1.Center for Hearing and Deafness, State University of New York at Buffalo;2.大连医科大学附属第一医院耳鼻咽喉头颈外科;3.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;4.中山大学附属第三医院耳鼻咽喉头颈外科;5.复旦大学附属华山医院耳鼻咽喉头颈外科;6.成都市第三人民医院耳鼻咽喉头颈外科
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）。
- DING Dalian 1
- SHI Lin 2
- XING Yazhi 3
- LI Peng 4
- QI Weidong 5
- ZHANG Jianhui 6
- SHI Haibo 3
Center for Hearing and Deafness, State University of New York at Buffalo
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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 采用出生后45天CBA/CaJ小鼠的颞骨，将2.5%戊二醛溶液灌入内耳腔并将颞骨浸泡其中6小时，再将颞骨浸入2%四氧化锇溶液2小时。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 / 0.0075mm2。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 / 0.0075mm2。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 the sagittal slices of temporal bones. Methods Temporal bones of 4 CBA/CaJ mice at 45 days of age were removed under deep anesthesia. Inner ear cavity in the temporal bones were perfused with 2.5% glutaraldehyde solution and immersed in the fixative for 6 hours. The temporal bones were then immersed in 2% osmium tetroxide solution for 2 hours. After decalcification, dehydration and embedding, semi-thin sections (2 μm) were cut along the sagittal plane of the temporal bones and stained with 0.5% toluidine blue. The hair cells, neurons and nerve fibers were quantitatively observed in a small field of view under an optical microscope. Results The collection and observation began with plane slices parallel to the medial wall of the temporal bone. When the medial wall of the temporal bone was cut to enter the cochlear nerve canal and the superior vestibular nerve canal, cross-sectional sections of the central end of cochlear nerve bundles and the inferior vestibular nerve bundles were obtained. When the cross-section of the macula of utricle and the crista ampullae of the superior and lateral semicircular canals is cut, the cross-section of the hair cells in above structures is obtained, and the cross-section of the cell bodies of the superior vestibular neurons and the nerve fibers at both peripheral and central ends are also obtained. When the cross-sections were cut to the macula of saccule and the crista of ampulla of the posterior semicircular canal, Cross-sectional slices of the hair cells contained therein were obtained, as well as cross-sectional slices of the cell bodies of the inferior vestibular neurons and the nerve fibers at both ends thereof. When the cross section of the bony spiral plate of the cochlea is cut, the cross section of the peripheral nerve fibers of the spiral ganglions is obtained. When the cross section of the modiolus is cut, the cross section of the cochlear hair cells and the body of spiral ganglion can be obtained. In this experiment, we found that the averaged density of hair cells in each vestibular sensory epithelium of normal mice was 18.67±1.88/180μm. The number of peripheral nerve fibers of the spiral ganglion in a single habenula perforata was 57.83±9.09. The averaged density of the spiral ganglion of the cochlea and the superior and inferior vestibular neurons was 20.39±2.23/0.0075mm2. The averaged density of the nerve fibers towards the central nervous system in the cochlear nerve, the inferior and superior vestibular nerve was 497.06±25.28/0.0075 mm2. Conclusion The observation angle of sagittal slices of the temporal bone is conducive to a comprehensive cytological quantitative evaluation of the inner ear nervous system.
