正常小鼠颞骨矢状面切片与内耳神经密度测定技术  

Sagittal sectioning of temporal bones and quantitative measurement of neural densities in the inner ear in normal mice

作　　者：丁大连[1,2,3,4] 石林 邢雅智[2] 李鹏 亓卫东[6] 张建辉 蒋海燕 时海波[2] DING Dalian;SHI Lin;XING Yazhi;LI Peng;QI Weidong;ZHANG Jianhui;JIANG Haiyan;SHI Haibo(State University of New York at Buffalo,NY 14214,USA;Department of Otolaryngology Head and Neck Surgery,the Sixth People’s Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200233,China;Department of Otolaryngology Head and Neck Surgery,the Third Affiliated Hospital of Sun Yat-sen University,Guangzhou 510630,China;Department of Otolaryngology Head and Neck Surgery,Chengdu Third People’s Hospital,Chengdu 610014,China;Department of Otolaryngology Head and Neck Surgery,the First Affiliated Hospital,Dalian Medical University,Dalian 116011,China;Department of Otolaryngology Head and Neck Surgery,Huashan Hospital,Fudan University,Shanghai 200040,China)

机构地区：[1]纽约州立大学布法罗大学,纽约布法罗14214 [2]上海交通大学医学院附属第六人民医院耳鼻咽喉头颈外科,上海200233 [3]中山大学附属第三医院耳鼻咽喉头颈外科,广东广州510630 [4]成都市第三人民医院耳鼻咽喉头颈外科,四川成都610014 [5]大连医科大学附属第一医院耳鼻咽喉头颈外科,辽宁大连116011 [6]复旦大学附属华山医院耳鼻咽喉头颈外科,上海200040

出　　处：《中国耳鼻咽喉颅底外科杂志》2024年第6期69-75,共7页Chinese Journal of Otorhinolaryngology-skull Base Surgery

基　　金：国家自然科学基金项目(82171147,82071050,81973913);科技部国家重点研发计划(2023YFC2410205);四川省自然基金(2023NSFC0621)。

摘　　要：目的从颞骨矢状面切片的观察角度,建立定量观察内耳毛细胞、神经元及其神经纤维的新方法。方法采用4只出生后45 d CBA/CaJ小鼠的颞骨,将2.5%戊二醛磷酸盐缓冲液灌入内耳腔并将颞骨浸泡固定6 h,再将颞骨浸入2%四氧化锇溶液中浸泡2 h。颞骨经脱钙、脱水及包埋后,沿着颞骨矢状面切取半薄切片(2μm)并用0.5%甲苯胺蓝染色。在光学显微镜下的小视野范围内,对毛细胞、神经元及神经纤维进行定量观察。结果从平行颞骨内侧壁的平面切片开始收集与观察。当切开颞骨内侧壁进入耳蜗神经孔和前庭上神经孔时,即可得到对应神经束及前庭下神经束中枢端神经纤维的横断切片。当切到椭圆囊斑和上半规管壶腹嵴及外半规管壶腹嵴的横断截面时,获得上述结构所含毛细胞的横断面切片,同时也获得前庭上神经元细胞体及其两端神经纤维的横断面切片。当切到球囊斑和后半规管壶腹嵴的横断截面时,获得其中所含毛细胞的横断面切片,同时也获得前庭下神经元的细胞体及其两端神经纤维的横断面切片。切到耳蜗骨性螺旋板的横断截面时,获得螺旋神经节外周端神经纤维的横断面切片。在切到蜗轴的横断截面,可获得耳蜗毛细胞和螺旋神经节的横断面切片。本实验发现正常小鼠各个前庭感觉上皮区的毛细胞平均密度为(18.67±1.88)/180μm。耳蜗底回中部单个疆孔内的螺旋神经节外周端神经纤维的数量为(57.83±9.09)根。耳蜗螺旋神经节和前庭上神经元及前庭下神经元的平均密度为(20.39±2.23)/0.0075 mm^(2)。耳蜗神经束和前庭下神经束及前庭上神经束的中枢端神经纤维的平均密度为(497.06±25.28)/0.0075 mm^(2)。结论颞骨矢状面切片的观察角度有利于对内耳神经系统做出全方位的细胞学定量评估。Objective To establish a new method for quantitative observation of inner ear hair cells,neurons and nerve fibers from sagittal sections of temporal bones.Methods Temporal bones of four CBA/CaJ mice at 45 days of age were removed under deep anesthesia.The inner ear cavity was perfused with 2.5%glutaraldehyde and then immersed in the fixative for 6 hours.The temporal bones were rinsed with PBS,and immersed in 2%osmium tetroxide for 2 hours.After routine decalcification,dehydration,and epoxy resin embedding,semi-thin sections(2μm)were prepared along the sagittal plane of the temporal bones and stained with 0.5%toluidine blue.Quantitative observations of hair cells,neurons and nerve fibers were performed in a small field of view under an optical microscope.Results Collection and observations began with planar sections parallel to the medial wall of the temporal bone.Upon sectioning through the medial wall to access the cochlear nerve canal and superior vestibular nerve canal,cross-sections of the central nerve fibers of the corresponding cochlear nerve bundles and inferior vestibular nerve bundles were obtained.Sections through the macula of the utricle,the crista ampullae of the superior and lateral semicircular canals revealed cross-sectional profiles of hair cells in these structures,as well as the soma of superior vestibular neurons and their peripheral and central nerve fibers.Similarly,sections through the macula of the saccule and the crista ampulla of the posterior semicircular canal were also profiled cross-sectional views of their hair cells,along with the soma of inferior vestibular neurons and their peripheral and central nerve fibers.Cross-sections of the bony spiral lamina of the cochlea exposed the peripheral nerve fibers of the spiral ganglion,while sections through the center axis of modiolus revealed the cross-section of cochlear hair cells and the soma of spiral ganglion neurons.Quantitative analysis demonstrated that the average density of hair cells in vestibular sensory epithelium of normal mi

关 键 词：颞骨 矢状面切片 耳蜗 前庭 神经 

分 类 号：R764[医药卫生—耳鼻咽喉科]