庆大霉素鼓室内注射后在脑干前庭与听觉中枢的分布和损伤的研究

发布时间：2018-01-18 15:15

本文关键词：庆大霉素鼓室内注射后在脑干前庭与听觉中枢的分布和损伤的研究　出处：《复旦大学》2011年博士论文　论文类型：学位论文

【摘要】：目的1)探索庆大霉素鼓室内注射后在脑干前庭中枢是否有分布及其分布特点2)探索鼓室内注射庆大霉素后在脑干听觉中枢是否有分布及其分布特点3)鼓室内注射庆大霉素后对于脑干前庭及听觉核团的影响方法1)本实验选用正常成年白化豚鼠作为研究对象,经鼓室注射30mg/ml庆大霉素作为动物模型；经卵圆窗注射HRP逆行示踪剂并结合既往文献,定位豚鼠前庭传出神经元；2)造模成功后,在不同时间点处死；行免疫组织化学、免疫荧光染色组,用多聚甲醛进行心脏灌注固定、取材,行冰冻切片,获得连续脑片；行透射电镜组,处死后用多聚甲醛和戊二醛混合液进行心脏灌注固定、取材,继而行振动切片；3)通过甲苯胺蓝染色标记脑干不同核团的相对位置,通过DAB染色,识别豚鼠前庭传出神经元；通过免疫组织化学单标、三标技术(anti-gentamicin标记庆大霉素,anti-betaⅢtubulin标记神经元,DAPⅠ标记细胞核),了解庆大霉素的分布情况；4)荧光显微镜、激光共聚焦扫描显微镜、透射电镜下观察并拍照,Adobe photoshop CS2处理图片,庆大霉素阳性细胞计数,并用SPSS 16.0进行统计分析。结果1)鼓室内注射后,庆大霉素在前庭传出神经元、上橄榄核、耳蜗核均有染色,在前庭神经节及前庭复合核无庆大霉素分布。在前庭传出神经元和上橄榄核,庆大霉素在给药侧及给药对侧均有染色,主要分布于神经元胞浆及其突起,神经核内未见庆大霉素染色。在耳蜗核,仅给药侧出现明显庆大霉素阳性标记,对侧耳蜗核未及庆大霉素染色；主要分布于神经元胞体以外,神经元胞体内未见任何阳性标记；背侧耳蜗核较腹侧耳蜗核分布强。2)庆大霉素给药后24小时,前庭传出神经元即有庆大霉素染色。至3天达到高峰,7天时仍保持等同水平,至14天时庆大霉素阳性细胞数明显减少,至30天时进一步减少。3)背侧耳蜗核内耳蜗神经纤维、进入脑干的前庭神经纤维以及上橄榄核下行传出神经通路全程均可及庆大霉素标记阳性。4)鼓室内注射庆大霉素3天后,前庭Ⅰ型、Ⅱ型毛细胞的传入及传出突触均可见显著的空泡样变性；支配Ⅱ型毛细胞的传入及传出突触发生空泡样变性后,无法区分。5)给药后3天,前庭传出神经核内的有髓神经纤维髓鞘板层松解,原浆型星形胶质细胞严重水肿；神经元突起出现显著的空泡样变性,内容物溶解。给药后30天,有髓神经髓鞘板层排列紧凑整齐；神经元胞质及核明显肿胀,电子密度降低,核内染色质缺失,胞浆内线粒体可见少许小泡。给药后3天,耳蜗核内可见有髓神经纤维髓鞘板层广泛松解,而神经元胞浆、树突、细胞核未见明显异常；给药后30天,有髓神经纤维髓鞘板层排列紧凑整齐,另外可见神经元外包绕的星形胶质细胞突起明显水肿。给药后3天,上橄榄核内有髓神经纤维髓鞘板层松解,树突内空泡样变性、胞浆内出现较多初级溶酶体；给药后30天,有髓神经髓鞘板层排列紧凑整齐,星形胶质细胞胞质、突起及核明显肿胀,核团内可见暗细胞。结论1)鼓室内注射庆大霉素后,不仅分布于耳蜗、前庭外周器官,相应的中枢核团也有分布,且保留庆大霉素一段时间；逆行性轴突运输是庆大霉素进入前庭传出神经元、上橄榄核的可能方式。顺行性轴突运输是庆大霉素进入耳蜗核的可能方式。2)前庭复合核无庆大霉素分布,可能原因包括：前庭神经节基本无庆大霉素分布,以及跨突触转运庆大霉素较不容易。3)庆大霉素对分布阳性的核团造成直接损伤,对传出神经核团的损伤较传入核团严重。给药后3天,传出神经核团神经纤维髓鞘板层松解、神经元突起空泡样变性、原浆型星形胶质细胞水肿；至30天,髓鞘病变可逆性恢复,但个别神经元发生严重肿胀,甚至凋亡；给药后,耳蜗核出现显著的神经纤维髓鞘变性,但神经元未见明显异常。
[Abstract]:1) to explore the intratympanic gentamicin injection in vestibular central whether distribution and distribution characteristics of 2) to explore the intratympanic gentamicin injection after brain stem auditory center whether distribution and distribution characteristics of 3) effect on vestibular and auditory nuclei after intratympanic injection of gentamicin
Method 1) used in the experiment of normal adult albino guinea pigs as the research object, through the tympanic injection of 30mg/ml gentamicin as animal model; the oval window injection HRP retrograde tracer technique combined with previous literature, localization of efferent vestibular neurons; 2) after the success of the model, in different time points were performed; immunohistochemistry, immunofluorescence staining group with paraformaldehyde perfusion fixation, were performed on frozen sections obtained in a continuous brain slice; for TEM group, after the death of paraformaldehyde and glutaraldehyde mixture for perfusion fixation, were then for vibration section; 3) the relative position of brainstem nuclei labeled by toluidine blue staining, by DAB staining, identification of efferent vestibular neurons; by immunohistochemical single standard, three standard technology (anti-gentamicin labeled gentamicin, anti-beta III tubulin labeled neurons, DAP 1 standard Remember the nucleus), understand the distribution of gentamicin. 4) fluorescence microscope, laser scanning confocal microscopy, transmission electron microscopy and photo taking, Adobe Photoshop CS2 image processing, gentamicin positive cell count, and SPSS 16 for statistical analysis.
Results 1) after intratympanic injection of gentamicin, in the efferent vestibular neurons, superior olivary nucleus, cochlear nucleus were stained in the vestibular ganglia and the vestibular nuclear complex without gentamicin distribution. In efferent vestibular neurons and the superior olivary nucleus, gentamicin in the medication administration and side stain are on the side, mainly distributed in the cytoplasm of neurons and their processes no, nerve nucleus staining. Gentamicin in the cochlear nucleus, only medication side appeared positive staining of gentamicin, cochlear nucleus than gentamicin; mainly distributed in other neurons, neurons were not found in any positive staining; the dorsal cochlear nucleus was ventral cochlear nucleus distribution of strong.2) after administration of gentamicin 24 hours, the vestibular efferent neurons staining. Gentamicin to 3 days to reach the peak, 7 days remain equivalent to 14 days, when gentamicin positive cells were significantly reduced to 30 days To further reduce.3) within the dorsal cochlear nucleus cochlear nerve fibers, vestibular nerve fibers enter the brainstem and the superior olivary nucleus descending efferent pathway can be full labeled.4) and gentamicin intratympanic injection of gentamicin for 3 days, vestibular type I, type II hair cells and afferent efferent synapses were vacuolar degeneration was dominant; type II hair cell afferent and efferent synapses had vacuolar degeneration, unable to distinguish.5) 3 days after administration, the efferent nerve nucleus of myelinated nerve fibers of myelin loose vestibular, protoplasmic astrocyte edema; neurites appeared vacuolar degeneration significantly, the contents of dissolved drug. After 30 days, myelin sheath arranged in neat compact; neuron cytoplasm and nuclear swelling, the electron density decreases, the nuclear chromatin deletion in mitochondria showed a few vesicles. 3 days after administration, In the cochlear nucleus visible myelin lamellar solution, and the cytoplasm of neurons, dendrites, nucleus had no obvious abnormalities; 30 days after administration of myelin lamellar compact arrangement is neat, also found in the neurons wrapped by astrocytic processes edema. 3 days after administration. Olivary nucleus of the myelinated nerve fibers in the myelin sheath lysis, dendrites of vacuolar degeneration, more lysosomes appeared in cytoplasm; 30 days after administration of myelin sheath compact arrangement neat, astrocyte cytoplasm and nuclear processes, obvious swelling, nuclei can be seen in the dark cell.
Conclusion 1) after intratympanic injection of gentamicin, not only distributed in the cochlear, vestibular peripheral organs, the central nuclei are also distributed, and the retention time of gentamicin; retrograde axonal transport of gentamicin is into the efferent vestibular neurons may be way superior olivary nucleus. Anterograde axonal transport is possible way into the cochlear nucleus of gentamicin.2) vestibular nuclear complex without gentamicin distribution, possible causes include: vestibular ganglion basically no gentamicin distribution, and trans synaptic transport is not easy to gentamicin.3) caused by gentamicin on the distribution of positive nuclei directly to the efferent nerve injury, nucleus afferent nucleus injury was serious. 3 days after administration of efferent nerve nucleus group of nerve fibers of myelin loose, neuronal vacuolation, protoplasmic astrocyte edema; to 30 days, reversible recovery of myelin lesions, but individual nerve There were severe swelling and even apoptosis in the elements. After the administration, the nucleus of the cochlea appeared significant degeneration of nerve fiber myelin sheath, but no obvious abnormalities were found in the neurons.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R764

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