自身免疫性内耳病动物模型的建立

目的　建立一种自身免疫性内耳病的动物模型 ,其具有可重复性高 ,适于进行深入免疫学分析的特点。方法　提取豚鼠内耳膜迷路组织为抗原 ,与等量完全弗氏佐剂 ,百日咳杆菌一次免疫C5 7BL/6小鼠。检测反应阈、血清免疫学、内耳形态学及免疫组织化学的改变。结果　免疫后小鼠听性脑干反应阈显著提高 ,内耳中出现显著的炎性细胞浸润、内淋巴积水和螺旋神经节细胞变性、数量减少等形态学改变 ,血清中可检测到抗内耳自身抗体 ,鼓阶内浸润细胞中的淋巴细胞主要为CD4 T细胞。结论　应用这种方法在C5 7BL/6小鼠可成功建立自身免疫性内耳病的动物模型     

纯化内耳P0蛋白诱发实验性自身免疫性内耳病动物模型

目的：用从内耳组织中纯化的P0蛋白免疫豚鼠,建立P0蛋白诱发的自身免疫性内耳病动物模型,研究其在自身免疫性内耳病中的作用。方法：采用制备性SDS-PAGE从内耳组织中分离、纯化P0蛋白。以纯化的豚鼠内耳P0蛋白作为抗原免疫豚鼠,观察其听性脑干反应阈,血清中抗体水平和内耳形态学的改变,并用免疫组织化学法确定P0蛋白在耳蜗的分布情况。结果：SDS-PAGE结果显示纯化的蛋白质只在分子量为30 000的位置上出现单一的蛋白染色带,Western blot结果示该蛋白即为P0蛋白。免疫后有22%豚鼠的听性脑干反应阈升高,对照组动物无变化。实验组血清IgG显著升高（F=6.48,P〈0.01）,反应阈提高豚鼠的螺旋神经节细胞均有不同程度的数目减少,蜗轴小血管周围有炎性细胞浸润。P0蛋白在耳蜗仅分布于螺旋神经节、蜗轴神经纤维的髓鞘上。结论：用制备性SDS-PAGE能够成功地从内耳纯化P0蛋白,用于自身免疫性内耳病的研究。P0蛋白在部分豚鼠能够诱发自身免疫性内耳病,可能是自身免疫性内耳病的自身抗原之一。     

自身免疫性内耳病的实验研究

用同种异体内耳抗原免疫豚鼠，观察听阈、血清免疫学、形态学及免疫病理学的改变，结果发现分豚鼠ＡＰ（Ｎ１）反应阈明显升高，血清中出现抗内耳自身抗体，且表现有膜迷路积水，蜗轴血管炎、螺旋神经元细胞空泡变性、细胞数减少，特别是部分动物蜗轴血管和血管纹毛细血管内皮有ＩｇＧ沉积。提示通过这种实验方法可在部分动物成功地建立实验性自身免疫性内耳病动物模型。此外，就该模型与该病临床表现的相似性进行了比较。     

自身免疫性内耳病的实验研究

用同种异体内耳抗原免疫豚鼠，观察听阈、血清免疫学、形态学及免疫病理学的改变，结果发现部分豚鼠ＡＰ（Ｎ＿１）反应阈明显升高、血清中出现抗内耳自身抗体，且表现有膜迷路积水、蜗轴血管炎、螺旋神经元细胞空泡变性、细胞数减少，特别是部分动物蜗轴血管和血管纹毛细血管内皮有ＩｇＧ沉积。提示通过这种实验方法可在部分动物成功地建立实验性自身免疫性内耳病动物模型。此外，就该模型与该病临床表现的相似性进行了比较。     

XIAP在年龄相关性听力减退C57BL/6J小鼠初级听皮质的表达

目的探讨幼年和10月龄C57BL／6J小鼠听力及初级听皮质（AI）中凋亡抑制蛋白XIAP的年龄相关变化。方法检测两组C57BL/6J小鼠听性脑干反应（ABR）;免疫组织化学法染色检测两组C57BL/6J初级听皮质神经元凋亡抑制蛋白XIAP的表达情况。结果与幼年C57BL／6J小鼠相比,10月龄C57BL/6J的ABR反应阈值更高,凋亡抑制蛋白XIAP的表达显著减少。结论随年龄增长C57BL/6J小鼠的听力减退,同时C57BL／6J小鼠大脑初级听皮质凋亡抑制蛋白表达减少,XIAP的表达水平可能与C57BL/6J小鼠听力减退有关。     

凋亡及其相关基因在实验性自身免疫性内耳病小鼠内耳组织中的表达

目的探讨凋亡及其相关基因在自身免疫性内耳病形成和发展中的作用。方法选用近交系C57BL／6小鼠随机数字表法分为正常对照组和免疫7、14、21、28d组，每组16只。提取豚鼠内耳膜迷路组织为抗原，与等量完令弗氏佐剂，百日咳杆菌一次免疫实验组动物，制备自身免疫性内耳病动物模型，应用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记技术（terminal deoxynucleotidyl transferase—mediated d-UTP nick end—labing，TUNEL）检测内耳中的细胞凋亡，应用免疫组化和逆转录聚合酶链反应（RT—PCR）技术检测Fas、FasL及bcl-2在内耳的表达。结果正常小鼠内耳组织中，TUNEL染色阳性细胞极为少见，偶尔在Corti器或球囊斑的支持细胞发现。免疫7d后，内毛细胞和少量的血管纹边缘细胞TUNEL染色阳性，14d后TUNEL染色阳性的细胞数量及种类显著增加，但外毛细胞、螺旋神经节细胞与前庭神经节细胞免疫前后均未见凋亡表达。免疫组化染色显示，正常小鼠内耳中Fas表达广泛，FasL存部分螺旋神经节细胞与前庭神经节细胞表达，bcl-2仅在螺旋神经节细胞、前庭神经节细胞有较强表达。免疫后FasL在各种组织均有较强表达，bcl一2在外毛细胞出现表达，在耳蜗神经无细胞的表达增加。RT—PCR检测正常小鼠内耳组织的Fas mRNA、FasL mRNA、bcl-2mRNA均为阳性，FasL mRNA低水平表达，免疫后升高，在2周达到高峰后逐渐下降；bcl-2 mRNA在免疫后进行性升高。结论Fas／FasL信号系统介导的凋亡与自身免疫性内耳病的发生、发展过程关系密切，bcl-2对内耳中Fas／FasL介导的凋亡有重要的调节作用。     

同种异体内耳组织免疫后豚鼠内耳形态学变化

目的建立一高阳性率、可供圆窗给药治疗内耳病研究用的自身免疫性内耳病动物模型。方法实验动物为86只豚鼠,其中32只用于制备粗制内耳抗原。其余动物随机分为实验组42只,对照组1、2各6只。将实验组动物腹腔注射环磷酰胺进行预处理,2d后用粗制内耳抗原行皮下多点接种,分别于接种后4、6、8、10、12、14、20d时用光镜观察动物内耳形态学变化,并检测其血清中IgG、IgM、C3、CH50、循环免疫复合物(CIC)水平。对照组1不行任何处理,对照组2不接种内耳抗原,余同试验组。结果接种后豚鼠耳蜗、前庭、内淋巴囊等部位出现以淋巴细胞为主的炎性细胞浸润,尤以前庭阶、鼓阶、螺旋神经节区域及耳蜗底圈等部位为重。接种后4d时,67%动物内耳有炎性细胞浸润;8~12d时,100%动物出现炎性细胞浸润;接种14d后,炎性细胞浸润明显减少。接种6~14d时,内耳尚有血管周围炎、螺旋神经节细胞变性、内淋巴积水等改变。结论将豚鼠采用环磷酰胺预处理及同种异体内耳抗原接种,可建立一高阳性率的自身免疫性内耳病动物模型。     

封闭环境对C57BL/6J小鼠听功能及听皮层GABA能神经元凋亡的影响

目的探讨封闭环境对老年性聋动物模型C57BL/6J小鼠听皮层GABA能神经元凋亡的影响。方法构建封闭环境,将2、10月龄C57BL/6J小鼠(实验组,各20只)放入其中,并将另2、10月龄C57BL/6J小鼠各20只设立为开放环境对照组,饲养2月后比较不同环境各组动物ABR反应阈变化,免疫荧光法及tunel法检测听皮层GABA能神经元凋亡水平。结果实验组各频率ABR阈值明显高于对照组,尤其以高频明显(P<0.05);tunel检测证实实验组动物听皮层GABA能神经元凋亡显著增多。结论封闭环境能促进老年性聋动物模型C57BL/6J小鼠听皮层GABA能神经元的凋亡,可能是老年性聋的重要环境因素。     

C57BL/10J小鼠内耳形态学观察

目的 探讨不同月龄C57BL／10J小鼠内耳形态学变化。方法 取C57BL/10J小鼠2月龄3只（6耳）、8月龄3只（6耳）、12月龄2只（4耳）、27月龄2只（4耳），采用耳蜗铺片和耳蜗图技术，观察耳蜗和前庭毛细胞的变化。结果 2月龄鼠在耳蜗底回和顶回可见有少量的外毛细胞缺失，内毛细胞正常。8月龄鼠外毛细胞缺失主要由耳蜗底回向顶回发展，少量内毛细胞缺失主要在耳蜗底回，对应于20kHz的基底膜区域显示外毛细胞缺失在20％左右。12月龄鼠外毛细胞缺失明显加重，耳蜗底回部分几乎完全缺失，外毛细胞损失区域发展到基底膜中部，并且蜗尖区域外毛细胞缺失达到40％，耳蜗底回内毛细胞缺失达到60％～80％，蜗尖部分内毛细胞基本正常，对应于20kHz的基底膜区域显示外毛细胞缺失近80％，内毛细胞缺失近30％。27月龄鼠耳蜗外毛细胞从基底膜中部至底回完全缺失，顶回缺失60％～80％，内毛细胞缺失逐渐向蜗尖扩展，对应于20kHz的基底膜区域显示外毛细胞完全缺失，内毛细胞缺失超过50％。前庭选取球囊斑，椭圆囊斑和壶腹嵴三个部位进行观察，不同月龄的C57BL／10J鼠前庭毛细胞均正常。结论 本研究首次对C57BL／10J小鼠的内耳形态学进行观察，发现该鼠同C57BL／6J小鼠比较在外毛细胞缺失规律和时间上有明显的差别，明确了内耳毛细胞的变化规律；同时首次观察了前庭毛细胞，为C57BL／10J小鼠作为遗传性和老年性聋研究的动物模型提供了形态学理论依据。     

自身免疫性内耳病动物模型的建立

利用粗制豚鼠内耳膜迷路抗原免疫豚鼠，观察内耳听功能及其形态学变化，检测血清中Ｉｇ，Ｃ３，ＣＩＣ及抗内耳自身抗体，并用免疫组织化学方法检测内耳切片中ＩｇＧ沉积物。结果表明，以本实验方法可在部分动物建立自身免疫性内耳损害的动物模型，为深入研究该病打下了基础。     

豚鼠环磷酰胺预处理后再免疫对其ABR阈值的影响

目的建制一高阳性率、可供圆窗给药治疗内耳病研究用的自身免疫性内耳病动物模型。方法采用270—370g重的白色红目豚鼠97只作为实验对象，其中32只用于制备粗制内耳抗原，47只经环磷酰胺腹腔注射预处理2d后，再用粗制内耳抗原行皮内多点接种。动物于接种后4、6、8、10、12、14、20d(分别为6、7、7、6、9,6、6只)接受听性脑干反应(ABR)检测。正常对照18只，组1(12只动物)不行任何处理，组2(6只动物)仅行环磷酰胺预处理，不行粗制内耳抗原接种。结果实验组动物接种后4d时ABR阈值升高10dB以上者为67％，8d时为86％．14d时仍为58％，接种后20d时所有动物ABR阈值恢复正常。结论将豚鼠用环磷酰胺预处理后，用粗制同种异体内耳抗原只需单次接种即可建立听力损害发生率高的自身免疫性内耳病动物模型。     

Experimental autoimmune lesions of the inner ear]

This paper presents the autoimmune lesions of the inner ear by using isologous inner ear antigen (IEAg) on the guinea pigs. The purposes of this study were to determine whether the inner ear is an immune response organ and what is the sequelae of the immune processes both on physiological and morphological changes in the inner ear. Animals were systemically sensitized with IEAg in complete Freund's adjuvant and boothed with IEAg in incomplete Freund's adjuvant three times in every 3-4 weeks and then allowed to survive 4-6 weeks. For the ABR measurement, 8 of 28 ears (28.6%) in the experimental group showed threshold enhancement greater than or equal to 10dB before the animal sacrifice and the mean threshold shift of the ABR 8 weeks post immunization and before sacrifice revealed significant difference. Histological specimens were processed for both light and electron microscopic study. In the LM study, it showed endolymphatic hydrops, local thickening of the Reissener's membrane, and spiral ganglion cells lost. In the SEM, disturbance and loss of the cilia of the outer hair cells of the cochlea and the type I and II sensory cells in the crista ampularis can be seen. Also, some otoconia showed numerous malformation.     

Passive transfer of experimental autoimmune labyrinthitis

The aim of the present study was to establish an animal model of autoimmune labyrinthitis using heterologous inner ear antigen (IEAg) and to elucidate whether the experimentally induced labyrinthitis could be passively transferred. Cochlear and vestibular membranous labyrinthine tissues from bovine temporal bones were used as IEAg. Donor mice were inoculated intracutaneously at multiple sites with an emulsion consisting of equal parts of IEAg and complete Freund's adjuvant. After 10 days, mononuclear cells were collected from lymph nodes, spleen and blood of the donor mice and injected intravenously into naive recipient mice. Cellular infiltration was observed in the perilymphatic space of the cochlea of all donor and recipient mice. Endolymphatic hydrops was also observed in 63% of donor and 42% of recipient mice. These findings suggest that the experimentally induced labyrinthitis observed in this animal model was probably due to an autoimmune reaction to the IEAg and was passively transferred by a cell-mediated immune reaction.     

同种异体内耳组织免疫后豚鼠听功能与内耳形态学变化的关系

目的 观察豚鼠接受环磷酰胺处理及免疫后其听功能与内耳形态学变化的关系。方法 采用86只白色红目豚鼠作为实验对象，其中32只动物提供粗制内耳抗原。实验组（42只）动物于腹腔注射环磷酰胺2d后用粗制内耳抗原接种，接种后4．6、8、10、12、14、20d时接受ABR检测及内耳形态光镜观察。对照组1（6只）不行任何处理，对照组2（6只）注射环磷酰胺，但不接种抗原。结果 接种后4d时67％动物ABR阈值明显提高，8d时为83％，14d时降为58％，20d时所有动物阈值恢复正常。光镜观察发现：接种后4d时，实验动物听功能受损耳出现炎性细胞浸润；8—12d时，所有实验动物内耳均有类似改变；6—14d时，内耳尚有血管周围炎、螺旋神经节细胞变性等改变，相关形态变化均以听功能受损耳明显为重；接种14d时，内耳炎性细胞浸润明显减轻，20d时，绝大多数动物内耳形态基本恢复正常。结论 豚鼠接受环磷酰胺预处理及同种异体内耳抗原接种后，其听功能与内耳形态学的变化基本相一致。     

Spontaneous remission in experimental autoimmune labyrinthitis.

We investigated the time course of hearing impairment and cellular infiltration into the inner ear after systemic sensitization of guinea pigs with a single intradermal injection of bovine inner ear antigen (IEAg) in complete Freund's adjuvant (CFA). Lymphocytes and polymorphonucleocytes appeared mainly in the scala tympani on days 7 to 14, and in addition there was thickening and cellular infiltration of the round window membrane at day 14. These cellular infiltrations resolved after day 28. The auditory brain stem response thresholds from IEAg-sensitized animals were significantly elevated after day 7. Some sensitized animals (n = 5) had spontaneous remissions after day 28; however, the hearing thresholds did not completely recover. These results demonstrate that experimental autoimmune labyrinthitis can be induced by a single inoculation of IEAg-CFA and that remission, as evidenced by clearing of the cochlear cellular infiltration and improved hearing thresholds, can occur spontaneously.     

Experimental autoimmune labyrinthitis induced by cell-mediated immune reaction

This study was designed to establish an experimental cell-mediated autoimmune labyrinthitis model in C57BL/6 mice, which could exhibit high reproducibility and be adopted for precise immunological analysis. Inner ear antigen (IEA) was prepared from bovine membranous labyrinth. Following pretreatment with cyclophosphamide (CP) and primary sensitization with IEA/FCA, many inflammatory cells infiltrated transiently into the perilymphatic and endolymphatic regions of the cochlea, vestibule and the endolymphatic sac, but not into the kidney, lung, brain or liver. This reaction occurred from day 7 and peaked on day 12 in all animals, and then rapidly reduced. This reaction occurred in all experimental mice during day 10 to day 12. The control mice showed no cellular reaction in the inner ear. These results suggest that the inner ear may possess cross-species organ-specific antigen and that a cell-mediated immune reaction may play an important role in the induction of autoimmune labyrinthitis.     

A rat T-cell line that mediates autoimmune disease of the inner ear in the Lewis rat.

In various patterns of sensorineural hearing loss including Ménière's disease, which may show improvement in auditory function following immunosuppressive therapy, an isolated autoimmune disease of the inner ear has been postulated. Because of the lack of well-defined diagnostic criteria to identify autoimmune processes within the inner ear and the fact that the human inner ear is one of the few organs of the body not amenable to diagnostic biopsy, there has been great interest in developing animal models that mimic these clinical entities. Previous studies have found evidence that this process might be cell mediated and that the endolymphatic sac functions as an immunodefensive organ for the inner ear. By heterologous immunization of inbred Lewis rats with inner ear tissue, an autoreactive inner-ear-specific T helper cell line was established. After passive transfer of these cells a labyrinthitis was induced in recipient animals. Immunohistochemically, T helper cells were first identified in the cochlea suggesting that this cell type might carry the autoantigenic epitope. Autoantibodies against inner ear tissue were demonstrated in animals with histologically evident labyrinthitis. We conclude that this experimental design can serve as an animal model for cell-mediated autoimmune disease of the inner ear and could be used to explain the etiology of certain types of sensorineural hearing loss such as Ménière's disease. With this approach the identification of the causative autoantigen should be possible and will lead to the development of appropriate clinical tests to diagnose autoimmune diseases of the inner ear in humans.