卡那霉素联合呋塞米快速诱导小鼠耳蜗损伤

目的 探讨卡那霉索和呋塞米联合应用对小鼠耳蜗的毒性作用,建立一种可靠的小鼠感音神经性聋模型.方法 选用3～4周龄的CBA/J小鼠为实验对象,按1 g/kg的剂量皮下注射卡那霉素,30～45 min后按0.4 g/kg的剂量腹腔注射呋塞米.在注射前、注射后12 h、24 h、48 h、7 d、2周、4周及12周分别应用听性脑干反应(ABR)检测小鼠听觉功能的改变;应用异硫氰酸荧光素标记的鬼笔环肽及碘化丙啶染色、半薄切片甲苯胺蓝染色、脱氧核苷酸末端转移酶介导的dUTP缺口末端标记技术、扫描电镜等观察小鼠毛细胞死亡的模式和程度;同时通过透射电镜观察记录耳蜗血管纹形态及厚度的变化.结果 序贯应用卡那霉素及呋塞米后12 h小鼠ABR阈值开始上升,随后的36 h内持续进行性上升,继而稳定在90 dB左右.应用激光共聚焦显微镜在药物注射后12 h观察到耳蜗底回外毛细胞开始出现死亡,24 h时底回外毛细胞基本全部消失,同时顶回外毛细胞开始出现死亡,至48 h时整个耳蜗外毛细胞绝大部分死亡;而内毛细胞的损伤至给药后7 d时才开始出现,随时间推移仍有部分内毛细胞完好无损.死亡的毛细胞均具有典型的凋亡细胞特征.扫描电镜观察发现在药物注射后受损毛细胞出现纤毛消失、表皮板塌陷,随后支持细胞增生并在该处形成瘢痕.血管纹表面边缘细胞在给药后出现部分胞体融合并且有坏死物自胞内排出,随后边缘细胞表面大部分微绒毛消失,胞体呈"石块"样改变.透射电镜结果 显示血管纹厚度在给药后进行性下降,主要为边缘细胞萎缩造成.结论 单次序贯应用卡那霉素及呋塞米能快速诱导小鼠耳蜗毛细胞大量死亡,适合应用于建立小鼠感音神经性聋模型.

Rapid cochlea lesion induced by co-administration of kanamycin and furosemide in mouse

Objective To investigate the ototoxicity of co-administration of kanamycin and furosemide in mouse and establish a reliable model to induce a sensorineural hearing loss.Methods CBA/J mice strain was selected,with the age around 3-4 weeks old,to be received a sigle subcutaneous injection of kanamycin at dose of 1 g/kg and another sigle intraperitoneal injection of furosemide at dose of 0.4 g/kg 30-45 min afterward.The auditory brainstem resperise(ABR)threshold shift was tested.The series of experimental methods including propidium iodide,phalloidin staining,semithin section toluidine blue staining,TUNEL,scanning electron microscopy and transmission electron microscopy were applied to observe the chareristics of the lesion of cochlea and hair cells.The time course wag set as following:before injection,12,24,48 hours and 1,2,4,12 weeks after injections,respectively.Results The ABR threshold shift was firstly presented a significant increase at 12 h after injection at 2,4,8 kHz,then the ABR threshold kept going up during next 36 h until it was presented a stable level around 90 dB.Pathological examination showed an absence of outer hair cells at basal turn rapidly since 12 h after treatment,and then by 48 h the most commonly observed lesion,where all outer hair cells throughout the length of the cochlea were killed,in the contrast,however,the inner hair cells loss were delayed and mild.TUNEL-positive nuclei demonstrated that most hair cells died via an apoptotic pathway.In scanning electron microscopy abundance of necrotic outer hair cells were detected by 24 h after treatment,in which reticular lamina were collapsed.Then all outer hair cells were replaced by expansion of heads of supporting cells.At 48 h after treatment.marginal cells presented a swollen and some of them were observed to be fused.In addition,spherical cell extrusion appeared to leak out from some marginal cells.By 2 weeks,nearly all microvillis were lost and marginal cells presented a shape of stone-like change.A significant and progressive decrease in strial vascularis thickness Wag found,of which the reason probably related with a reduction in volume of marginal cells.Conclusion This systemic protocol eliminates hair cells extensively in vivo,and it could be a reliable model to examine different aspects of cochlear pathology in transgenie or mutant mice strains.