Oral administration of an herbal medicine to prevent progressive hearing loss in a mouse model of diabetes.

OBJECTIVE: Tsumura Suzuki Obese Diabetes (TSOD) mice exhibit early age-associated hearing loss. Histopathological analysis of these mice shows narrowing of capillaries in the stria vascularis and chronic reduction of blood flow in the cochlea. In this study, we investigated the effect of oral administration of a herbal medicine or calorie restriction on hearing in TSOD mice. METHODS: TSOD mice were divided into 4 groups: CR (calorie restriction), BF and DS (treated with the herbal medicines, Bofutsushosan and Daisaikoto, respectively), and the control group. Body weight, blood glucose levels, and auditory brainstem responses (ABRs) were measured. The cochleae were excised and evaluated histopathologically. RESULTS: Blood glucose levels were suppressed in the CR, BF, and DS groups. In addition, the elevation of ABR thresholds was inhibited in the CR, BF, and DS groups. Cochlear blood vessels remained wide in the three treatment groups compared with the control group. These results suggested that the administration of these herbal medicines improved glucose tolerance and yielded results similar to those on calorie restriction. CONCLUSION: Oral administration of 2 herbal medicines can prevent hearing function disorder in a model mouse of diabetes. The results may clarify the possibility of clinical application.

Isolation, cultivation, and characterization of primary bovine cochlear pericytes: A new in vitro model of stria vascularis.

The study of strial pericytes has gained great interest as they are pivotal for the physiology of stria vascularis. To provide an easily accessible in vitro model, here we described a growth medium-based approach to obtain and cultivate primary bovine cochlear pericytes (BCP) from the stria vascularis of explanted bovine cochleae. We obtained high-quality pericytes in 8-10 days with a > 90% purity after the second passage. Immunocytochemical analysis showed a homogeneous population of cells expressing typical pericyte markers, such as neural/glial antigen 2 (NG2), platelet-derived growth factor receptorß (PDGFRß), α-smooth muscle actin (α-SMA), and negative for the endothelial marker von Willebrand factor. When challenged with tumor necrosis factor or lipopolysaccharide, BCP changed their shape, similarly to human retinal pericytes (HRPC). The sensitivity of BCP to ototoxic drugs was evaluated by challenging with cisplatin or gentamicin for 48 hr. Compared to human retinal endothelial cells and HRPC, cell viability of BCP was significantly lower ( p < 0.05) after the treatment with gentamicin or cisplatin. These data indicate that our protocol provides a simple and reliable method to obtain highly pure strial BCP. Furthermore, BCP are suitable to assess the safety profile of molecules which supposedly exert ototoxic activity, and may represent a valid alternative to in vivo tests.

Reduction of cisplatin ototoxicity in rats by oral administration of pomegranate extract.

The aim of this study was to investigate the effectiveness of the oral administration of pomegranate extract (PE) as a protective agent against cisplatin-induced ototoxicity. The study included a prospective, controlled animal study Group 1 (n = 6), received no cisplatin or PE, and group 2 (n = 6) received cisplatin at 8 mg/kg/day for 3 consecutive days. Group 3 (n = 6) received not only cisplatin at 8 mg/kg/day for 3 consecutive days, but also received PE (100 µL/day) via gavage for 5 days prior to the cisplatin injection and for 3 days concomitantly with the cisplatin injections. To measure cisplatin ototoxic effects, "distortion product otoacoustic emissions" (DPOAE) were analyzed 3 days before and after the cisplatin injections. Histological changes in the cochleas were observed by light microscopy. Compared with group 3, the DPOAE amplitudes of group 2 decreased significantly. Among the groups, there was a statistically significant difference in basal and mid turn external ciliated cells (ECC) number, but there was no statistically significant difference in apical turn. Differences in stria vascularis (SV) changes were statistically significant between the groups, and the median score for SV injury was significantly greater in group 2 than in group 3. Differences in the median scores for SGC changes being significantly greater in group 2 than in group 3. In conclusion, these results indicated that oral administration of PE afforded statistically significant protection to the cochlea in rats from cisplatin toxicity, and thus, oral experimental dose of PE administration may have a protective effect against cisplatin ototoxicity in rats.

Trafficking of systemic fluorescent gentamicin into the cochlea and hair cells.

Aminoglycosides enter inner ear hair cells across their apical membranes via endocytosis, or through the mechanoelectrical transduction channels in vitro, suggesting that these drugs enter cochlear hair cells from endolymph to exert their cytotoxic effect. We used zebrafish to determine if fluorescently tagged gentamicin (GTTR) also enters hair cells via apically located calcium-sensitive cation channels and the cytotoxicity of GTTR to hair cells. We then examined the serum kinetics of GTTR following systemic injection in mice and which murine cochlear sites preferentially loaded with systemically administered GTTR over time by confocal microscopy. GTTR is taken up by, and is toxic to, wild-type zebrafish neuromast hair cells. Neuromast hair cell uptake of GTTR is attenuated by high concentrations of extracellular calcium or unconjugated gentamicin and is blocked in mariner mutant zebrafish, suggestive of entry via the apical mechanotransduction channel. In murine cochleae, GTTR is preferentially taken up by the stria vascularis compared to the spiral ligament, peaking 3 h after intra-peritoneal injection, following GTTR kinetics in serum. Strial marginal cells display greater intensity of GTTR fluorescence compared to intermediate and basal cells. Immunofluorescent detection of gentamicin in the cochlea also revealed widespread cellular labeling throughout the cochlea, with preferential labeling of marginal cells. Only GTTR fluorescence displayed increasing cytoplasmic intensity with increasing concentration, unlike the cytoplasmic intensity of fluorescence from immunolabeled gentamicin. These data suggest that systemically administered aminoglycosides are trafficked from strial capillaries into marginal cells and clear into endolymph. If so, this will facilitate electrophoretically driven aminoglycoside entry into hair cells from endolymph. Trans-strial trafficking of aminoglycosides from strial capillaries to marginal cells will be dependent on as-yet-unidentified mechanisms that convey these drugs across the intra-strial electrical barrier and into marginal cells.

[Effect of injectio Salvia Miltiorrhiza on gentamicin ototoxicity-induced activity of nitric oxide synthase in cochlear stria vascularis of guinea pig].

AIM: to investigate the change of nitric oxide synthase (NOS) activity in cochlear stria vascularis (SV) of guinea pig after gentamicin (GM) and Salvia Miltiorrhiza (SM) injection, and to explore the protective role of injectio SM on GM ototoxicity. METHODS: NADPH-diaphorase histochemistry staining and image quantitative analysis technique, combined with auditory brainstem response (ABR) measurement. RESULTS: SM + GM significantly reduced NOS activity in cochlear SV and ABR threshold as compared with CM along (P < 0.01); and ABR threshold shift was in high correlation with NOS activity (rControl = -0.9464; rGM = -0.9117; rSM + GM = -0.8958; P < 0.01). CONCLUSION: SM can reduce NOS activity in cochlear SV so as to alleviate GM ototoxicity, thus ameliorate hearing function.

Effect of cisplatin on the basement membrane anionic sites in the ampulla, macula, and stria vascularis of guinea pigs.

Our objective was to compare changes in the basement membrane anionic sites (BMAs) in the ampulla, macula, and stria vascularis following the infusion of cisplatin (CDDP). After CDDP was administered to anesthetized Hartley guinea pigs, the bony labyrinth was immersed in a solution of polyethyleneimine (PEI). The size and distribution of PEI particles associated with BMAs in the stria vascularis and in the dark cell and sensory cell areas of the vestibular labyrinth were determined by electron microscopy. A significant reduction in the number and size of PEI particles was observed on CDDP-treated strial vessels. The number and size of PEI particles on the basement membranes of the vestibular labyrinth did not differ from those in the control. Our findings suggest that the BMAs of the vestibular labyrinth were not significantly affected by the administration of a single dose of CDDP.

The ototoxicity of 3,3'-iminodipropionitrile: functional and morphological evidence of cochlear damage.

Previous reports have suggested that IDPN may be ototoxic (Wolff et al., 1977; Crofton and Knight, 1991). The purpose of this research was to investigate the ototoxicity of IDPN using behavioral, physiological and morphological approaches. Three groups of adult rats were exposed to IDPN (0-400 mg/kg/day) for three consecutive days. In the first group, at 9-10 weeks post-exposure, thresholds for hearing of 5.3- and 38-kHz filtered clicks were measured electrophysiologically and brainstem auditory evoked responses (BAERs) were also recorded to a suprathreshold broadband click stimulus. A second set of animals was tested at 9 weeks for behavioral hearing thresholds (0.5- to 40-kHz tones) and at 11-12 weeks post-exposure for BAER thresholds (5- to 80-kHz filtered clicks). A third group of animals was exposed (as above), and killed at 12-14 weeks post-exposure for histological assessment. Kanamycin sulfate was used as a positive control for high-frequency selective hearing loss. Surface preparations of the organ of Corti were prepared in order to assess hair cells, and mid-modiolar sections of the cochlea were used to examine Rosenthal's canal and the stria vascularis. Functional data demonstrate a broad-spectrum hearing loss ranging from 0.5 kHz (30 dB deficit) to 80 kHz (40 dB deficit), as compared to a hearing deficit in kanamycin-exposed animals that was only apparent at frequencies greater than 5 kHz. Surface preparations revealed IDPN-induced hair cell loss in all turns of the organ of Corti, with a basal-to-apical gradient (more damage in the basal turns) at the lower dosages. At higher dosages there was complete destruction of the organ of Corti. There was also a dosage-related loss of spiral ganglion cells in all turns of the cochlea, again with a basal-to-apical gradient at the lower dosages. These data demonstrate that IDPN exposure in the rat results in extensive hearing loss and loss of neural structures in the cochlea.

Neuropeptide Y and behaviour: effects of intrastriatal NPY on circling behaviour of rats.

Neuropeptide Y (NPY) is a member of the pancreatic polypeptide family and consists of 36 amino acids, sharing sequence homologies with the putative gut hormone peptides YY and PP. NPY dose-dependently stimulates food intake when administered icv into the paraventricular hypothalamic nucleus of rats. Icv NPY has also been reported to decrease locomotor activity, rearing and grooming behaviour. Little behavioural research focusing on other unconditioned behaviours has been conducted. However, intrastriatal injections of NPY have been shown to increase dopamine (DA) turnover there. As unilateral manipulations of central DA result in turning away from the side of higher DA activity, it is of interest to evaluate the effects of intrastriatal NPY on this behaviour. Preliminary results indicate that NPY produces a significant contralateral turning bias when injected directly into the striatum. This raises the intriguing possibility that contralateral turning induced by intrastriatal NPY may be mediated by DA.

Simultaneous investigations of elemental changes in individual cells of the stria vascularis and in endolymph.

Using the microprobe for energy dispersive X-ray microanalysis, the elemental compositions of both the individual cells of the stria vascularis and of the endolymph were followed simultaneously under normal conditions and after the administration of 120 mg/kg ethacrynic acid (EA). Marginal cells and intermediate cells showed reversible increases in potassium and decreases in sodium concentrations. Shifts in the ionic composition of endolymph occurred later than after elemental changes in the strial cells. The present results indicate that the marginal and the intermediate cells are the primary target for EA-induced ototoxicity. However, generalized toxic effects of EA are also indicated, with a general leakage of different elements occurring during the 30-60 min period after EA administration.

Principles in cochlear toxicity.

The hair cells of the cochlea (neuroepithelium) represent the primary target in most drug-induced ototoxic adverse effects on hearing (e.g. aminoglycoside antibiotics). To what extent an exogenically-induced morphologic damage to hair cells is reversible is not known. In aging structurally altered hair cells can persist for years likewisely not any longer participating in sensory transduction as the hair cells degenerate, secondary changes occur in the spiral ganglion cells and the neuronal pathways. Following heavy metal poisoning an adverse effect is observed on both central and peripheral innervation of the cochlea and only minor primary changes occur in the receptor cells. The link between function and morphology in the cochlea is very obvious regarding the high and middle frequencies with a distinct tonotopic localisation whereas for low frequencies (below 1 khz) such a specific morphologic correlation is lacking. Ototoxic effects primarily affecting the source for the production of endolymph, i.e. the stria vascularis, become manifest at all frequencies and at a rather early stage. Independent of type of substance penetrating into the inner ear, the substance has a considerably slower elimination rate as compared with all other compartments in the body. The toxicity of the drugs seems to be more related to its tissue binding capacity and saturation of receptor sites than related to the concentration of the drug in endo-or perilymph.

Changes in cation contents of stria vascularis with ouabain and potassium-free perfusion.

Perfusion of the perilymphatic space of guinea pig cochleae with K-free medium leads to a gradual decline of the endocochlear potential (EP) over 30-50 min to a negative value (mean: -12 mV). The input resistance of scala media does not decrease during this time. The ATP and K content of the stria vascularis are reduced by similar amounts (26 and 34%, respectively) during this period. Perfusion of 1 mM ouabain produces a different pattern of response: strial ATP remains normal while strial K content is strongly reduced (by 77%). Strial Na rises in a complementary way to the K loss. These results demonstrate that a reduction of the K concentration of the perilymph leads to an inhibition of the generator of the positive component of the EP rather than to a general increase of cochlear duct membrane conductance. In addition, they suggest, in concert with other considerations (such as the slower rate of decline of the EP during K-free vascular perfusion (Wada, J., Kambayashi, J., Marcus, D.C. and Thalmann, R (1979): Arch. Otorhinolaryngol. 225, 79-81)), that the mode of action may be different from that of ouabain. In spite of the lack of teleological support, we offer the hypothesis that the strial generator of the EP may primarily utilize K from perilymph and that vascular K may not have access to the generator.