexperimented.Fromthepotentialofstemcellsre generatingdeadorweakenedhaircellstoalternativestoaminoglycosidesmaybe emergingawayfromthelaboratorytestingandtoclinicaltrialsinthenearfuture.

Blanca 5 Source: Taleb,M.,Brandon,C.,Lee,F.,Harris,K.,Dillmann,W.,etal.(2009).Hsp70inhibits aminoglycosideinducedhearinglossandcochlearhaircelldeath.CellStress& Chaperones,14(4),427437. References: Bremer,H.,deGroot,J.,Versnel,H.,&Klis,S.(2011).Combinedadministrationof kanamycinandfurosemidedoesnotresultinlossofvestibularfunctioninguinea pigs.Audiology&NeuroOtology,17(1),2538. Tabuchi,K.,Nishimura,B.,Nakamagoe,M.,Hayashi,K.,Nakayama,M.,etal. (2011).Ototoxicity:Mechanismsofcochlearimpairmentanditsprevention.Current MedicinalChemistry,18(31),48664871. JulianE.Davies ReviewsofInfectiousDiseases,Vol.5,Supplement2.AClinicalPerspectiveof AntibioticTherapy:Aminoglycosidesvs.BroadSpectrumLactams(MayJun., 1983),pp.S261S267 (ResistancetoAminoglycosides:MechanismsandFrequency[withDiscussion]) Publishedby:OxfordUniversityPress ArticleStableURL:http://0www.jstor.org.opac.sfsu.edu/stable/4453102