Shunichi Tomiyama

Development of Endolymphatic Hydrops Following Immune Response in the Endolymphatic Sac of the Guinea Pig

The present study investigated immune injury associated with endolymphatic hydrops (e.hydrops) following locally mounted immune reaction in the endolymphatic sac (e.sac) of guinea pigs. E.hydrops occurred, progressing rapidly within the first week post secondary Keyhole limpet hemocyanin (KLH) challenge in the e.sac and developed into two phases, acute and chronic. On the other hand, primary KLH challenge of the e.sac, PBS inoculation into the e.sac or intradural secondary KLH challenge were incapable of inducing e.hydrops. These results indicate that reversible and irreversible e.hydrops are induced by the immune response of the e.sac, suggesting that local immunological events of the e.sac may provide an animal model of Menieres disease.

Immunological Approach to Ménière’s Disease: Vestibular Immune Injury following Immune Reaction of the Endolymphatic Sac

Following direct antigen challenge to the endolymphatic sac (sac), the relation of caloric test results and spontaneous nystagmus to the histological changes of the sensory epithelium of the vestibular organ and perilymph antibody levels was investigated in the guinea pig. In a secondary keyhole limpet hemocyanin (KLH) challenge to the sac, irritative nystagmus was followed by paralytic nystagmus and a suppression of caloric reaction. These findings correlated well with the degree of degeneration of the sensory epithelium of the vestibular organ and the levels of perilymph antibody. On the other hand, neither phosphate-buffered saline inoculation nor primary KLH challenge to the sac nor a secondary KLH challenge to the intradural space resulted in a disturbance of the vestibular function. These results suggest that the immune response of the sac may possibly be an important key to the pathogenesis of Ménières disease.

Endolymphatic Hydrops Induced by Immune Response of the Endolymphatic Sac: Relation to Perilymph Antibody Levels

This study investigated the effect of a secondary immune response in the endolymphatic sac on the development of endolymphatic hydrops in guinea pigs. Following secondary keyhole limpet hemocyanin (KLH) challenge directly to the sac in systemically presensitized animals, hydrops rapidly developed within 1 week, then gradually reduced by 4 weeks. After 5 weeks, hydrops gradually recurred and developed again. Mean perilymph anti-KLH antibody levels rose from day 1 to 4 weeks and then decreased. The elevation of perilymph anti-KLH antibody levels on day 2 was due to an increased vascular permeability and there was a local regeneration by the challenge of the sac after day 7. The development of hydrops correlated well with mean perilymph anti-KLH antibody levels in the period from day 1 to day 7. After 2 weeks, there was no correlation between them. These results suggest that an immune response of the sac may induce an excess secretion of endolymph in the initial stage and may further lead to irreversible dysfunction of the sac at a later stage.

Fluctuating Hearing Loss following Immune Reaction in the Endolymphatic Sac of Guinea Pigs

This study has investigated immune injuries to the inner ear auditory system of guinea pigs. Following secondary antigen challenge to the endolymphatic sac, the mean hearing threshold significantly increased in the early phase from day 1 to day 3 and thereafter recovered. In the early phase, hearing threshold significantly increased simultaneously to the elevation of perilymph antibody levels. The size of hydrops was not the only factor that causes an increase in hearing loss as well as in AP/SP ratio. Scale-out hearing loss was seen in 2 animals with severe degeneration of the stria vascularis as well as the organ of Corti associated with the inflammatory cellular infiltration especially in the perilymphatic space, even in the absence of keyhole limpet hemocyanin antigen in the cochlea. On the other hand, control animals did not suffer hearing loss. These results suggest that an immune reaction in the endolymphatic sac is a possible pathogenic etiology of Ménières disease or sudden deafness.

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 raction.

Expression of Cochlin in the Vestibular Organ of Rats

The COCH gene mutated in autosomal dominant sensorineural deafness (DFNA9) encodes cochlin, a major constituent of the inner ear extracellular matrix. Cochlin constitutes 70% of the inner ear protein and cochlin isoforms can be classified into three subgroups, p63s, p44s and p40s. Symptoms of some DFNA9 patients are consistent with those of Ménière’s disease. Here, we report the expression of cochlin in the vestibular organ of rats using isoform-specific antibodies that recognize all three isoforms. Cochlin is highly expressed in the stromata of the maculae of otolithic organs and cristae of semicircular canals, and in the channels in the bony labyrinth that transmit the dendritic innervation to the cristae and maculae. Cochlin cannot be detected in the sensory cells, dark cells, nor in the acellular structures, otolithic membrane or in the cupula. These findings support the theory that deposition of acidophilic substance in the inner ear caused by mutation of cochlin can induce a secondary retrograde dendritic degeneration of the vestibular nerves.

INTERCELLULAR ADHESION MOLECULE-1 EXPRESSION IN THE INNER EAR OF RATS FOLLOWING SECONDARY IMMUNE REACTION IN THE ENDOLYMPHATIC SAC

An immunological aetiology for inner ear diseases has long been proposed. The endolymphatic sac (ES) is the only immunoprivileged site in the inner ear with a resident population of immunocompetent cells. By keyhole limpet hemocyanin (KLH) challenge into the ES of systemically pre-immunized guinea pigs, we previously demonstrated an infiltration of inflammatory cells into the perilymphatic space of the cochlea. In order to understand the mechanisms involved in the recruitment of immunocompetent cells into the inner ear, and their relation to the development of endolymphatic hydrops (EH), we investigated the expression and time-kinetics of intercellular adhesion molecule 1 (ICAM-1) in the inner ear of systemically pre-immunized rats after antigen (KLH) challenge into the ES, its relation to cell infiltration in the cochlea and subsequent development of EH. By immunohistochemistry, strong ICAM-1 expression was detected in the spiral ligament, suprastrial region, spiral prominence, spiral modiolar veins, spiral collecting venules, surface membrane of the perilymphatic compartment, perilymphatic space and ES of immunized rats, but not of control rats. ICAM-1 expression was detected at 5-6 h, peaked at 10-15 h, and gradually reduced by 2 weeks. Cell infiltration into the cochlea started at 6-12 h and peaked at day one. By 6 h, 50% of challenged rats developed EH. This figure rose to 70% at 12 h, and then gradually reduced. However, immunoreactivity for KLH (antigen) was only detected in the ES. These results emphasize that the sac is the central immunological organ of the inner ear, and suggest that ICAM-1 may play a pivotal role in the aetiology of immune-mediated inner ear diseases through the recruitment of immunocompetent cells into the inner ear and subsequent development of EH.

Distribution of Endothelin-1-like Activity in the Cochlea of Normal Guinea Pigs

Distribution of endothelin-1 (ET-1) in the cochlea of normal guinea pigs was determined by immunohistochemistry. ET-1 activity was identified with the mouse anti-human ET-1 IgG1 monoclonal antibody. ET-1 activity was distributed in the modiolus, spiral ligament, stria vascularis, spiral prominence. Reissners membrane, supporting cells of the organ of Corti and spiral ganglion cells. These findings suggest that ET-1 may be involved in the regulation of fluid volume and ions of the cochlea.

Inner ear autoantibodies in patients with sensorineural hearing loss.

In order to identify the inner ear autoantibodies, sera of 195 patients with sensorineural hearing loss and/or vertigo were reached with bovine inner ear antigen by western blotting method. Positive reaction was seen at several molecular weights, in which 33-35 kD, 42 kD and 68 kD were highly reactive. In moderate to severe hearing loss patients, positive reactions with 33-35 kD, 42 kD, 68 kD and others were 25.8%, 26.8%, 26.8% and 71.5% respectively. On the other hand, a positive rate did not clearly correlate with diagnostic entities. Further study to purify inner ear antigen may evaluate inner ear autoimmune disease.

Vestibular disorders following immune response of the endolymphatic sac in the guinea pig

The effect of a direct antigen challenge to the endolymphatic sac on vestibular function was investigated in guinea pigs. Following keyhole limpet hemocyanin (KLH) challenge to the sac in systemically presensitized guinea pigs, caloric responses were examined in 18 animals on days 1, 7, 14, 21, and 28. Caloric responses were significantly suppressed in 13 animals by day 7; of these, 5 animals had recovered by day 14 and 8 animals had not yet recovered by day 28. The behavior of spontaneous nystagmus was examined every hour in 10 animals at intervals of 3 to 56 hours after sac challenge. Irritative spontaneous nystagmus preceding paralytic nystagmus appeared in 5 animals, for which the mean onset was 14.6 ± 3.1 hours and the mean duration was 4.4 ± 6.5 hours. Paralytic spontaneous nystagmus appeared in all animals, for which the mean onset time was 23.3 ± 12.3 hours. Neither direct KLH primary challenge of the sac nor phosphate-buffered saline injection to the sac caused significant changes in the vestibular function. These results suggest that an immune response of the sac induces a vestibular disorder and may produce an attack of vertigo similar to that of Menieres disease.