Ken Jinnouchi

Expression of caspase-activated deoxyribonuclease (CAD) and caspase 3 (CPP32) in the cochlea of cisplatin (CDDP)-treated guinea pigs

Cisplatin, an anti-cancer drug, is known to induce apoptosis. During apoptosis, double-stranded DNA is broken into single-stranded DNA by the action of caspases and caspase activated deoxyribonuclease (CAD). We immunohistochemically examined the cochlea of guinea pigs for signs of the apoptosis after the administration of cisplatin. Cisplatin (10 mg/kg b.w.) was intraperitoneally injected to guinea pigs and 3 days later, the animals were sacrificed by intracardiac perfusion of 4% paraformaldehyde. The temporal bones were then removed and immunohistochemically stained for CAD and caspase 3, using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labelling method. CAD was observed in the stria vascularis and the spiral ligament. Caspase 3 was also detected in the stria vascularis, the spiral ligament and the supporting cells of the organ of Corti. These findings suggest that apoptosis is involved in the cochlear damage observed in cancer patients treated with cisplatin.

Carboplatin Induces Less Apoptosis in the Cochlea of Guinea Pigs than Cisplatin

Background: Cisplatin (CDDP) is known to cause inner ear damage while carboplatin (CBDCA) induces less ototoxicity than CDDP. We examined apoptotic changes in the cochlea of guinea pigs after injection of CDDP or CBDCA using immunohistochemical and electrophysiological techniques. Methods: Three days after the injection of each solution, the cochleas were immunohistochemically examined for the presence of fragments of single-stranded DNA (ssDNA). The auditory brain stem response was recorded before and three days after the injection. Results: We detected fragments of ssDNA in the stria vascularis and the spiral ligament of the CDDP-treated cochlea. In this group, the threshold of the auditory brainstem response was significantly elevated, however, in the CBDCA group, no apparent change of the threshold was detected. In the CBDCA group, fragments of ssDNA were detected in the stria vascularis and the spiral ligament. The number of cells that stained positive for ssDNA, was less than that in the CDDP group. Conclusions: Our findings indicate that CBDCA induces less apoptosis than CDDP and that this phenomenon contributes to the ototoxicity of CDDP.

Detection of apoptotic change in the lipopolysaccharide (LPS)-treated cochlea of guinea pigs

This study was undertaken to examine, electrophysiologically and immunohistochemically, the effect of endotoxin on the guinea pig cochlea. A bacterial endotoxin (lipopolysaccharide, LPS, 5 mg/ml, 0.2 ml) was injected into the middle ear trans-tympanically. The electrocochleograms were continuously recorded from before to 48 h after the injection with an electrode inserted into the facial canal. Then, the animals were sacrificed by intracardiac perfusion of a fixative, temporal bones were removed and immunohistochemically stained for single-stranded DNA (ssDNA) and caspase 3 (CPP32). ssDNA was detected at 48 h in the stria vascularis and spiral ligament. CPP32 was observed in the stria vascularis, the spiral ligament and the organ of Corti. The threshold of the compound action potential increased significantly at 48 h in the LPS group. These results suggest that the activation of CPP32 and fragmentation of DNA are involved in the dysfunction of the cochlea observed under inflammatory conditions.

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.

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.

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.

Expression of caspase-activated deoxyribonuclease (CAD) and caspase 3 (CPP32) in the hydropic cochlea of guinea pigs: second report

Abstract Apoptosis was induced in the cochlea by the injection of keyhole limpet hemocyanin (KLH) into the endolymphatic sac of guinea pigs and immunohistochemically examined. Keyhole limpet hemocyanin was injected into the right endolymphatic sac. The temporal bones were fixed via cardiac infusion of fixative and immunohistochemically stained for caspase-activated deoxyribonuclease or caspase 3. Endolymphatic hydrops became evident in the cochlea 1 day after the injection of keyhole limpet hemocyanin (n=6). The temporal bones in the control group did not show any caspase-activated deoxyribonuclease or caspase 3 immunoreactivity (n=6). Immunoreactivity for caspase 3 was detected in the supporting cells of the organ of Corti, the stria vascularis and the spiral ganglion cells. Caspase-activated deoxyribonuclease was also detected in the same areas. These findings suggest that apoptosis is involved in the pathogenesis of endolymphatic hydrops. This phenomenon could lead to cochlear dysfunction, as seen in endolymphatic hydrops.

Expression of myeloperoxidase and cochlear dysfunction in the lipopolysaccharide-treated guinea pig.

Abstract In this study, the effect of endotoxin on the guinea pig cochlea was examined by electrophysiology and immunohistochemistry. Bacterial lipopolysaccharide (LPS) was injected into the middle ear transtympanically. Electrocochleograms were measured immediately and 48 h after the injection with an electrode inserted into the facial canal. After the electrophysiological measurement, the animals were killed with an intracardiac perfusion of fixative and the temporal bones were removed and processed for immunohistochemistry with anti-myeloperoxidase (MPO) antibody. MPO could be detected after 48 h in the lateral wall and the organ of Corti. After injection of LPS, the threshold of the compound action potential worsened significantly at 48 h in the LPS group. These results suggest that MPO and reactive oxygen species are involved in cochlea dysfunction under inflammatory conditions.