Th1: mediator lymphocytes in experimental autoimmune labyrinthitis.

A previous study on experimental autoimmune labyrinthitis (EAL) consistently demonstrated transient infiltration of lymphocytes only into the inner ear of mice. To clarify the profile of lymphocytes in the initiation of EAL, the present study investigated cell surface antigens, as well as cytokines, from Day 4 to Day 35, using immunohistochemical techniques. Many CD4+ cells mainly infiltrated the endolymphatic sac as early as Day 4 and gradually spread to the rest of the inner ear. Infiltration peaked on Day 12 and persisted in most animals until Day 35, although the number of cells gradually decreased. In contrast, very few CD8+ cells were found to have appeared in the inner ear of all animals on Day 10, and the number of cells rapidly decreased. Many cells positive for IFN-gamma and IL-2 were identified in the endolymphatic sac on Day 4. These results suggest that helper T1 lymphocytes, rather than cytotoxic T lymphocytes, may play a central role in the initiation of EAL.     

The endolymphatic sac: its importance in inner ear immune responses

The present study investigated the role of the endolymphatic sac in the generation of inner ear immune responses. The inner ear immune response to KLH antigen challenge was examined in animals whose endolymphatic sac was surgically obliterated and in a sham operated control group. In primary inner ear immune responses, endolymphatic sac obliteration resulted in reduced serum anti-KLH levels, compared to controls, while no difference was observed for perilymph anti-KLH levels. In secondary inner ear immune responses, endolymphatic sac obliteration resulted in significantly suppressed perilymph anti-KLH levels and greatly reduced cellular infiltration within the cochlea. In contrast, the sham operated animals showed a greatly increased number of plasma cells and lymphocytes in the perisaccular space and the endolymphatic sac lumen. These findings suggested that the endolymphatic sac must play an integral role in the generation of both systemic and local antibody responses following inner ear antigen challenge. The endolymphatic sac, like the gut, appears to have an immunological as well as a resorptive role in the host.     

Elevation of inner ear antibody levels following direct antigen challenge of the endolymphatic sac

The effect of direct antigen challenge of the endolymphatic sac (e.sac) on inner ear antibody levels was investigated in guinea pigs. Two weeks following e.sac antigen inoculation, perilymph antibody levels to keyhole limpet hemocyanin (KLH) were found to be significantly elevated in the challenged ear of those animals compared to the unchallenged, opposite ear. Moreover, when the endolymphatic duct (e.duct) in a group of animals were obstructed prior to antigen challenge of their e.sac then the rise in perilymph antibody was prevented. The local accumulation of plasma cells and lymphocytes in the perisaccular space and e.sac lumen was seen following these antigen injections. These experiments suggest that inner ear antibody emanates from the e.sac and reaches the perilymphatic compartment along the route of the e.duct. Furthermore, these findings emphasize the central role of the e.sac in inner ear immunity.     

The Endolymphatic Sac Receives Antigenetic Information from the Organs of the Mucosa-Associated Lymphatic System

The endolymphatic sac holds the entire arrangement of immunocompetent cells and functions as an immunological potent control organ for the inner ear. The evidence of secretory immunoglobulin A and other features of lymphocyte subtypes characterizes the endolymphatic sac as an organ of the mucosa-associated lymphatic system (MALT). In this system a permanent recirculation of sensitized memory lymphocytes from one organ to the other has been demonstrated experimentally as serving to dispose memory lymphocytes after renewed antigenetic stimulus. The aim of this study was to prove the possible recirculation of antigen-sensitized lymphocytes to the endolymphatic sac after antigenic stimulus of another part of the mucosa-associated lymphatic system. The results are evidence that the endolymphatic sac is provided with immunocompetent cells which derive from the lymphatic tissue of the nasopharynx. While the origin of immunocompetent cells in the endolymphatic sac still remains uncertain, this study underlines the role of lympho-epithelial tissue of the nasopharynx as a possible cell source for the endolymphatic sac. The results might explain the altered or disturbed function of the endolymphatic sac as a possible cause of certain inner ear diseases.     

Analysis of immunocompetent cells following inner ear immunostimulation

Immunohistochemical methods were used to investigate the development of inner ear immunocompetent cells in healthy, BALB/c mice over a 3-week period as a result of a secondary inner ear response against keyhole limpet hemocyanin. Antibodies against murine macrophages and granulocytes (anti-Mac-1), T-cells (anti-Lyt-1, anti-Lyt-2), and immunoglobulins (anti-IgM, anti-IgG, anti-IgA) were used. Mac-1 positive (Mac-1+) cells were observed at 6 hours post-challenge in the endolymphatic sac and cochlea and rapidly increased in both sites. Lyt-1+ cells gradually increased in the endolymphatic sac after challenge, peaking at 2 and 3 weeks post-challenge. In the cochlea, Lyt-1+ cells were detected at 1 day post-challenge and then increased. Lyt-2+ cells were detectable in the endolymphatic sac and the cochlea by 3 weeks post-challenge. The predominant immunoglobulin-bearing cell in the endolymphatic sac was IgG, followed by IgM, with IgA seen late in the response. We conclude that the inner ear has the capacity to mount an immune response through the accumulation of the needed immunocompetent cells for antigen processing, antibody production, and modulation of the response through T-helper and suppressor cell activity.     

Cell proliferation in the endolymphatic sac in situ after the rat Waldeyer ring equivalent immunostimulation

OBJECTIVES/HYPOTHESIS: It has been recognized that immunological mechanisms could be involved in the pathogenesis of different inner ear disorders, such as progressive sensorineural hearing loss, Meniere's disease, and even sudden deafness. The endolymphatic sac acts as an immune control organ for the inner ear and has been considered as an effector site of the mucosa-associated lymphoid tissue. The purpose of the study was to determine the potentially immunological relationship between endolymphatic sac and Waldeyer ring equivalent, one of the most important affector organs in mucosa-associated lymphoid tissue. STUDY DESIGN: Animal model. METHODS: Thirty-six rats were employed. Two animals were killed for histological observation of Waldeyer ring equivalent, and another 34 animals were randomly divided into experimental and control groups and received bilateral intranasal immunizations with keyhole limpet hemocyanin or Freund adjuvant, respectively. The ears of immunized animals and control animals were examined for keyhole limpet hemocyanin-positive memory cells and immunoglobulin G-positive plasma cells with the technique of immunohistochemical analysis. The histopathological changes and cell proliferation in those ears were also assessed. RESULTS: There were paired and organized lymphoid tissues in the lateral wall of the first choana in the rat. Keyhole limpet hemocyanin-positive lymphocytes appeared within the endolymphatic sac at 3 days after the first anti-genetic stimulus of the Waldeyer ring equivalent. Endolymphatic hydrops in the cochlea, elevated amounts of immunocompetent cells, and increased activity of cell proliferation within the endolymphatic sac were also considered after four intranasal injections of keyhole limpet hemocyanin. CONCLUSION: Presumably, endolymphatic sac is supplied with immunocompetent cells from Waldeyer ring equivalent and has an ability of co-reaction with Waldeyer ring equivalent.     

T cells infiltrating from the systemic circulation proliferate in the endolymphatic sac

It has been reported that autoimmune mechanisms are involved in the development of inner ear disorders such as Meniere's disease and steroid-responsive sensorineural hearing loss. In the present study, using an animal model for graft-versus-host disease, we investigated the immune regulatory mechanism in the endolymphatic sac and demonstrated that donor T cells injected into the systemic circulation of recipients infiltrate and proliferate in the perisaccular region. These findings suggest that immunocompetent cells are supplied from the systemic circulation through blood-labyrinth and blood-endolymph barriers into the endolymphatic sac, and that the endolymphatic sac allows these cells to proliferate locally as a local immune defense. It therefore seems likely that the endolymphatic sac plays a crucial role in not only graft-versus-host disease but also autoimmune inner ear disorders.     

An immunohistochemical study of the endolymphatic sac in patients with acoustic tumors

Intraosseous endolymphatic sacs obtained from patients with acoustic neuromas who had undergone total labyrinthectomy during tumor removal were examined for the presence of T helper/inducer and T suppressor/cytotoxic lymphocytes, B lymphocytes, plasma cells, and macrophages. Immunoperoxidase staining of cryostat sections revealed the presence of T helper/inducer lymphocytes, T suppressor/cytotoxic lymphocytes, and macrophages. The number of B lymphocytes and plasma cells was much smaller than the number of T lymphocytes. The number of T suppressor/cytotoxic lymphocytes was higher than the number of T helper/inducer lymphocytes. This study supports the notion of local immune responsiveness in the human inner ear. This is the first immunohistochemical study to analyze lymphocyte subpopulations; specifically, to provide insight into T-cell function in the endolymphatic sac.     

Time-dependent alterations of 3-fucosyl-N-acetyl-lactosamine (CD15) expression in the endolymphatic sac of adult guinea pigs after glycerol administration

The present study examined the effect of glycerol administration on 3-fucosyl-N-acetyl-lactosamine (CD15) epitope expression in the endolymphatic sac (ES) of the guinea pig's inner ear. Adult guinea pigs were injected intravenously with glycerol (2 g/kg body wt.). CD15 expression was studied at 80 min up to 5 h after treatment. In untreated animals single cells and cell groups in the ES expressing CD15 epitope intra- and intercellularly were identified by immunohistochemistry to be mainly in the epithelial layer of the rugosal and distal part of the sac. Glycerol administration modulated the expression of CD15 epitope. In the epithelial layer, expression decreased and was nearly depleted after 3 h. After 4 h of glycerol administration, CD15 expression reappeared and reached the comparable level of controls. The numbers of CD15-positive cells in the lumen of the ES increased steadily and arrived at their the highest levels in 2-h specimens. The localization of CD15-epitope expression and its modulation after glycerol administration within the ES implies that this molecule may play a role in re-establishing the sac's normal function. In addition, we speculate that CD15 may be associated with processes of an immune response in the inner ear.     

In vivo visualization of endolymphatic hydrops in guinea pigs: magnetic resonance imaging evaluation at 4.7 tesla

In order to find out whether it is possible to visualize experimental endolymphatic hydrops in the cochlea with magnetic resonance imaging (MRI) at 4.7 T, we used 11 guinea pigs. Five normal guinea pigs were used as controls. Early manifestation of endolymphatic hydrops was evaluated in endolymphatic sac (ES)-intact animals (n = 6), and advanced manifestation in ES-damaged animals (n = 5) by means of MRI with gadolinium-diethylenetriaminepentaacetate-bismethylamide (Gd-DTPA-BMA) contrast agent. Hearing was tested with electrocochleography. The surface area of 3 partitions of the cochlea was used to quantify endolymphatic hydrops. The fine structure of the 3 partitions of the cochlea was visualized with MRI in all animals, as Gd-DTPA-BMA appeared mainly in the scala tympani and scala vestibuli. As early as 5 days after endolymphatic sac surgery, endolymphatic hydrops started to appear as visualized by MRI and also verified with histology. Severe damage to the inner ear barrier with Gd-DTPA-BMA leakage into the scala media was detected with MRI in 1 ES-damaged animal that had a 60-dB hearing loss. To conclude, endolymphatic hydrops can be visualized with high-resolution MRI by means of Gd-DTPA-BMA, and it is possible to quantify the extent of endolymphatic hydrops. Damage to the inner ear barrier or possible rupture of membranes can be shown with the assistance of Gd-DTPA-BMA.     

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。     

用4.7特斯拉磁共振成像活体内观测豚鼠实验性内淋巴积水

目的探讨用4.7特斯拉试验用磁共振成像系统能否在豚鼠中检测内淋巴积水.方法20只白色或者杂色豚鼠用于该研究.5只正常豚鼠作为对照组,15只豚鼠用于制作内淋巴积水模型.9只内淋巴囊破坏组中的5只和6只内淋巴囊完整组(与乙状窦游离)动物采用gadolinium(Gd)-DTPA-BMA增强MRI检测内淋巴积水.结果由于Gd-DTPA-BMA主要进入鼓阶和前庭阶,耳蜗的三个阶可在所有动物中由MRI清晰显示.在内淋巴囊完整组,内淋巴囊手术后6天MRI即可检测到内淋巴积水,并且由组织学证实.在内淋巴囊破坏组中的1只动物,因内耳屏障的严重破坏而使Gd-DTPA-BMA快速漏入中阶,MRI可检测到该变化,其听力损失为60dB.结论用Gd-DTPA-BMA增强的高分辨MRI可检测出内淋巴积水,有可能对积水程度进行定量测试.在Gd-DTPA-BMA的帮助下,内耳屏障损伤或可能的膜破裂可以被检出.     

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     

Immunohistochemical study of the endolymphatic hydrops due to secondary endolymphatic sac immune reaction

Secondary endolymphatic sac (ES) immune response against keyhole limpet hemocyanin (KLH) were immunohistochemically investigated in guinea pigs by analysing the distribution of IgG and C3 complement in the inner ear over a 4-week period. After systemic sensitization with KLH and Freund's adjuvant, KLH was inoculated into the right ES by trans-cranial approach. KLH was found in the ES immediately after KLH challenge and then disappeared on the 7th day. On the 2nd and 3rd day moderate endolymphatic hydrops and a massive cell infiltrations within the ES were observed, and they gradually decreased their severity with time. Diffuse stainings with IgG and C3 were found in the ES, the epithelial and subepithelial regions of the vestibule. Similar findings were obtained in the spiral ligament and the hair cells after antigen challenge. IgG bearing cells were detected within the endolymphatic sac alone. Our results suggested that endolymphatic hydrops may be produced by the imbalance of in-out flow of the endolymph and the immunologically mediated inflammation through IgG-complement system.     

Inner ear anomalies are frequent but nonobligatory features of the branchio-oto-renal syndrome

OBJECTIVE: To summarize the syndromic features and evaluate the presence of inner ear anomalies in 35 patients with branchio-oto-renal (BOR) syndrome from 6 families. DESIGN: Retrospective evaluation of magnetic resonance imaging of the temporal bones and clinical features in patients with BOR syndrome. SETTING: Tertiary referral center. PATIENTS: The study population comprised 35 clinically affected patients with BOR syndrome from 6 families. Most of these families were followed for over 25 years. MAIN OUTCOME MEASURES: Twenty-four patients underwent high-resolution, heavily T2-weighted 3-dimensional magnetic resonance imaging of the temporal bones for evaluation of inner ear anomalies. Special attention was paid to the endolymphatic duct and sac. RESULTS: A total of 7 enlarged endolymphatic ducts and sacs (3 bilaterally and 4 unilaterally) and 5 enlarged endolymphatic ducts only (2 bilaterally and 3 unilaterally) were observed. Eight hypoplastic cochleas and 6 hypoplastic labyrinths were seen bilaterally. Seven family members had normal inner ears. CONCLUSION: These findings suggest that inner ear anomalies are frequent but nonobligatory features of BOR syndrome.     

Expression of inducible nitric oxide synthase in the cochlea following immune response in the endolymphatic sac of guinea pigs

Immunohistochemical study for inducible nitric oxide synthase (iNOS or NOS II) in the cochlea of guinea pigs was performed after the injection of keyhole limpet hemocyanin (KLH) into the endolymphatic sac. Morphological changes were observed in the cochlea of all animals after the injection of KLH. Increased iNOS expression was detected in the lateral wall, organ of Corti and ganglion cells. It is known that high levels of nitric oxide can lead to inner ear dysfunction. Our results suggest that iNOS may mediate the inner ear disturbance as seen in endolymphatic hydrops.     

Electrocochleographic changes in relation to cochlear histopathology in experimental endolymphatic hydrops

The relation between electrocochleographic and histological changes in experimental endolymphatic hydrops was studied 1, 2, 4, or 8 months after obliteration of the endolymphatic sac. An increase in the compound Action Potential (AP) threshold was found 2, 4, and 8 months after obliteration. This increase was strongly correlated with loss of outer hair cells, nerve fibres and spiral ganglion cells. An enhanced negative Summating Potential (SP) and an enhanced SP-AP ratio were found mainly in animals with an endolymphatic hydrops without further cochlear pathology. A normal or decreased SP and SP-AP ratio was regularly recorded in animals with both an endolymphatic hydrops and a variety of other histopathological changes in the inner ear. An increased second peak (N2) in the AP waveform was recorded from 63% (15/24) of the hydropic ears, equally divided over the four groups. There was no obvious correlation between the increased N2 and other electrophysiological or histological findings.     

Somatostatin (somatostatinlike) immunoreactive cells in the human inner ear

Certain epithelia of the human inner ear and human endolymphatic sac display somatostatin and/or somatostatin-like immunoreactivity. Histologic sections from 13 human temporal bones and from 15 endolymphatic sacs were studied using the unlabeled antibody peroxidase-antiperoxidase technique. The somatostatin and/or somatostatin-like immunoreactive cells were located exclusively in the covering epithelium of the spiral prominence and in the epithelium of the intermediate and rugosal part of the endolymphatic sac. In the epithelium of the spiral prominence and endolymphatic sac, secretory granules of the same size and appearance as those of intestinal or pancreatic somatostatin-producing cells were demonstrated ultrastructurally. The findings are consistent with a local exocrine, paracrine, and/or endocrine system of the inner ear.     

内淋巴囊对外源性TD抗原的清除作用

目的:探讨内淋巴囊对外源性胸腺依赖性(TD)抗原的吞噬和清除作用。方法:用S-D大鼠30只,以TD抗原钥孔(?)血蓝蛋白(KLH)全身免疫后2周,再次在内耳接种相同抗原,分别于此后1 h、3 h、1 d、3 d、7 d和14 d,取颞骨作组织学处理、冰冻切片,用抗KLH单克隆抗体,运用免疫组织化学技术,观察内耳免疫应答过程中内淋巴囊腔内KLH的动态变化。结果:内耳抗原接种后3 h,内淋巴囊出现KLH;第1～7 d在内淋巴囊的抗原被捕捉吞噬和递呈;第14天内淋巴囊仅发现微弱的抗原染色。结论:在内耳的二次应答中,内淋巴囊能够吞噬、处理和清除外源性抗原说明其具有免疫防御和免疫应答功能。