本文關鍵詞：慶大霉素鼓室內(nèi)注射后在腦干前庭與聽覺中樞的分布和損傷的研究　出處：《復旦大學》2011年博士論文　論文類型：學位論文

  更多相關文章： 慶大霉素 前庭傳出神經(jīng)元 耳蝸核 上橄欖核 軸突運輸 藥物作用

【摘要】：目的1)探索慶大霉素鼓室內(nèi)注射后在腦干前庭中樞是否有分布及其分布特點2)探索鼓室內(nèi)注射慶大霉素后在腦干聽覺中樞是否有分布及其分布特點3)鼓室內(nèi)注射慶大霉素后對于腦干前庭及聽覺核團的影響 方法1)本實驗選用正常成年白化豚鼠作為研究對象,經(jīng)鼓室注射30mg/ml慶大霉素作為動物模型；經(jīng)卵圓窗注射HRP逆行示蹤劑并結合既往文獻,定位豚鼠前庭傳出神經(jīng)元；2)造模成功后,在不同時間點處死；行免疫組織化學、免疫熒光染色組,用多聚甲醛進行心臟灌注固定、取材,行冰凍切片,獲得連續(xù)腦片；行透射電鏡組,處死后用多聚甲醛和戊二醛混合液進行心臟灌注固定、取材,繼而行振動切片；3)通過甲苯胺藍染色標記腦干不同核團的相對位置,通過DAB染色,識別豚鼠前庭傳出神經(jīng)元；通過免疫組織化學單標、三標技術(anti-gentamicin標記慶大霉素,anti-betaⅢtubulin標記神經(jīng)元,DAPⅠ標記細胞核),了解慶大霉素的分布情況；4)熒光顯微鏡、激光共聚焦掃描顯微鏡、透射電鏡下觀察并拍照,Adobe photoshop CS2處理圖片,慶大霉素陽性細胞計數(shù),并用SPSS 16.0進行統(tǒng)計分析。 結果1)鼓室內(nèi)注射后,慶大霉素在前庭傳出神經(jīng)元、上橄欖核、耳蝸核均有染色,在前庭神經(jīng)節(jié)及前庭復合核無慶大霉素分布。在前庭傳出神經(jīng)元和上橄欖核,慶大霉素在給藥側及給藥對側均有染色,主要分布于神經(jīng)元胞漿及其突起,神經(jīng)核內(nèi)未見慶大霉素染色。在耳蝸核,僅給藥側出現(xiàn)明顯慶大霉素陽性標記,對側耳蝸核未及慶大霉素染色；主要分布于神經(jīng)元胞體以外,神經(jīng)元胞體內(nèi)未見任何陽性標記；背側耳蝸核較腹側耳蝸核分布強。2)慶大霉素給藥后24小時,前庭傳出神經(jīng)元即有慶大霉素染色。至3天達到高峰,7天時仍保持等同水平,至14天時慶大霉素陽性細胞數(shù)明顯減少,至30天時進一步減少。3)背側耳蝸核內(nèi)耳蝸神經(jīng)纖維、進入腦干的前庭神經(jīng)纖維以及上橄欖核下行傳出神經(jīng)通路全程均可及慶大霉素標記陽性。4)鼓室內(nèi)注射慶大霉素3天后,前庭Ⅰ型、Ⅱ型毛細胞的傳入及傳出突觸均可見顯著的空泡樣變性；支配Ⅱ型毛細胞的傳入及傳出突觸發(fā)生空泡樣變性后,無法區(qū)分。5)給藥后3天,前庭傳出神經(jīng)核內(nèi)的有髓神經(jīng)纖維髓鞘板層松解,原漿型星形膠質(zhì)細胞嚴重水腫；神經(jīng)元突起出現(xiàn)顯著的空泡樣變性,內(nèi)容物溶解。給藥后30天,有髓神經(jīng)髓鞘板層排列緊湊整齊；神經(jīng)元胞質(zhì)及核明顯腫脹,電子密度降低,核內(nèi)染色質(zhì)缺失,胞漿內(nèi)線粒體可見少許小泡。給藥后3天,耳蝸核內(nèi)可見有髓神經(jīng)纖維髓鞘板層廣泛松解,而神經(jīng)元胞漿、樹突、細胞核未見明顯異常；給藥后30天,有髓神經(jīng)纖維髓鞘板層排列緊湊整齊,另外可見神經(jīng)元外包繞的星形膠質(zhì)細胞突起明顯水腫。給藥后3天,上橄欖核內(nèi)有髓神經(jīng)纖維髓鞘板層松解,樹突內(nèi)空泡樣變性、胞漿內(nèi)出現(xiàn)較多初級溶酶體；給藥后30天,有髓神經(jīng)髓鞘板層排列緊湊整齊,星形膠質(zhì)細胞胞質(zhì)、突起及核明顯腫脹,核團內(nèi)可見暗細胞。 結論1)鼓室內(nèi)注射慶大霉素后,不僅分布于耳蝸、前庭外周器官,相應的中樞核團也有分布,且保留慶大霉素一段時間；逆行性軸突運輸是慶大霉素進入前庭傳出神經(jīng)元、上橄欖核的可能方式。順行性軸突運輸是慶大霉素進入耳蝸核的可能方式。2)前庭復合核無慶大霉素分布,可能原因包括：前庭神經(jīng)節(jié)基本無慶大霉素分布,以及跨突觸轉運慶大霉素較不容易。3)慶大霉素對分布陽性的核團造成直接損傷,對傳出神經(jīng)核團的損傷較傳入核團嚴重。給藥后3天,傳出神經(jīng)核團神經(jīng)纖維髓鞘板層松解、神經(jīng)元突起空泡樣變性、原漿型星形膠質(zhì)細胞水腫；至30天,髓鞘病變可逆性恢復,但個別神經(jīng)元發(fā)生嚴重腫脹,甚至凋亡；給藥后,耳蝸核出現(xiàn)顯著的神經(jīng)纖維髓鞘變性,但神經(jīng)元未見明顯異常。
[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.

【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R764

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