三组固定方式对应小鼠耳蜗组织染色效果的研究  

Effects of Fixation Methods on Cochlear Staining in Mice

作　　者：位格格 邱士伟 熊巍 袁永一 聂卉一 戴朴 WEI Gege;QIU Shiwei;XIONG Wei;YUAN Yongyi;NIE Huiyi;DAI Pu(Sixth Medical Center of the PLA General Hospital,National Clinical Research Center for Otolaryngologic Diseases,College of Otolaryngology Head and Neck Surgery,Beijing 100048,China)

机构地区：[1]中国人民解放军总医院第六医学中心耳鼻咽喉头颈外科医学部,国家耳鼻咽喉疾病临床医学研究中心,北京100048

出　　处：《中华耳科学杂志》2024年第1期141-145,共5页Chinese Journal of Otology

基　　金：国家自然科学基金(82271185,82271177)。

摘　　要：目的研究不同固定方式对耳蜗结构及细胞形态的影响、探索耳蜗内特定细胞类型的最佳固定方式,最大限度保存耳蜗不同细胞类型以及细胞内细胞器的形态结构,为生理和病理条件下的耳蜗细胞形态学研究提供一种更为个性化的免疫荧光染色方法。方法选择野生型C57BL/6小鼠9只,将小鼠随机分为一组、二组及三组,每组3只。一组采用0.9%氯化钠溶液+4%多聚甲醛(polyformaldehyde,PFA)心脏灌流、二组采用直接4%PFA心脏灌流、三组采用耳蜗4%PFA灌流。对3组固定后的耳蜗采用常规切片免疫荧光染色,激光扫描共聚焦显微镜在低倍镜、高倍镜以及超高分辨率等模式下观察并比较3组耳蜗内不同细胞和细胞内形态结构。结果3种染色方法在不同观察模式下均能清楚显示毛细胞、神经丝形态结构,3组毛细胞、神经丝形态结构比较,差异无统计学意义(P>0.05)。血管纹区域在一组的固定方式下,其形态结构相比于其他两种固定方式保存的更为完整。结论3组耳蜗固定方式均可以用来研究Corti's器、螺旋神经节区域的形态结构,而一组的固定方式更适合用于研究血管纹区域内的细微结构。Purpose To report effects of fixation techniques on demonstration of general structures and cell morphology in the cochlea with preservation of cellular structures and intracellular organelles,to improve current protocols for best immunostaining results.Method One-month-old wild-type C57BL6J mice with normal hearing(n=9)were randomly divided into three groups with three mice in each group.Under general anesthesia,animals were perfused with saline+4%paraformaldehyde(PFA)(Group 1)or 4%PFA directly into the heart(Group 2),or decapitated with the cochlea harvested and directly perfused with 4%PFA.The fixed cochlea was cryosectiond and stained for immunofluorescence,followed by laser confocal scanning microscopy imaging at low and ultra-high magnification.Results Structures of hair cells and auditory nerves were clearly revealed by all three fixation protocols with similar qualities(P>0.05).However,the structure of stria vascularis was better preserved in group 1 than the other two groups.Conclusions The three fixation methods in this study are all adequate for morphology studies of the organ of Corti and spiral ganglia,while saline+4%PFA perfusion may be preferable for studying ultrastructure of the stria vascularis.

关 键 词：心脏灌流 耳蜗灌流 Corti's器 血管纹 螺旋神经节 

分 类 号：R764[医药卫生—耳鼻咽喉科]