Apoptotic and necrotic cell death in cochlea treated with ototoxic chemicalsDalian Ding, Haiyan Jiang, Peng Li, Kelei Gao, Hong Sun, Richard Salvi

Center for Hearing & Deafness, University at Buffalo

2015

Fig 1B z-radial image: Cochlear culture treatedwith 10 µM cisplatin for 48 h. Hair cells weremissing. Some supporting cell labeled green forapoptosis and some labeled red for necrosis.

Fig. 1C z-radial image: Cochlear culture treatedwith 500 µM gentamicin treatment for 24 h causedsevere hair cell damage. Red necrotic and greenapoptotic cells seen in organ of Corti; many greenapoptotic supporting cells in inner sulcus ; bluehealthy cells deeper in epithelium (Fig.1C).

In Vivo Acute KM/EA DamageFig. 2 z-radial image:Control (A-D) Only blue heathy cells present, noapoptotic or necrotic labelingKM/EA (E-F) Both apoptotic and necrotic signalingpresent in inner hair cells (IHC) and outer hair cells(OHC). Intact supporting cells in blue.

AbstractBackground: Cell death can occur by apoptosis ornecrosis. Apoptosis, an orchestrated intracellular deathprogram, is triggered by a wide range of stimuli thatregulate a network of apoptosis-related proteins thatleads to the removal of damaged or excess cells. Themajor morphological characteristics of apoptosis areblebbing, cell shrinkage, nuclear fragmentation,chromatin condensation and chromosomal DNAfragmentation. In contrast, necrosis is triggered byfactors that cause cell swelling, membrane rupture andthe uncontrolled release of the cell’s contents. Weidentified cochlear cells undergoing apoptosis andnecrosis in vitro and in vivo after treatment withseveral ototoxic drugs. Cochlear explants were treatedwith gentamicin (0.5 mM) for 24 h or cisplatin (50µM) for 48 h. In adult rats, acute cell death wasinvestigated after co-administration of kanamycin(KM, 800 mg/kg, I.M.) plus ethacrynic acid (EA, 50mg/kg, I.V.); chronic cell death in adults rats wasinvestigated by treating adult rats for 9 d with dailyinjection of KM (800 mg/kg, I.M.).Methods: Cochlea hair cell death was identified usingan Apoptosis-Necrosis Quantification Kit. Apoptoticcells were identified by annexin V conjugated withFITC (green fluorescence) which specifically binds tothe phosphatidylserine exposed on the outermembrane leaflet. Necrotic cells were identified bylabeling with ethidium homodimer III (EthD-III, redfluorescence), a highly charged nucleic acid probe.Healthy cells were labeled with the membrane-permeant blue fluorescent DNA dye, Hoechst 33342.Results: In acute KM/EA damage, apoptosis wasmore common than necrosis. In contrast, duringchronic damage with KM injection for 7d necrosiswas more common than apoptosis.Conclusions: Chronic hair cell death from KM canoccur either by an apoptosis or necrosis. Potentialintervention strategies to promote hair cell survivalwill likely be most effective during KM/EA-inducedapoptosis provided that the intervention is potent andoccurs early enough to block the cell death cascade.

MethodsExperimental Models•Cisplatin or gentamicin treatment in vitroCochlear organotypic cultures were treated with 10µM cisplatin for 48 h or 500 µM gentamicin for 24h.•Acute damage in vivo with KM and EAAdult rats were treated with EA (50 mg/kg, i.v.) plusKM (800 mg/kg, i.m.) and sacrificed 24 h later.•Chronic damage in vivo with KMAdult rats were treated with KM (800 mg/kg/d x 8 d,i.m.) and sacrificed 24 h after the last injection.Apoptotic, Necrotic and Healthy Cell (ANH)Labeling KitThe “Apoptotic & Necrotic & Healthy CellsQuantification Kit” (Biotium, Inc.) was used toidentify apoptotic, necrotic and healthy cells.Cochlear explants were labeled with workingsolution in the ANH kit for 60 min and then fixedwith 10% formalin for 2 h. For adult rats, animalswere anesthetized, the middle ear cavity opened andANH working solutions in the kit perfused into thecochlea for 60 min. Afterwards, the cochlea wasperfused with 10% formalin in PBS.Cochlear surface preparations and observationsAfter fixation, the cochlear basilar membrane wasmicro-dissected out from the bony spiral plate of themodiolus. The basilar membrane was mounted inantifade solution on glass slides, and examinedunder a confocal microscope.ResultsIn Vitro Cochlear CulturesFig. 1A z-radial image: Normal organ of Corticultured for 48 h; only blue healthy cells in organ ofCorti

In Vivo Chronic KM DamageFig. 3 surface image organ CortiControl (A-D) No apoptotic or necrotic cells inControl; only blue healthy cellsKM 9 d (E-H) Both apoptotic and necrotic signalingoccurred in hair cells, but necrotic cell death seemedto predominate.

Conclusions•Ototoxic drugs can destroy the sensory hair cellsand supporting cells in the organ of Corti by eitherapoptosis or necrosis depending on the drug, doseand mode of treatment.•In cochlear cultures treated for 48 h with 10 mMcisplatin, there was considerable hair cell death thatwas accompanied by signs of apoptotic and necroticcell death in the supporting cells•In cochlear cultures treated with gentamicin for 24there were signs of both apoptotic and necrotic cellsignaling in both hair cells and supporting cells in theinner sulcus.•In vivo, rats treated with KM/EA showed bothapoptotic and necrotic cell death with necroticlabeling being the more common of the two.•In vivo, rats treated for 9 d with KM, both necroticand apoptotic labeling was present with apoptoticlabeling being more prominent.

Supported in part by NIH grant 5R01DC011808

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