A Mouse Model For Rapid Cochlear Lesions

PART 1 Two regimes for cochlear lesions in miceObjective:To compare the effects of two regimes for cochlear lesions in mice.Methods:Experimental mice were received either 700 mg of kanamycin/kg body weight by subcutaneous injection twice daily for 14 days or a sigle subcutaneous injection of kanamycin at 1000 mg/kg body weight, followed 30-45 min later by a sigle intraperitoneal injection of furosemide at 400 mg/kg body weight. The auditory brainstem response were performed prior to the beginning of the study,1 day and 7 days after drug adminstration. Succinate dehydrogenase (SDH) activty was examined at 7 days after drug adminstration to assesse the mitochondrial energetic function in hair cells.Results:After 14 days of kanamycin treatment, animals developed significant threshold shifts and the threshold shifts increased further during the third week. In animals treated with kanamycin and furosemide, threshold shifts were also elevated at 1 day posttreatment and continuted to increase during the second week. The threshold shifts in the latter group were larger than those in the former group. SDH staining results showed there was a significant reduction of SDH activity in outer hair cells (OHCs) in the basal turn in the animals treated with kanamycin alone. In contrast, SDH activity was reduced in OHCs in both basal turn and apical turn in the animals treated with kanamycin and furosemide. In both cases, SDH activity showed no change in inner hair cells.Conclusions:Cochlear lesions can be induced by both regimes but co-administration of kanamycin and furosemide can cause a more severe damage to the cochlea and this precedure is easy to perform. PART 2 Rapid cochlear lesions induced by co-administration of kanamycin and furosemide in miceObjective:To investigate the ototoxicity of co-administration of kanamycin and furosemide in mice and present a reliable approach for induing a rapid and profound sensorineural hearing loss.Methods:CBA/J mice,3-4 weeks old received a sigle subcutaneous injection of kanamycin at 1000 mg/kg body weight, followed 30-45 min later by a sigle intraperitoneal injection of furosemide at 400 mg/kg body weight. The auditory brainstem response (ABR) threshold shifts, extent and defining characteristics of the cochlear lesions were assessed and verified by propidium iodide and phalloidin staining, toluidine blue staining, TUNEL, scanning electron microscopy (SEM) prior to the beginning of the study,12 hours,1,2,7,14,28 and 112 days after injections.Results:This drug combination resulted in an significant increased ABR threshold shifts firstly at 12h posttreatment, which increased further in the first 2 days posttreatment and then stabilized around 90 dB SPL thereafter. Histopathological examination showed an absence of outer hair cells (OHCs) at basal turn rapidly from 12 hours posttreatment. By 2 days the most commonly observed lesion was that all OHCs throughout the length of the cochlea were killed, while inner hair cells (IHCs) loss were delayed and mild. TUNEL-positive nuclei demonstrated that most hair cells died via an apoptotic pathway. In SEM abundance of damaged OHCs were detected by 1 day posttreatment, in which reticular lamina were collapsed. Then all OHCs were repalced by expansion of heads of the supporting cells. Spiral ganglion neurons also exhibited remarkable degeneration.Conclusion:This reliable systemic protocol eliminated hair cells extensively in vivo and is a means with which to examine different aspects of cochlear pathology in transgenic or mutant strains. PART 3 Morphological and functional response of cochlear lateral wall following a sensorineural hearing loss in miceObjective:To investigate the alteration of the endocochlear potential (EP) and experssion pattern of some key potassium transporters in the cochlear lateral wall following a long-term sensorineural hearing loss (SNHL).Methods:CBA/J mice were administered a single dose of kanamycin followed by furosemide. The alteration of EP, morphological change of the lateral wall, expression of the a 1 and a 2 isoforms of Na,K-ATPase, Na-K-2Cl-Cotransporter-1 (NKCC1) and potassium channel KCNQ1 were assessed.Results:The EP displayed a significant decline at 12 hours posttreatment followed by complete recovery by 2 days posttreatment. Then the EP maintained at near normal levels in animals deafened for periods up to 112 days. By 2 days posttreatment marginal cells were swollen and some of them were observed to be fused. By 14 days nearly all microvillis were lost and marginal cells presented a sign of stone-like change. There was also a significant and progressive decrease in stria vascularis thickness, which was predominantly due to atrophy of marginal cells. Meanwhile both protein and mRNA expression of a 1 and a 2 isoforms of Na,K-ATPase and NKCC1 in the lateral wall were dramatically reduced following a long-term deafening but KCNQ1 expression remained unchanged.Conclusion:The EP remained at normal levels following a long-term aminoglycoside-induced hearing loss. Simultaneously reduced NKCC1 and Na,K-ATPase expression in the cochlear lateral wall may contribute to such conservation of EP.