Neutrophils infiltrate into the spiral ligament but not the stria vascularis in the cochlea during lipopolysaccharide-induced inflammation.

It has been challenging to apply intravital imaging for monitoring the inner ear, as the anatomical location and intricate structure hamper the access of imaging instruments to the inner ear of live mice. By employing intravital imaging of the cochlea in live mice with two-photon microscopy, we investigated neutrophil infiltration into the cochlea tissue and its characteristics under a lipopolysaccharide (LPS)-induced inflammatory state. Methods: Cochlea inflammation was induced by LPS injection to the middle ear. Using two-photon intravital microscopy with specifically designed surgical exteriorization of the cochlea in live mice, we investigated the dynamic features of neutrophils in the lateral wall of the cochlea. The molecular expression pattern of the cochlea lateral wall was also investigated during the LPS-induce inflammation. Results: Despite the contention of whether neutrophils are recruited to the spiral ligament (SL) during inflammation, we observed that LPS-induced inflammation of the middle ear, which mimics acute otitis media, triggered neutrophil migration to the SL in the lateral wall. Notably, massive neutrophil infiltration to the SL occurred 2 days after LPS inoculation, but there was no neutrophil infiltration into the stria vascularis (SV) region. At 1 day after LPS-induced cochlear inflammation, increased mRNA expression of interleukin-1ß, interleukin-6 were identified in both the SL and SV, while the ICAM-1 mRNA expression increased only in the SL. The differential reactivity of ICAM-1 is likely responsible for the different neutrophil recruitment pattern in the cochlea. Conclusion: Intravital imaging of the cochlea revealed that neutrophil recruitment and infiltration during inflammation are spatially controlled and exclusively observed in the SL but not in the SV and organ of Corti.

An experimental study of inner ear injury in an animal model of eosinophilic otitis media.

CONCLUSION: As the periods of intratympanic injection of ovalbumin (OVA) to the middle ear became longer, marked eosinophil infiltration in the perilymphatic space was observed. Moreover severe morphological damage of the organ of Corti was observed in the 28-day antigen-stimulation side. These results indicate that eosinophilic inflammation occurred in the inner ear and caused profound hearing loss. OBJECTIVE: The purpose of the present study was to elucidate the inner ear damage in a new animal model of eosinophilic otitis media (EOM) which we recently constructed. METHODS: We constructed the animal model of EOM by intraperitoneal and intratympanic injection of OVA. Infiltrating cells and the inner ear damage were examined by histological study. RESULTS: In the inner ear, a few eosinophils were seen in the scala tympani of the organ of Corti and the dilation of capillaries of the stria vascularis was observed in the 7-day stimulation side. In the 14-day antigen stimulation side, some eosinophils and macrophages were seen in not only the scala tympani but also the scala vestibule. In the 28-day antigen-stimulation side, severe morphological damage of the organ of Corti and many eosinophils, red blood cells, and plasma cells infiltrating the perilymph were observed.

Macrophage invasion contributes to degeneration of stria vascularis in Pendred syndrome mouse model.

BACKGROUND: Pendred syndrome, an autosomal-recessive disorder characterized by deafness and goiter, is caused by a mutation of SLC26A4, which codes for the anion exchanger pendrin. We investigated the relationship between pendrin expression and deafness using mice that have (Slc26a4+/+ or Slc26a4+/-) or lack (Slc26a4-/-) a complete Slc26a4 gene. Previously, we reported that stria vascularis of adult Slc26a4-/- mice is hyperpigmented and that marginal cells appear disorganized. Here we determine the time course of hyperpigmentation and marginal cell disorganization, and test the hypothesis that inflammation contributes to this tissue degeneration. METHODS: Slc26a4-/- and age-matched control (Slc26a4+/+ or Slc26a4+/-) mice were studied at four postnatal (P) developmental stages: before and after the age that marks the onset of hearing (P10 and P15, respectively), after weaning (P28-41) and adult (P74-170). Degeneration and hyperpigmentation stria vascularis was evaluated by confocal microscopy. Gene expression in stria vascularis was analyzed by microarray and quantitative RT-PCR. In addition, the expression of a select group of genes was quantified in spiral ligament, spleen and liver to evaluate whether expression changes seen in stria vascularis are specific for stria vascularis or systemic in nature. RESULTS: Degeneration of stria vascularis defined as hyperpigmentation and marginal cells disorganization was not seen at P10 or P15, but occurred after weaning and was associated with staining for CD68, a marker for macrophages. Marginal cells in Slc26a4-/-, however, had a larger apical surface area at P10 and P15. No difference in the expression of Lyzs, C3 and Cd45 was found in stria vascularis of P15 Slc26a4+/- and Slc26a4-/- mice. However, differences in expression were found after weaning and in adult mice. No difference in the expression of markers for acute inflammation, including Il1a, Il6, Il12a, Nos2 and Nos3 were found at P15, after weaning or in adults. The expression of macrophage markers including Ptprc (= Cd45), Cd68, Cd83, Lyzs, Lgals3 (= Mac2 antigen), Msr2, Cathepsins B, S, and K (Ctsb, Ctss, Ctsk) and complement components C1r, C3 and C4 was significantly increased in stria vascularis of adult Slc26a4-/- mice compared to Slc26a4+/+ mice. Expression of macrophage markers Cd45 and Cd84 and complement components C1r and C3 was increased in stria vascularis but not in spiral ligament, liver or spleen of Slc26a4-/- compared to Slc26a4+/- mice. The expression of Lyzs was increased in stria vascularis and spiral ligament but not in liver or spleen. CONCLUSION: The data demonstrate that hyperpigmentation of stria vascularis and marginal cell reorganization in Slc26a4-/- mice occur after weaning, coinciding with an invasion of macrophages. The data suggest that macrophage invasion contributes to tissue degeneration in stria vascularis, and that macrophage invasion is restricted to stria vascularis and is not systemic in nature. The delayed onset of degeneration of stria vascularis suggests that a window of opportunity exists to restore/preserve hearing in mice and therefore possibly in humans suffering from Pendred syndrome.

Bone marrow cells as an origin of immune-mediated hearing loss.

The MRL/lpr mouse, which is homozygous for the recessive lpr genes and has a mutation in the Fas gene encoding a cell-surface receptor for apoptosis, exhibits severe lymphadenopathy and develops systemic lupus erythematosus (SLE)-like disease. It has recently been reported that this mouse also manifests sensorineural hearing loss (SHL) with cochlear pathology at 20 weeks of age. We examined the effects of reconstituting severe combined immunodeficient (SCID) mice with MRL/lpr bone marrow on the development of SHL. These mice normally develop neither SHL nor cochlear pathology. Immune-mediated SHL and cochlear pathology did, indeed, occur following transfer of MRL/lpr bone marrow into SCID mice. These findings suggest that the development of SHL and cochlear pathology observed in MRL/lpr mice and in SCID mice receiving MRL/lpr bone marrow are the result of bone marrow defects rather than the result of a problem intrinsic to the cochlea.

Clinical associations of serum antiendothelial cell antibodies in patients with sudden sensorineural hearing loss.

OBJECTIVES/HYPOTHESIS: The role of antiendothelial cell antibodies in systemic vasculitis has been reported. The aim of the study was to define the clinical associations of serum antiendothelial cell antibodies in patients with sudden sensorineural hearing loss. STUDY DESIGN: A prospective study in patients with sudden sensorineural hearing loss. METHODS: Serum samples were taken from 59 consecutive patients with sudden sensorineural hearing loss at time of presentation and from 28 normal control subjects. Indirect immunofluorescence assay was used to detect antiendothelial cell antibodies. RESULTS: The prevalence of antiendothelial cell antibody detection was 54% (32 of 59 patients), with a statistically significant difference between patients and control subjects (P =.0004). Antiendothelial cell antibody positivity was significantly associated with absent recovery of hearing loss (P =.0020). CONCLUSIONS: The cytotoxicity to endothelial cells of the inner ear by antiendothelial cell antibody-positive sera might play a role in causing the stria vascularis damage in immune-mediated sudden sensorineural deafness. The appearance of antiendothelial cell antibody is related to the poor outcome of hearing loss, and its detection could be helpful in the selection of particular patients with sensorineural hearing loss for specific immunosuppressive treatments.

Mpv37 mouse strain--a model for the relationship between the kidney and the inner ear.

Using the Mpv17-negative mouse strain, which developed inner ear and kidney dysfunction, we confirm a strong relationship between the kidney and the inner ear. Both organs have specialized epithelia involved in active ion transport, which are separated from the vessels by a basement membrane of similar composition. Our recent results indicate that the glomerular and the stria vascularis basement membrane are simultaneously affected in early stages. Concomitant deposits of IgG during the progressive development of the disease support the idea of a shared antigen. Understanding the pattern of the development of the degeneration will provide a basis towards understanding the essential role of the Mpv17 protein in the structures of both organs and may provide a basis for future therapeutic intervention.

Effects of immunosuppression on the development of cochlear disease in the MRL-Fas(lpr) mouse.

OBJECTIVES: The MRL-Fas(lpr) mouse, an animal that spontaneously develops multisystemic autoimmune disease, has been proposed as model of immune-mediated inner ear disease. Previous studies revealed that this mouse manifested elevated auditory brainstem response thresholds, hydropic degeneration of strial cells, and antibody deposition within strial capillaries. As the etiology of the observed strial disease may be immune, genetic, or uremic, a study was designed to attempt to delineate between these possible etiologic factors. STUDY DESIGN: Prospective, controlled animal study. METHODS: Dexamethasone, which is known to suppress autoantibody production and glomerulonephritis in these animals, was administered systemically on a daily basis to experimental animals, beginning at 6 weeks of age. Control animals received no treatment. Animals were allowed to age, with control animals predictably manifesting systemic disease at 20 weeks of age, at which point all animals were sacrificed. RESULTS: Animals receiving dexamethasone treatment manifested a significant reduction in serum immunoglobulin levels, lymphoid hyperplasia, and a significant improvement in the level of renal function. However, morphologic analysis revealed a persistence of strial disease despite the elimination of strial antibody deposition. CONCLUSION: The results of this experiment support the hypothesis that genetic mechanisms may be responsible for the observed strial disease. Further studies are under way to confirm these findings.

Antibody deposition in the stria vascularis of the MRL-Fas(lpr) mouse.

The MRL-Fas(lpr) mouse, a model of multisystemic, organ non-specific autoimmune disease, has been proposed as a model of immune-mediated inner ear disease. Preliminary studies indicate that it develops cochlear pathology focused in the stria vascularis including intracellular edema and degeneration which develops in the absence of an inflammatory infiltrate but in the presence of antibody deposition. It was thus hypothesized that the antibodies found in the stria were mediating a direct pathologic effect on this structure, without recruiting classical inflammatory mediators. It was further hypothesized that the antibodies deposited within the stria would be derived from the non-complement fixing isotypes and subclasses, which are known to be able to mediate direct pathologic effects on target tissues. This study utilized immunohistologic techniques to identify the antibody isotypes and subclasses deposited within the stria vascularis of the MRL-Fas(lpr) mouse. Results indicate that all antibody isotypes and subclasses can be identified within the stria vascularis in the absence of complement. Thus, antibody deposition was not restricted to non-complement fixing antibodies. While it is possible that antibodies are mediating direct pathologic effects within the stria, the non-specific nature of the antibody deposition may indicate that these antibodies are not responsible for the observed pathology. Rather, other mechanisms, such as metabolic and genetic etiologies, must also be considered.

Hearing in the MRL/lpr mouse as a possible model of immune-mediated sensorineural hearing loss.

In order to clarify the possible mechanism of hearing loss in immune-mediated sensorineural hearing loss, basic research needed includes animal model studies. In the present investigation, we examined hearing thresholds and cochlear histologies of the MRL/lpr mouse which is now well-known as a model for pathology consistent with systemic lupus erythematosis (SLE). Present findings demonstrated that there were no statistically significant differences in auditory brainstem response (ABR) thresholds between 4- to 6-week-old "young" and 20- to 25-week-old "old" MRL mice. These differences were not sex-dependent. Under light microscopy, there were no abnormal morphological findings in the cochleas of either young or old MRL mice. With immunohistochemistry, mouse IgG was detected around the capillary walls in the stria vascularis in both young and old MRL mice. Serum IgG level of the MRL mice significantly decreased after predonisolone (PSL) administration. However, expression of mouse IgG in the stria vascularis was not observed in the MRL mice after PSL administration. From these results, we speculate that the hearing of the MRL mouse does not always deteriorate, and the deposition of mouse IgG on the capillary wall in the stria vascularis is not a sufficient factor to induce hearing loss. At this point, we conclude that the MRL mouse should not be considered a useful model for immune-mediated sensorineural hearing loss.

Cochlear immunoglobulin in the C3H/lpr mouse model for autoimmune hearing loss.

Immunoglobulin staining was conducted in cochlear tissue from the C3H/lpr autoimmune strain mouse to better understand the local immune processes underlying autoimmune inner ear disease. This mouse is a model for spontaneous systemic lupus erythematosus with coincident elevated cochlear thresholds. Cochleas were examined from C3H/lpr mice at 2 months of age, before disease onset, and at 8 months of age, when systemic disease and hearing loss are manifested. Sections of these cochleas, along with cochlear sections from age-matched C3H/HeJ nonautoimmune controls, were immunocytochemically stained for IgG and IgM to identify areas of abnormal immunoglobulin activity. IgM immunoreactivity was similar in control and autoimmune cochlear tissue and did not appear to vary with disease progression. Staining was limited to the inside of capillaries in the stria vascularis and other areas within the cochlea. Similar staining patterns were seen in control animals stained for IgG. However, C3H/lpr mice with autoimmune disease showed extensive IgG immunoreactivity spreading out from the stria vascularis capillaries into the extracapillary spaces. This increased permeability suggested that breakdown of the blood labyrinth barrier was coincident with systemic autoimmune disease.

Immunoglobulin deposition in thickened basement membranes of aging strial capillaries.

The presence of immunoglobulins in the thickened basement membrane (BM) of aging strial capillaries was investigated as a possible indicator of autoimmunity in the genesis of atypical BM. Cochleas from young and old Mongolian gerbils raised in quiet were examined by immunostaining at the light microscopic level for IgG and IgM and for the BM components laminin (La) and type IV collagen (IV-C). Another age-graded series of cochleas was stained for IgG at the ultrastructural level. No immunoreactive IgG was detected in specimens from animals less than 6 months old. In contrast, 2 of 12 cochleas from 20- to 28-month-old gerbils and 11 of 20 cochleas from gerbils 30 months or older showed positive staining for IgG in strial capillary BM. IgM was not detected at any age. At the electron microscope level, no immunoreactive IgG was detected in the stria of cochleas younger than 30 months. However, labeling demonstrative of IgG was observed in the thickened BM of some strial capillaries in all six cochleas from gerbils older than 33 months. Lysosome-like granules in endothelial cells and the superiormost marginal cells also stained for content of IgG as did fibrillar material in edematous regions in the intrastrial space. In addition to showing accumulation of IgG, the findings confirm our prior demonstration of increased La deposition in the thickened strial capillary BM of all cochleas from old gerbils. The BM alterations appear confined to strial capillaries in old gerbils, since morphological observations and immunostaining for La and IgG failed to detect changes in BMs at any other site in a wide survey of aged gerbil organs including vessels in other regions of the affected cochleas. The results point more towards the development of an age-dependent permeability to IgG selectively in strial capillaries than to autoimmunity as an explanation of the IgG in BM.

The localization and specificity of guinea pig inner ear antigenic epitopes.

In this study, we investigated the relative localization of some antigenic epitopes in the inner ear. The inner ear protein antigens were extracted from various parts of the guinea pig inner ear. Brain, kidney, lung, heart and liver extracts were also obtained. We found by SDS-polyacrylamide gel electrophoresis that total inner ear extracts separated into three high concentration polypeptide bands with molecular weights of approximately 30, 42, 58 kd and three low density bands of 20, 25 and 35 kd. The 30 kd band was found mainly in the extract of the spiral ganglion and the acoustic nerve in the modiolus. The 42 and 58 kd bands were detected in the extract of the spiral ligament and the stria vascularis. The Organ of Corti and the basilar membrane extract gave rise to three bands of 30, 42 and 58 kd. Twenty-eight of the 75 sera from patients with inner ear disease reacted with the 30 and 58 kd bands of the inner ear protein extracts by immunoblotting. Sixteen of these 28 positive sera were then used to probe immunoblots of the brain, kidney, lung, heart and liver extracts. The 58 kd band was also found in protein extracts of the brain, the lung and the liver. This study suggests that the 30 kd antigenic epitope may be mainly related to the acoustic nerve and that the 58 kd antigenic epitope is not cochlear specific.

Mechanisms of hearing disturbance in an autoimmune model mouse NZB/kl.

A subline of the NZB mouse, NZB/kl, was found to develop severe hearing disturbances at high frequency sound at the age of 4 to 6 months. Deposition of IgG was observed on the capillary wall of the stria vascularis of the mice, but the concentration of circulating immune complex did not seem to be correlated to the deposition. Electron microscopic examination revealed that the capillaries had a thick basement membrane, and in severe cases the membrane contained foamy structures of various size. In some cases the base membrane was so thick that the capillary lumen was narrowed, and the intermediate cells seemed to be damaged. No pathological findings were found in other inner ear tissues. These results suggest that the changes in the stria vascularis were possibly caused by an autoimmune mechanism which resulted in hearing disturbance.

Indirect immunofluorescence in the investigation of rapidly progressive sensorineural hearing loss and Menière's disease.

Being aware of the conservation of antigenicity, we performed indirect immunofluorescence on non fixed non decalcified frozen hamster's cochlea. Once killed, the heads of the hamsters were immediately dipped into liquid nitrogen and then stored at -20 degrees C until cutting. The sections were done at the same temperature, with Tissue-Tek embedding the heads to avoid breaking, using a sharp tungsten knife. The immunofluorescence itself was performed with the sera of the patients which were revealed through donkey fluorescein-conjugated antiserum to human immunoglobulins. For counterstaining, we used Evans blue. Twenty patients with rapidly progressive sensorineural hearing loss (according to the clinical criteria of MacCabe) and 6 with Menière's disease were tested. Five had a specific fluorescence on the stria vascularis. A correlation with the lymphocyte transformation test on human inner ear antigen has been found (p = 0.055).

Alterations of charge barrier in the inner ear following immune reactions.

We investigated electron microscopically the changes of anionic sites of a charge barrier in the capillary basement membrane of the stria vascularis and endolymphatic sac following inner ear immune reactions. Hartley guinea pigs were immunized with bovine type II collagen, keyhole limpet hemocyanin, or horseradish peroxidase, with boosted and challenged antigens through the stylomastoid foramen. Animals were killed painlessly from several days up to 56 days after the antigen challenge. Polyethylenimine was used as a cationic tracer in order to observe the localization of anionic sites of the charge. In the animals immunized with bovine type II collagen or horseradish peroxidase, a significant decrease of anionic charge in the stria and the sac was found in the early stage of immunization. However, the keyhold limpet hemocyanin immunization group did not show any remarkable changes in the charge because of its lesser transfer into the inner ear due to of its high molecular weight and negative surface charge. A decrease of the charge under immunologic conditions may induce a hyperpermeability of vessels and a malabsorption of endolymph, and thus may cause endolymphatic hydrops.

Stria vascularis ultrastructural pathology in the C3H/lpr autoimmune strain mouse: a potential mechanism for immune-related hearing loss.

The stria vascularis in the C3H/lpr autoimmune strain mouse was ultrastructurally examined in order to better understand the potential mechanisms by which systemic autoimmune disease affects the ear. The inner ear from C3H/lpr mice before disease onset and C3H/HeJ controls showed no apparent pathology. However, the stria vascularis from older C3H/lpr mice after systemic autoimmune disease onset showed considerable intercellular edema around the stria capillaries and thickening of the capillary basement membrane, compared to controls. These observations suggest that perivascular abnormalities, which are the hallmark of systemic autoimmune diseases, may underlie the stria dysfunction and hearing loss seen in autoimmune diseases in humans.

The effect of anaphylatoxin component on inner ear damage.

It has already been reported that anaphylatoxin (AT) is closely related to inner ear lesions. In this study the pathology of inner ear damage caused by AT was investigated in Hartley strain and C4 deficient guinea pigs (C4D-GP). Although the biological activity of C3a and C5a is strong, it is of short duration, and the resulting ear damage is considered to be reversible. Because of the cellular degeneration caused by this slight damage, the damage owing to continuously activated C4a is thought to be irreversible, that is, atrophy in the stria vascularis, degeneration and sloughing of the cochlear neurons, and stretching of Reissner's membrane, and C4a is therefore considered to be requisite for the manifestation of inner ear damage.