实验性自身免疫性内耳病耳蜗热休克蛋白70的表达

目的　探讨自身免疫性内耳病模型动物耳蜗热休克蛋白的表达。方法　利用同种异体内耳抗原免疫豚鼠 ,以建立自身免疫性内耳病 (autoimmuneinnereardisease ,AIED)动物模型 ,并应用免疫组织化学及原位杂位技术 ,研究热休克蛋白 (heatshockprotein ,hsp) 70在正常对照组及AIED模型动物实验组耳蜗中的表达情况。结果　对照组豚鼠螺旋神经节细胞中存在hsp70样蛋白基础表达 ;实验组 2 8耳中 10耳 (35 7% )听阈提高≥ 10dB ;此组动物螺旋神经节细胞和血管纹、螺旋韧带hsp70及其mRNA表达明显增强。结论　以粗制膜迷路抗原免疫豚鼠 ,听阈提高动物hsp70在螺旋神经节细胞和血管纹、螺旋韧带合成增加。     

The role of heat shock protein 70 and its autoantibody in experimental autoimmune inner ear disease]

OBJECTIVE: To examine the effect of antigen stimulation on induction of the heat shock protein 70(HSP70) in the guinea pig cochlea and to explore the role of HSP70 and its autoantibody in the pathogenesis and diagnosis of autoimmunize inner ear disease (AIED). METHODS: To establish the animal model of AIED, homologous crude inner ear antigen was used to immunize the guinea pig. The expression of HSP70 was detected by immunocytochemistry and the relative staining densities were quantified with a light microscope image analysis system. The autoantibodies against HSP71 in the plasma of animals were tested by Western blot using purified recombinant human HSP71 as antigen. RESULTS: HSP70 presented in the normal guinea pig cochlea at a lower level. After immunological challenge, the levels of HSP70 immunoreactivity in the immunized animals were significantly increased as compared with control animals. Optical densities of cochleae of immunized animals were significantly greater than those of animals in the control group (P < 0.01). Most of the immunized animals had developed autoantibodies against HSP71. The incidence of autoantibody in the experimental group was significantly different (P < 0.01) from the control group. CONCLUSION: Antigen stimulation could lead to an increase in the synthesis of HSP70 in the guinea pig cochlea. The detection of autoantibody against HSP71 might have significance in the diagnosis of AIED.     

Effect of inner ear immune response on vestibular function in guinea pigs]

We examined vestibular dysfunction and histological damage caused by direct antigen challenge to the endolymphatic sac in guinea pigs. We observed spontaneous nystagmus every eight hours and performed caloric testing every week following endolymphatic sac secondary KLH challenge. Spontaneous nystagmus was seen in 12 of 18 animals, and nystagmus in all directed toward the unchallenged ear (paralytic). The caloric response time courses were classified into two types, which were irreversible type and reversible type after endolymphatic sac KLH challenge. The immune injury of animals with irreversible type was thought to be stronger than that of these with reversible type. The spontaneous nystagmus of irreversible type animals was longer than that of reversible type animals. The temporary vestibular dysfunction was thought to be similar to that observed in Meniere's disease.     

Detection and localization of heat shock protein 70 in the normal guinea pig cochlea

Normal guinea pig cochlear sections were treated with an antibody against heat shock protein (HSP70). HSP70-like immunoreactivity was observed in Claudius' cells and in the interdental cells of the spiral limbus. In the organ of Corti, immunoreactivity was confined to pillar cells, as well as Hensen's and Deiters' cells.     

内耳免疫反应中细胞凋亡的研究

目的探讨内耳免疫反应过程是否引起细胞凋亡以及Fas和FasL、Bcl-2和Bax的表达情况。方法选用雌性白色豚鼠16只，随机分为实验组和对照组各8只，以钥孔蛾血蓝蛋白全身免疫后，实验组以相同抗原进行内耳免疫，对照组内耳注射等量的磷酸盐缓冲生理盐水，在内耳免疫7d后处死动物，取内耳免疫侧耳蜗做石蜡切片。通过电镜和脱氧核糖核苷酸末端转移酶介导的缺口末端标记技术(terminal-deoxynucleotidyl transferase mediated nick end labeling，TUNEL)检测内耳凋亡细胞，免疫组化检测内耳Fas和FasL以及Bcl-2和Bax的表达。结果透射电镜观察发现实验组术后7d内耳外毛细胞、血管纹细胞及螺旋神经节细胞都出现了凋亡细胞的特征性改变，而对照组未发现具有上述特征的细胞。实验组内耳Corti器毛细胞，血管纹的缘细胞和螺旋神经节细胞存在TUNEL染色阳性细胞，TUNEL染色阳性细胞具有凋亡细胞的典型形态学特征，对照组内耳的任何结构中都没发现TUNEL染色阳性细胞。免疫组化染色实验组Corti器、螺旋神经节细胞、血管纹和螺旋韧带Fas和FasL蛋白表达阳性，而对照组只有螺旋神经节细胞和血管纹有较弱的Fas蛋白表达，FasL蛋白表达阴性。实验组Corti器、螺旋神经节细胞、侧壁Bcl-2蛋白表达阴性，对照组的Corti器、侧壁和螺旋神经节细胞Bcl-2蛋白表达阳性。实验组Corti器、侧壁和螺旋神经节细胞Bax蛋白表达阳性，对照组只有螺旋神经节细胞Bax蛋白表达弱阳性，Corti器、侧壁表达阴性。结论内耳免疫反应可诱导细胞凋亡发生，Fas-FasL是此过程的信号转导途径之一，Bcl-2和Bax蛋白在其中起了重要调节作用。     

细胞间粘附分子—1在内耳免疫反应中的表达

探讨细胞间占附分子－１在内耳免疫反应中的作用。方法 采用钥孔戚血蓝蛋白激发已全身致敏的２６只大鼠内耳，诱发其内耳免疫反应，然后通过免疫组化技术检测内耳扔ＩＣＡＭ－１的表达。结果 内耳激发后６小时，螺旋轴静脉及其回流小静脉即有ＩＣＡＭ－１表达，１２小时内淋巴囊及其下周区出现ＩＣＡＭ－１的表达，以２４－４８小时内各部位ＩＣＡＭ－１表达达最高峰。     

Distribution of beta-tubulin in guinea pig inner ear

Our previous research had suggested that beta-tubulin might be an autoantigen for autoimmune inner ear disease. In this study, the expression of beta-tubulin in inner ears of normal and tubulin-immunized guinea pigs was examined by immunohistochemical staining. Strong immunoreactivity to beta-tubulin monoclonal antibody was found in stria vascularis, neurons of the spiral ganglion, cochlear nerve fibers and spiral ligament. Diffuse staining was found in the stria vascularis and the neurons of the spiral ganglion, while dense network staining was found in the spiral ligament, the nerve fibers and the vestibular end organs. The semicircular canals, endolymphatic duct and sac were also positively stained. In inner ears of guinea pigs challenged with beta-tubulin, staining intensity was diminished in the stria vascularis, the spiral ligament, and the neurons of the spiral ganglion. The results suggest that beta-tubulin is distributed to most structures of guinea pig inner ear. A challenge to the inner ear by tubulin could change the beta-tubulin distribution and cause degeneration in the spiral ganglion. The results support the hypothesis that beta-tubulin might be an autoantigen for autoimmune inner ear disease.     

Effect of inner ear immune response on the caloric nystagmus in guinea pigs

The effect of direct antigen (KLH) challenge of endolymphatic sac (e. sac) on caloric response was investigated in guinea pigs. The caloric response was not significantly suppressed both in the primary KLH challenged ears and in the PBS challenged ears as compared to the unchallenged contralateral ears. In the animal of secondary KLH challenge of the e. sac, the caloric response was significantly reduced in the challenged ears as compared to the unchallenged contralateral ears and to the PBS challenged ears. These result suggested that local immune reaction mounted in the e. sac caused immune injury not only to the e. sac but also to the vestibular function.     

Antigen-specific immune response in the inner ear

The specificity of inner ear immune responses was investigated by challenging each inner ear of presensitized animals with different antigens. Animals presensitized systemically with keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) were challenged with KLH in the right and BSA in the left inner ears. Two weeks later perilymph anti-KLH levels were increased significantly in the right inner ears compared to the levels in the left inner ears. In contrast, perilymph anti-BSA levels were increased significantly in the left inner ears compared to the levels in the right inner ears. These results suggested that the rise in perilymph antibody following inner ear antigen challenge was predominantly the result of an antigen-specific immune response in the inner ear and not simply the result of an increase in vascular permeability of serum contamination from the experimental procedure itself.     

Effects of inner ear immune responses on auditory function in guinea pigs]

Following direct challenge with KLH antigen primary or secondary (after systemic immunization) to the ES (endolymphatic sac) in guinea pigs, ECoG (electrocochleograms) were recorded from the round window induced by click and tone pips. The recordings were carried out on the 2nd, 7th and 21st days after local antigen challenge. There were no abnormal findings in the ECoG of the primary antigen challenged animals. On the other hand, prolongation of latencies, decrease in amplitudes of APs (compound action potentials) and increases in SP/AP ratios were observed in the 2nd day recordings of the secondary antigen challenged animals. However, all parameters of ECoG in the 7th day recordings were normal. Decreases in AP amplitudes were again found in the 21st day recordings. The ECoG findings with click and tone pip stimulation showed similar results. From these findings, in conjunction with morphological observations, it is speculated that these ECoG findings are caused by immuno-injury to the ES and cochlea, as well as the resultant endolymphatic hydrops which develops acutely and gradually subsides after secondary challenge.     

内耳免疫反应中细胞凋亡的研究

目的　探讨内耳免疫反应过程是否引起细胞凋亡以及Fas和FasL、Bcl 2和Bax的表达情况。方法　选用雌性白色豚鼠 16只 ,随机分为实验组和对照组各 8只 ,以钥孔血蓝蛋白全身免疫后 ,实验组以相同抗原进行内耳免疫 ,对照组内耳注射等量的磷酸盐缓冲生理盐水 ,在内耳免疫 7d后处死动物 ,取内耳免疫侧耳蜗做石蜡切片。通过电镜和脱氧核糖核苷酸末端转移酶介导的缺口末端标记技术 (terminal deoxynucleotidyltransferasemediatednickendlabeling ,TUNEL)检测内耳凋亡细胞 ,免疫组化检测内耳Fas和FasL以及Bcl 2和Bax的表达。结果　透射电镜观察发现实验组术后7d内耳外毛细胞、血管纹细胞及螺旋神经节细胞都出现了凋亡细胞的特征性改变 ,而对照组未发现具有上述特征的细胞。实验组内耳Corti器毛细胞 ,血管纹的缘细胞和螺旋神经节细胞存在TUNEL染色阳性细胞 ,TUNEL染色阳性细胞具有凋亡细胞的典型形态学特征 ,对照组内耳的任何结构中都没发现TUNEL染色阳性细胞。免疫组化染色实验组Corti器、螺旋神经节细胞、血管纹和螺旋韧带Fas和FasL蛋白表达阳性 ,而对照组只有螺旋神经节细胞和血管纹有较弱的Fas蛋白表达 ,FasL蛋白表达阴性。实验组Corti器、螺旋神经节细胞、侧壁Bcl 2蛋白表达阴性 ,对照组的Corti器、侧壁和     

Immunolocalization of beta-lipotropin in the inner ear of the guinea pig

The occurrence of beta-lipotropin (beta-LTH) immunoreactive material was investigated in the inner ear of newborn and juvenile guinea pigs by means of Sternberger's PAP technique. Unlike met5-enkephalin and endorphin, beta-LTH could not be found in the organ of Corti but was identified in the spiral ganglion and the neuroepithelium of the crista ampullaris.     

细胞间粘附分子-1在内耳免疫反应中的表达

目的探讨细胞间粘附分子１（ｉｎｔｅｒｃｅｌｕｌａｒａｄｈｅｓｉｏｎｍｏｌｅｃｕｌｅ１，ＩＣＡＭ１）在内耳免疫反应中的作用。方法采用钥孔血蓝蛋白激发已全身致敏的２６只大鼠内耳，诱发其内耳免疫反应，然后通过免疫组化技术检测内耳的ＩＣＡＭ１的表达。结果内耳激发后６小时，螺旋轴静脉及其回流小静脉即有ＩＣＡＭ１表达，１２小时内淋巴囊及其囊周区出现ＩＣＡＭ１表达，在２４～４８小时内耳各部位ＩＣＡＭ１表达达最高峰。７２小时各种炎性细胞浸润到内耳的各个部位。随后ＩＣＡＭ１表达逐渐减弱，２８天完全消失。结论内耳免疫反应时，ＩＣＡＭ１在炎性细胞从循环系统进入内耳的过程中发挥着重要作用，调控ＩＣＡＭ１表达的细胞因子也可能还来自内淋巴囊以外的其他细胞。     

Distribution of gentamicin by immunofluorescence in the guinea pig inner ear

Summary We studied the distribution of gentamicin in the inner ear, brain and kidney of the guinea pig following intraperitoneal administration or perfusion of gentamicin through the perilymphatic space. The resulting histopathologcial changes were examined by immunofluorescence using antigentamicin antiserum. After perfusion of gentamicin through the perilymphatic space, specific fluorescence was found in the cochlea, and was especially prominent in the outer hair cells, basilar membrane and basilar crest. Although no fluorescence was observed in the cochlea following intraperitoneal administration of high doses of gentamicin, type I hair cells in the vestibule were seen to be selectively stained with the antibody. Furthermore, some of the vestibular ganglion cells, Purkinje cells and unidentified nuclei in the brain stem were also stained. In particular, fine granules showing relatively intense fluorescence were recognized in the cytoplasm of the stained cells. In the cortex of kidney, only proximal tubular cells were stained with intense fluorescence. Our results suggest that the aminoglycoside antibiotics have two sites of action: one is the cell membrane of the sensory hair cells and the other is the cytoplasm.This study was supported in part by a grant from the Ministry of Education, Science and Culture of Japan, and by a Research Grant for Specific Diseases from the Ministry of Health and Welfare to the Acute Profound Deafness Research Committee of Japan     

Volumetric and dimensional analysis of the guinea pig inner ear

The objective of this study was to provide accurate volumetric data on the fluid spaces and soft tissue in the guinea pig inner ear by measuring all histologic serial sections by means of Metamorph Imaging Software at 400x to 1,000x magnification. The total endolymph volume of the inner ear was 4.691 mm3, of which 1.501 mm3 was in the cochlea, 3.090 mm3 in the vestibular labyrinth, and 0.100 mm3 in the endolymphatic duct and sac. The total perilymph volume was 15.938 mm3, of which 8.867 mm3 was in the cochlea and 7.071 mm3 in the vestibular labyrinth. The volume of the organ of Corti per millimeter length increased toward the apex, but the volumes of the stria vascularis, spiral ligament, and spiral limbus decreased. The volume of the macula utriculi was larger than that of the macula sacculi. The measurement of the luminal surface area of the stria vascularis was 3.944 mm2, and that of the vestibular dark cells was 5.772 mm2.     

Distribution of gentamicin by immunofluorescence in the guinea pig inner ear

We studied the distribution of gentamicin in the inner ear, brain and kidney of the guinea pig following intraperitoneal administration or perfusion of gentamicin through the perilymphatic space. The resulting histopathological changes were examined by immunofluorescence using antigentamicin antiserum. After perfusion of gentamicin through the perilymphatic space, specific fluorescence was found in the cochlea, and was especially prominent in the outer hair cells, basilar membrane and basilar crest. Although no fluorescence was observed in the cochlea following intraperitoneal administration of high doses of gentamicin, type I hair cells in the vestibule were seen to be selectively stained with the antibody. Furthermore, some of the vestibular ganglion cells, Purkinje cells and unidentified nuclei in the brain stem were also stained. In particular, fine granules showing relatively intense fluorescence were recognized in the cytoplasm of the stained cells. In the cortex of kidney, only proximal tubular cells were stained with intense fluorescence. Our results suggest that the aminoglycoside antibiotics have two sites of action: one is the cell membrane of the sensory hair cells and the other is the cytoplasm.     

Cell proliferation in endolymphatic sac during the immune response of inner ear]

OBJECTIVE: To study the cell proliferation in endolymphatic sac(ES) during the secondary immune response in inner ear. METHODS: Fifteen healthy, female SD rats were employed in the experiment. Sensitized systematically with keyhole limpet hemocyanin (KLH), the animals were inoculated with the same antigen through cochlea basal turn into the labyrinth. Administrated 5-bromo-2'-deoxyuridine (BrdUrd) intraperitoneally, the rats were sacrificed and the temporal bones were harvested at 3, 7, 14 day after labyrinth vaccination respectively. The frozen sections of the decalcified samples were dealt with H-E staining and immunohistochemical methods to investigate the cellular infiltration, BrdUrd and IgG positive cells in the ES. RESULTS: While the ES lumen and perisaccular region were infiltrated with mononuclear-phagocyte at the 3rd day post-vaccination, plasmocyte and lymphatic cells become the predominant infiltrating cells in the ES at the 7th day. The KLH in the ES lumen were phagocytized at the 3-7th day post-vaccination. S-phase cells and IgG positive cells in ES increased markedly in the 3rd day and 7-14 days post-vaccination respectively with similar localization. CONCLUSION: The activity of cell proliferation in the ES enhance and local proliferated cells may differentiate in situ into immunocompetent cells during the secondary immune response against TD antigen in the inner ear. This indicates that ES plays an important role in immune response of inner ear.     

Immunolocalization of β-lipotropin in the inner ear of the guinea pig

Summary The occurrence of -lipotropin (-LTH) immunoreactive material was investigated in the inner ear of newborn and juvenile guinea pigs by means of Sternberger's PAP technique. Unlike met⁵-enkephalin and endorphin, -LTH could not be found in the organ of Corti but was identified in the spiral ganglion and the neuroepithelium of the crista ampullaris.     

Free radicals in the guinea pig inner ear following gentamicin exposure

The purpose of this study was to investigate the occurrence of free radicals, nitric oxide (NO), superoxide (O-2) and peroxynitrite, in the inner ear of the guinea pig following intratympanic injection with 5 mg of gentamicin (GM). Forty-eight hours after GM injection, varying degrees of degeneration of the inner ear were observed. Immunohistochemical study revealed immunoreactivity to NO synthase II (which generates NO) and to xanthine oxidase (which generates O-2) in both the vestibular organ and the organ of Corti. Immunohistochemical investigation, using a specific antinitrotyrosine antibody, also showed intense staining, suggesting formation of peroxynitrite in the inner ear through the reaction of NO with O-2. Scanning electron-microscopic study showed that the ototoxic effects could be blocked with N-nitro-L-arginine methylester, a competitive inhibitor of NO synthase, with superoxide dismutase, an O-2 scavenger, and with ebselen, a scavenger of peroxynitrite. On the basis of these findings, it can be concluded that NO together with O-2, which form more reactive peroxynitrite, play an important role in GM ototoxicity in the guinea pig.     

Expression of intermediate filament proteins in the mature inner ear of the rat and guinea pig.

The expression of intermediate filament proteins was studied in the mature inner ear of the rat and guinea pig, using a panel of polyclonal and monoclonal antibodies directed against cytokeratins, desmin, neurofilament proteins and glial fibrillary acidic protein (GFAP). The epithelial lining of the endolymphatic space displayed a complex expression pattern of cytokeratin filament proteins, suggesting greater cell diversity than was known sofar from morphological studies. The cytokeratin antibodies when applied to the inner ear tissues revealed the presence of only cytokeratin polypeptides which are typical of simple epithelia (i.e. nos. 7, 8, 18, and 19). Profound differences in cytokeratin expression patterns were, however, found in the various cell types of both the cochlear and vestibular partition. Remarkably, the sensory cells appeared to be devoid of both cytokeratins and neurofilament proteins. Staining with a 200 kDa neurofilament antibody displayed the presence of different populations of ganglion cells in the spiral ganglion and the vestibular ganglion. There was no reaction with antibodies directed against desmin and GFAP. The great resemblance of the intermediate filament protein expression patterns in the inner ear of the rat and guinea pig indicates a close similarity between the different epitopes.