Gary Zajic

Comparison of isolated outer hair cells from five mammalian species

Live outer hair cells were isolated from guinea pig, chinchilla, rat, mouse, and gerbil. The organ of Corti from selected turns of the cochlea was briefly incubated with collagenase and outer hair cells were separated from the tissue by micromanipulation under microscopic observation. Morphological criteria for cell viability were: cylindrical cell shape without swelling or distortion of the membrane; location of the nucleus in its normal position near the base of the cell; cytoplasm devoid of Brownian motion and granulation. Both yield and quality (as judged by these morphological criteria) of isolated hair cells varied with the species and the turn from which the isolation was attempted. Consistently high yields and cells of good morphology were obtained from guinea pigs and chinchillas. Fewer cells were obtained from rats and mice, and their quality was less consistent. Gerbils gave the poorest yield and quality of outer hair cells. In all species, the preparation was more successful from the apical than from the basal turn. The length of apical hair cells varied almost 4-fold from 60 to 80 microns in guinea pig and chinchilla to 20 to 40 microns in the other species, while their diameter only varied 1.5-fold from 7 (mouse) to 10 microns (chinchilla). Outer hair cells could be maintained in vitro in good condition for several hours. Typical early signs of degeneration were increased Brownian motion and granulation in the cytoplasm, upward movement of the nucleus, or distortion of cell shape. Degeneration was always accompanied by a shortening along the long axis.

Overexpression of Copper/Zinc- Superoxide Dismutase Protects from Kanamycin-Induced Hearing Loss

The participation of reactive oxygen species in aminoglycoside-induced ototoxicity has been deduced from observations that aminoglycoside-iron complexes catalyze the formation of superoxide radicals in vitro and that antioxidants attenuate ototoxicity in vivo. We therefore hypothesized that overexpression of Cu/Zn-superoxide dismutase (h-SOD1) should protect transgenic mice from ototoxicity. Immunocytochemistry confirmed expression of h-SOD1 in inner ear tissues of transgenic C57BL/6-TgN[SOD1]3Cje mice. Transgenic and nontransgenic littermates received kanamycin (400 mg/kg body weight/day) for 10 days beginning on day 10 after birth. Auditory thresholds were tested by evoked auditory brain stem responses at 1 month after birth. In nontransgenic animals, the threshold in the kanamycin-treated group was 45–50 dB higher than in saline-injected controls. In the transgenic group, kanamycin increased the threshold by only 15 dB over the respective controls. The effects were similar at 12 and 24 kHz. The protection by overexpression of superoxide dismutase supports the hypothesis that oxidant stress plays a significant role in aminoglycoside-induced ototoxicity. The results also suggest transgenic animals as suitable models to investigate the underlying mechanisms and possible strategies for prevention.

Differences in the distribution of F-actin in outer hair cells along the organ of Corti

There is evidence of differences in the structure, innervation and physiological responses between outer hair cells (OHCs) of the basal and apical turns of the mammalian cochlea. In this study we have used rhodamine-labelled phalloidin to investigate the differential distribution of F-actin in OHCs along the organ of Corti of the guinea pig. Isolated OHCs and surface preparations and cryosections of the organ of Corti were studied. F-actin was observed in stereocilia and the cuticular plate of all OHCs. In addition, some OHCs had a network of F-actin extending from the cuticular plate towards the nucleus. This infracuticular network was observed in most OHCs of the apical cochlear turns but was not seen in any OHCs of the basal turn. These microstructural differences between OHCs of the base and apex could be related to differences in OHC function between the apical and basal portions of the cochlea.

Differential motile response of isolated inner and outer hair cells to stimulation by potassium and calcium ions

Inner and outer hair cells were mechanically isolated from the guinea pig cochlea and subjected to stimuli known to induce shape changes in outer hair cells. Depolarization by 70 mM KCl which causes osmotic swelling of outer hair cells also swelled inner hair cells by approximately 8% of their volume. The application of the calcium ionophore ionomycin which induces cortical contractions and elongation of outer hair cells, did not affect the shape of inner hair cells. Since ionomycin increased free intracellular calcium levels in both inner and outer hair cells, the results demonstrate that inner hair cells do not possess the mechanisms necessary for a contractile response to calcium. Thus, calcium is a specific regulator of outer hair cell motility making this mechanism a likely physiological modulator of a transduction feedback process.

Gentamicin alters membrane structure as shown by freeze-fracture of liposomes.

Freeze-fracture has been used to examine the effects of gentamicin on membrane structure in liposomes of different anionic phospholipids combined with a neutral phospholipid, phosphatidylcholine. The molar ratios of neutral: anionic lipid were 1:1 (high anionic lipid ratio) and 4:1 (low anionic lipid) and the liposomes were incubated with 0.1 mM (low) and 1 mM (high) gentamicin. With the anionic phospholipid phosphatidylinositol bisphosphate, an identifiable disruption of the membrane bilayer was observed as well as aggregation of liposomes leading to membrane fusion. These effects occurred both at low gentamicin concentration and low anionic lipid content of the liposomes; these responses were not inhibited by 1 mM Ca2+. With the other anionic lipids tested (phosphatidylserine, phosphatidylinositol and phosphatidylinositol monophosphate), only aggregation and fusion of liposomes was observed and this effect only occurred at high gentamicin concentration and high anionic lipid content. Further, 1 mM Ca2+ inhibited the responses of these other anionic lipids to gentamicin. The results demonstrate the unique character of the interaction between gentamicin and phosphatidylinositol bisphosphate and provide further support for the hypothesis that a specific binding to this lipid is a key step in the ototoxic action of aminoglycoside antibiotics. They also suggest that such an interaction in vivo might cause alterations to the structure and properties of cell membranes in the inner ear.

Assessment of ultrastructure in isolated cochlear hair cells using a procedure for rapid freezing before freeze-fracture and deep-etching

SummarySeparated cochlear outer hair cells and isolated strips of organ of Corti containing hair cells and supporting cells have been rapidly frozen before freeze-fracture and deep-etching by immersion of samples sandwiched between two copper plates into liquid nitrogen-cooled propane: isopentane. Assessment of this procedure has shown that no significant freezing damage occurs. The ultrastructure of the hair cells revealed by freeze-fracture of these non-chemically fixed preparations was generally very similar to that seen in fixed material. This indicates that the processing of cochlear tissue normally used for electron microscopy produces few obvious structural artefacts. It also demonstrated that procedures for isolating cochlear hair cells generally do not affect cell structure significantly. However, some isolated hair cells did show abnormalities within the membranes of the lateral cisternae. Such membrane alterations, which would not be identified by light microscopy, occurred to a variable extent but were more commonly present after prolonged periods in maintenance medium. Deep-etching of the preparations to examine extracellular features around stereocilia revealed clearly lateral cross-links between stereocilia. However, tip-links could not be positively identified in either unfixed or prefixed preparations.

Species differences in the distribution of infracuticular F-actin in outer hair cells of the cochlea

The distribution of filamentous actin (F-actin) in outer hair cells has been examined in several mammalian species using tetramethylrhodamine phalloidin, a specific marker for F-actin. The stereocilia and cuticular plates of the OHC in all species examined (pigmented guinea pig, hooded rat, chinchilla and squirrel monkey) contained F-actin; however, an infracuticular network of F-actin was present in OHC of the apical turns of the guinea pig cochlea but could not be identified in any other species examined.

Cytochemical demonstration of adenylate cyclase with strontium chloride in the rat pancreas.

A cytochemical procedure for the localization of adenylate cyclase with Sr2+ as the capture ion and adenylyl imidodiphosphate as the specific substrate was evaluated in the rat pancreas. Incubation medium was unaffected by the addition of 5 mM strontium ions but became turbid in the presence of lead or strontium plus 10 mM NaF. Tissues were prefixed in 2% formaldehyde/0.5% glutaraldehyde and incubated, and the cytochemical precipitate was converted to the Pb2+ salt. Enzymatic activity was demonstrated on the plasma membrane of pancreatic acinar cells and responded to stimulation by secretin. Controls frequently contained Pb2+ sequestered in mitochondria, but otherwise only a few randomly distributed grains were observed. The controls were 1) omission of substrate from the medium; 2) incubation of tissue for 1 min in complete medium; and 3) tissue previously inactivated by microwave irradiation and incubated for 30 min in complete medium including secretin.

Living isolated cells from inner ear vessels: A new approach for studying the regulation of cochlear microcirculation and vascular permeability

The spiral modiolar artery with its proximal branches and the microvessels in the spiral ligament and the stria vascularis were microdissected from the guinea pig cochlea. After incubation with proteolytic and collagenolytic enzymes the mixed cell suspension was fractionated by gradient centrifugation. The cells migrated according to their buoyant densities into the fractions of 1.04 g/ml (endothelial cells), 1.06 g/ml (vascular smooth muscle cells obtained from the spiral modiolar artery; strial pericytes) and 1.08 g/ml (pericytes obtained from the spiral ligament). To test for viability cells were loaded with a fluorescent vital stain (BCECF-AM); for identification, cell-specific stains were used. Identity of endothelial cells (ECs) was confirmed using acetylated low density lipoprotein fluorescently labeled with dioctadecyl-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL). Pericytes were identified immunofluorescently using the method according to Nayak et al. (1988). Vascular smooth muscle cells were stained for F-actin with rhodamin-phalloidin. This in vitro technique may open new approaches to study local factors involved in microcirculation and vessel permeability of various cochlear vascular beds.

CHARACTERISTICS OF THE MEMBRANE OF THE STEREOCILIA AND CELL APEX IN COCHLEAR HAIR-CELLS

SummaryFreeze-fracture has been used to examine the membrane of the cell apex and of the stereocilia in cochlear hair cells. The apical (non-stereociliary) membrane of inner hair cells (IHCs) exhibited a lower density of intramembrane particles (IMP) than that of the outer hair cells (OHCs) but in both cell types the apical membrane responded to the effects of filipin. The distribution of IMP and of filipin-induced membrane deformations was uniform over the apical membranes in both IHC and OHC, thus, providing no evidence for local membrane differentiation on the non-stereociliary part of the hair cell apex. The stereociliary membranes of IHC and of OHC differed not only in the density of IMP, but also in their responses to filipin and to tomatin. IHC stereocilia responded intensely to both agents. OHC stereocilia showed a significantly lower density of filipin-induced lesions and appeared almost unaffected by tomatin. This suggests that the OHC stereocilial membrane may be structurally specialized. The membrane at the apical end of stereocilia appeared to be differentiated from the membrane of the stereociliary shaft. The tip region was free of the usual IMP and showed no filipin-induced lesions. The differentiation at the apical end was also apparent in samples which have been rapidly frozen without prior chemical fixation or cryoprotection, showing that the particle-free area was not an artefact induced by glutaraldehyde fixation. Close examination of the membrane at the apical-most tip of the stereocilium revealed the presence of a small number of large particles of 10.5–11.0 nm diameter. The occurrence of membrane differentiation localized to the tip of the stereocilium may be consistent with the suggestion that transduction channels in hair cells are situated at this point.