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

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(2 μm)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)/180 μm。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)/0.007 5 mm²。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)/0.007 5 mm²。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。

Abstract:

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 (2 μm) 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 mice was (18.67±1.88)/180 μm. The number of peripheral nerve fibers within a single habenula perforata in the middle turn of cochlea was 57.83±9.09. The average density of spiral ganglion neurons of the cochlea, superior and inferior vestibular ganglion neurons was (20.39±2.23)/0.007 5 mm². The average density of central nerve fibers in the cochlear nerve, inferior and superior vestibular nerve was (497.06±25.28)/0.007 5 mm². Conclusion The sagittal plane of the temporal bone provides a favorable perspective for comprehensive cytological and quantitative assessments of the inner ear nervous system.