J.C.M.J. de Groot

Human Cochlear Pathology in Aminoglycoside Ototoxicity—A Review

The pathological findings due to aminoglycoside ototoxicity in man, as reported in the literature, is reviewed. 21 cases (40 ears) have been studied after serial sectioning, 8 cases (12 ears) have been investigated by the microdissection and surface preparation technique. The combination of both methods provides maximal information. OHCs are primarily affected, followed by IHC loss. Degeneration starts at the basal coil and proceeds towards the apex. The stria vascularis becomes involved in all turns. It is not clear whether stria degeneration is primary or secondary. Supporting cells, nerve fibres and ganglion cells degenerate secondary to hair cell loss. In some cases ingrowth of myelinated nerve fibres in areas with complete destruction of the organ of Corti has been observed. Ototoxic lesions can be asymmetric. Attention is drawn to the audiometric Z-curve in moderate lesions.

An improved fixation method for guinea pig cochlear tissues

The influence of different fixation methods and of various primary fixatives on the ultrastructural preservation of guinea pig cochlear tissues was investigated. No differences in fixation quality were observed between cochleas fixed by intravascular perfusion and cochleas fixed by intralabyrinthine perfusion. Tri-aldehyde primary fixation resulted, in contrast to other formulae investigated, in an excellent, uniform preservation of all cochlear tissues without obvious fixation artefacts. The influence of OSO4/K4Ru(CN)6- and OSO4/K4Fe(CN)6 postfixation was also tested. Cochlear tissues postfixed with OSO4/K4Ru(CN)6 or OSO4/K4Fe(CN)6 exhibited more cellular detail (e.g., membrane- and glycogen contrast) as compared to tissues postfixed with OSO4 alone. Tri-aldehyde primary fixation followed by OSO4/K4Ru(CN)6- or OSO4/K4Fe(CN)6 postfixation therefore is recommended as a multipurpose procedure for optimal preservation of labyrinthine tissues.

The ototoxic effects of ethyl benzene in rats

Exposure to organic solvents has been shown to be ototoxic in animals and there is evidence that these solvents can induce hearing loss in humans. In this study, the effects of inhalation of the possibly ototoxic solvent ethyl benzene on the cochlear function and morphology were evaluated using three complementary techniques: (1) reflex modification audiometry (RMA), (2) electrocochleography and (3) histological examination of the cochleas. Rats were exposed to either ethyl benzene (800 ppm, 8 h/day for 5 days) or to control conditions. The RMA threshold increased significantly by about 25 dB, 1 and 4 weeks after the exposure, irrespective of the stimulus frequency tested (4-24 kHz). Electrocochleography was performed between 8 and 11 weeks after exposure to the organic solvent. The threshold for the compound action potential increased significantly by 10-30 dB at all frequencies tested (1-24 kHz). Histological examination of the cochlea showed outer hair cell (OHC) loss, especially in the upper basal and lower middle turns (corresponding to the mid-frequency region) to an extent of 65%. We conclude that exposure to 800 ppm ethyl benzene for 8 h/day during 5 days induces hearing loss in rats due to OHC loss.

Ultrastructure of the stria vascularis and Reissner's membrane in experimental hydrops

A time-sequence study was made of the early ultrastructural changes of the stria vascularis and Reissners membrane in the guinea pig after obliteration of the endolymphatic sac and duct. Pathological alterations of both the stria vascularis and Reissners membrane were found to start in the apex of the cochlea. The morphological changes of the stria vascularis were characterized by an increase of vesicles in the marginal cells and by intercellular edema, followed by vacuolization and atrophy of marginal and intermediate cells. In Reissners membrane extensive gaps in the mesothelial cell layer were observed together with intracellular pathology of the epithelial cells. The significance of these ultrastructural changes in the stria vascularis and Reissners membrane with regard to the pathophysiology of the endolymphatic hydrops is discussed.

Cisplatin ototoxicity and the possibly protective effect of α-melanocyte stimulating hormone

It is known that adrenocorticotrophic hormone (ACTH)-derived peptides, the so-called melanocortins, can reduce cisplatin-induced neurotoxicity. Recently, our group has found that cisplatin-induced ototoxicity can also be reduced or prevented by treatment with the synthetic melanocortin-like peptide, ORG 2766 (Hamers et al., 1994; De Groot et al., 1997). The present study was designed to investigate the possibly ameliorating effects of the physiologically more relevant naturally occurring neuropeptide α-melanocyte stimulating hormone (α-MSH) upon cisplatin ototoxicity and to compare its protective effects to those of ORG 2766. For eight consecutive days guinea pigs were treated with cisplatin at a concentration of either 1.5 mg/kg/day or 2 mg/kg/day. Animals were co-treated with either α-MSH (75 μg/kg/day), ORG 2766 (75 μg/kg/day), or a sham injection containing physiological saline. Electrocochleography and hair cell counts were performed. Treatment with 1.5 mg/kg/day cisplatin resulted in a large variability of the morphological and electrophysiological data, a variability that might have masked possible effects of ORG 2766 and α-MSH. Treatment with 2 mg/kg/day cisplatin caused less variable, severe reductions in the compound action potentials and cochlear microphonics combined with basal and middle-turn outer hair cell loss in five out of six animals. However, in the α-MSH co-treated groups, two out of six animals could be classified as normal, two animals as moderately affected and two animals as severely affected. In the ORG 2766 co-treated group we found three animals that were not affected and three animals that were severely affected. We conclude that the protective effects of α-MSH and ORG 2766 co-treatment are comparable and that α-MSH might be clinically useful in protecting against cisplatin-induced ototoxicity.

Early effects of gentamicin on inner ear glycocalyx cytochemistry

The early effects of gentamicin treatment on the inner ear glycocalyx were investigated using two cationic probes, colloidal thorium and cationized ferritin. Gentamicin treatment resulted in a diminished thorium reactivity of both the endolymphatic and perilymphatic glycocalyx of the hair cells after 1 day and complete abolishment of reactivity after 5 days. Cationized ferritin reactivity of the perilymphatic and endolymphatic glycocalyx was not significantly influenced. The cytochemistry of the inner ear glycocalyx and the possible biochemical changes induced by gentamicin are discussed.

Early Ultrastructural Effects of Gentamicin Cochleotoxicity

Ultrastructural changes in the cochlear hair cells during the early stages of gentamicin intoxication were investigated after tri-aldehyde primary fixation and OSO4/K4Ru(CN)6 post-fixation. In cochleas treated for 5, 10 and 15 days with gentamicin the individual outer hair cells (OHC1, OHC2 and OHC3) were randomly affected. Degeneration of the inner hair cells was not observed at this stage. Ultrastructurally, the earliest identifiable changes were an increase in lysosomes, proliferation of the endoplasmic reticulum, and formation of Hensens bodies. This was followed by dilatation of the endoplasmic reticulum and the nuclear envelope, giving rise to extensive cytoplasmic vacuolation. These results demonstrate that gentamicin-induced changes primarily involve the cells synthetic apparatus. All further intracellular changes occur at a much later stage and are therefore to be considered secondary events.

Induction of endolymphatic hydrops in the guinea pig by perisaccular deposition of sepharose beads carrying and not carrying immune complexes

We tried to induce endolymphatic hydrops in guinea pig cochleas by unilateral, perisaccular deposition of sepharose beads carrying immune complexes. Controls consisted of the deposition of sepharose beads without immune complexes and the contralateral, untreated ear. The effects of the treatment were studied by light microscopy and electrophysiological recordings of the gross cochlear potentials 1, 2, and 6 weeks after treatment. Each condition included six animals. Analysis of variance of the morphometric data concerning the ears treated with deposition of the beads showed a statistically significant difference (P = 0.04) between the degree of hydrops found for the beads with immune complexes and for those without. The difference between the treated ears and the contralateral untreated ears was significant (P = 0.01) for the beads with immune complexes and not significant (P = 0.8) for those without immune complexes while there was no significant effect of post-treatment time interval. Analysis of variance of the electrophysiological data, collected in response to tone bursts at the apex of the cochlea, showed no significant differences between the results for the beads with and without immune complexes. Therefore these results were pooled. One week after treatment the pooled results for the compound action potential showed a small decrease in amplitude, just significant at 2 kHz, but not at 4 and 8 kHz. This decrease disappeared completely after 6 weeks. The pooled results for the negative summating potential (SP) showed a significant increase in magnitude at all frequencies decreasing with post-treatment interval. The cochlear microphonics did not demonstrate any change in amplitude after treatment. The results indicate that deposition of sepharose beads with immune complexes induces endolymphatic hydrops. Also, deposition of the sepharose beads itself may have induced hydrops together with enhancement of the SP. SP enhancement may be related to the development of endolymphatic hydrops rather than to the presence of hydrops as such. This may be based on pressure build-up while hydrops develops.

Ultrastructure of the Organ of Corti in Experimental Hydrops

The early ultrastructural changes of the organ of Corti in the guinea pig after obliteration of the endolymphatic sac and duct were examined in a time-sequence study. Initial loss of outer hair cells was followed by inner hair cell degeneration, both starting at the apical part of the cochlea. Morphological changes of the sensory cells were found to start at the endolymphatic surface and were characterized by a variety of sterociliary pathologies, distortion of the cuticular plate and a less prominent contrast-staining of glycocalyx. Further degeneration was marked by pathological changes of intracellular organelles and the afferent and efferent nerve endings. Details of the ultrastructural changes in the organ of Corti are discussed with regard to the pathophysiology of experimental hydrops.

Structure and composition of stereocilia cross-links in normal and hydropic cochleas of the guinea pig.

SummaryStructure and composition of stereocilia cross-links were investigated cytochemically in normal and hydropic cochleas of the guinea pig. The electron-dense markers colloidal thorium and cationized ferritin were used for visualization. Side links as well as tip links were visualized using both markers. Cationized ferritin allowed a better visualization of the delicate cross-link substructure than did colloidal thorium. Following digestion with neuraminidase, cross-link reactivity for colloidal thorium was virtually abolished. However, the basic structure of the cross-links could still be observed as a result of routine post-fixation and contrast staining. In both 3- and 6-month hydropic cochleas glycocalyx reactivity of the stereocilia appeared to be unaltered, provided that stereocilia were still present. However, loss of cross-links of the outer hair cells — resulting in disarrangement of the stereociliary bundles — was observed in hydropic cochleas. Our results suggest that cross-links are a separate morphological and cytochemical entity, which is different from the glycocalyx. Furthermore, loss of stereocilia cross-links, with concomitant disarrangement of the outer hair cell stereociliary bundle, appears to be one of the early pathological features of surgically induced endolymphatic hydrops, which might be responsible for permanent sensorineural hearing loss.