Eri Hashino

Endocytosis of aminoglycoside antibiotics in sensory hair cells

Immuno-gold electron microscopy was used to assess the uptake pathways of aminoglycoside antibiotic kanamycin (KM) in sensory hair cells. Accumulation of gold particles was evident on the plasma membrane as well as in large smooth vesicles beneath the apical surfaces of hair cells 12 h after a systemic administration of KM. Immuno-gold was exclusively localized in the vesicles 27 h post-injection. Cationic ferritin, a membrane-bound insoluble marker, was colocalized with KM in the vesicle structures after their simultaneous in vitro application. These results strongly suggest that KM is taken up into sensory hair cells via receptor-mediated endocytosis at their apical surfaces. In addition, the profound time lag between KM uptake and hair cell death suggests involvement of targeting mechanisms in cytotoxic signalling pathways of the drugs.

Lysosomal targeting and accumulation of aminoglycoside antibiotics in sensory hair cells.

Our recent study demonstrated that aminoglycoside antibiotics are taken up into sensory hair cells of the inner ear by receptor-mediated endocytosis (E. Hashino, M. Shero, Endocytosis of aminoglycoside antibiotics in sensory hair cells, Brain Res. 704 (1995) 135-140). To elucidate the intracellular trafficking pathway of aminoglycosides following endocytotic uptake, we administered kanamycin to neonatal chicks for 1 or 5 days (400 mg/kg/day) and determined the location of kanamycin within the hair cells at various time points using immunogold electron microscopy. Quantitative and qualitative analysis of immunogold staining revealed that: (1) kanamycin was primarily localized in vesicles beneath the cuticular plate 27 h postinjection; (2) the number of vesicles per hair cell and the number of gold particles per vesicle increased over time; (3) individual vesicles tended to increase in size over time, presumably due to aggregation of smaller vesicles; and (4) in pathological hair cells, immunogold was dispersed throughout the entire subcellular region. Light microscopic observations of the basilar papilla stained with the same antibody confirmed the temporal changes in the kanamycin distribution. Moreover, results obtained from acid phosphatase cytochemistry indicated that vesicles accumulating kanamycin were mainly lysosomes. These results suggest that internalized aminoglycosides are transported via vesicular traffic into lysosomes where they accumulate over time and lead to disruption of lysosomes. The time of diffusion of kanamycin was closely related to the time of cell death, suggesting that lysosomal rupture could be a direct trigger for the hair cell degeneration.

Hair cell damage and recovery following chronic application of kanamycin in the chick cochlea

Three-day old chicks were given kanamycin at a dose of 200 mg/kg/day for 10 days and their cochleae were processed for scanning electron microscopy at 1, 3, 7 and 14 days following the last injection. Both hair cells and supporting cells were damaged by kanamycin in the basal 35% of the basilar papilla. By 14 days post-treatment, however, most of the damaged region had been replaced with regenerating hair cells and supporting cells. The base-to-apex gradient of morphological development along the cochlea was observed in the process of regeneration. Kinocilium and microvilli were observed on the apical surfaces of the regenerating hair cells.

Hair cell regeneration in the adult budgerigar after kanamycin ototoxicity.

Adult budgerigars were given kanamycin at a dose of 200 mg/kg/day for 10 successive days. At 1, 7, 14 and 28 days after the drug treatment, the cochleae of the birds were processed for scanning electron microscopy (SEM). Complete degeneration of sensory hair cells was observed in the basal 55-75% of the basilar papilla immediately after the treatment. Regenerating hair cells, characterized by clusters of microvilli and small apical surfaces, were present in the basal end of the papilla as early as one day post-treatment. During the 28 day recovery period, the number of hair cells progressively increased beginning at the base and spreading toward the apex. Although the appearance of the basilar papilla had improved considerably by 28 days post-treatment, the sensory epithelium still contained a number of pathologies, most noticeably, incomplete restoration of hair cell number in the most apical part of the damaged region and the disorganization of hair cell packing. These remaining pathologies may be responsible for the permanent threshold shifts observed in budgerigars exposed to the same dose of kanamycin treatment (Hashino and Sokabe, 1989).

Discharge patterns of chicken cochlear ganglion neurons following kanamycin-induced hair cell loss and regeneration

Hair cells in the basal, high frequency region (>1100 Hz) of the chicken cochlea were destroyed with kanamycin (400 mg/kg/d × 10 d) and allowed to regenerate. Afterwards, single unit recordings were made from cochlear ganglion neurons at various times post-treatment. During the first few weeks post-treatment, only neurons with low characteristic frequencies (<1100 Hz) responded to sound. Despite the fact that the low frequency region of the cochlea was not destroyed, neurons with low characteristic frequencies had elevated thresholds, abnormally broad U-shaped or W-shaped tuning curves and low spontaneous discharge rates. At 2 days post-treatment, the spontaneous discharge rates of some acoustically unresponsive units fluctuated in a rhythmical manner. As recovery time increased, thresholds decreased, tuning curves narrowed and developed a symmetrical V-shape, spontaneous rate increased and neurons with higher characteristic frequencies began to respond to sound. In addition, the proportion of interspike interval histograms with regularly spaced peaks increased. These improvements progressed along a low-to-high characteristic frequency gradient. By 10–20 weeks post-treatment, the thresholds and tuning curves of neurons with characteristic frequencies below 2000 Hz were within normal limits; however, the spontaneous discharge rates of the neurons were still significantly lower than those from normal animals.

Kanamycin induced low‐frequency hearing loss in the budgerigar (Melopsittacus undulatus)

The chronic effects of kanamycin (KM) on hearing in the budgerigar were investigated by behavioral audiometry. The birds received a daily intramuscular injection of KM (100 mg/kg or 200 mg/kg) for 10 successive days, and absolute thresholds between pre- and post-treatment were compared. KM induced both transient and permanent low-frequency specific hearing loss; i.e., the elevation of threshold for frequencies below 1 kHz was greater than that for frequencies above 1 kHz. Moreover, the degree of hearing loss was dose dependent. The low-frequency selective effects of KM in the present study were contrary to the high-frequency specificity of aminoglycoside ototoxicity in mammals. To assess the effect of KM on auditory frequency resolution, critical ratios were estimated in pathological birds with low-frequency specific hearing loss. There was a linear relation between shift in critical ratio and shift in absolute threshold, suggesting that the increase in the critical ratio is due to a decrease in the efficiency of the detector mechanism rather than a change in the spectral resolving power of the birds.

Recovery of CAP threshold and amplitude in chickens following kanamycin ototoxicity.

Chickens were given a dose of kanamycin (400 mg/kg/d x 10 d) which destroyed hair cells over the basal 37-58% of the basilar papilla. Afterwards, the threshold and amplitude of the compound action potential were measured at recovery times ranging from 2 days to 10-20 weeks post-kanamycin treatment. At 2 days post-treatment, the thresholds at 1000, 2000 and 4000 Hz were elevated 40-60 dB while the thresholds at 250 and 500 Hz were elevated only 25 dB. By 10-20 weeks post-treatment, the threshold at 250 and 500 Hz had completely recovered whereas a residual threshold shift of 5 dB to 25 dB was present between 1000 to 4000 Hz. The maximum amplitude of the compound action potential was also reduced by more than 60% at all frequencies at 2 days post-treatment; however by 10-20 weeks post-treatment, the amplitude of the compound action potential had completely recovered at 500, 1000 and 2000 Hz. By contrast, the amplitude of the compound action potential at 4000 Hz was still reduced by more than 50% of its normal value 10-20 weeks post-treatment. The results of the present study indicate that the time course of recovery of the compound action potential is extremely slow and may lag behind the regeneration of hair cells by many weeks. The permanent deficits observed at the high frequencies could conceivably be due to functional deficits in regenerated hair cells, their afferent synapses or the loss of cochlear ganglion cells.

Reversible and irreversible damage to cochlear afferent neurons by kainic acid excitotoxicity

Kainic acid (KA) selectively damages afferent synapses that innervate, in chickens, mainly tall hair cells. To better understand the nature of KA‐induced excitotoxic damage to the cochlear afferent neurons, KA, at two different concentrations (0.3 or 5 mM), was injected directly into the inner ear of adult chickens. Pathologic changes in the afferent nerve ending and cell body were evaluated with light and transmission electron microscopy at various time points after KA application. The compound action potential (CAP) and cochlear microphonic (CM) potential were recorded to monitor the physiologic status of the afferent neurons and hair cells, respectively. Hair cell morphology and function were essentially normal after KA treatment. However, afferent synapses beneath tall hair cells were swollen within 30 minutes after KA at both low (KA‐L) and high (KA‐H) doses. In the KA‐L group, the swelling disappeared within 1 day and the morphology of the postsynaptic region returned to near normal condition. In the KA‐H group, by contrast, the vacant region beneath tall hair cells remained evident even 20 weeks after KA. The number of cochlear ganglion neurons in the KA‐H group decreased progressively from 1 to 8–20 weeks, whereas hair cells in the basilar papilla remained morphologically intact out to 20 weeks after KA. There was no significant change in neuron number in the KA‐L group. Temporal changes in the CAP amplitude paralleled the anatomic changes, although the CAP only partially recovered. These results suggest that KA induces partially reversible damage to cochlear afferent neurons with low KA concentration; above this level, KA triggers irreversible, progressive neurodegeneration. J. Comp. Neurol. 430:172–181, 2001.

Base-to-apex gradient of cell proliferation in the chick cochlea following kanamycin-induced hair cell loss

In order to elucidate the mechanisms that drive cell proliferation in the avian cochlea, we investigated the spatio-temporal relationship between hair cell degeneration and cell proliferation after aminoglycoside ototoxicity. Neonatal chicks were given a daily intramuscular injection of kanamycin (KM) at 400 mg/kg per day for 10 consecutive days. At various times during or after KM administration, proliferating cells were labeled over a period of 2 days with bromodeoxyuridine (BrdU) and visualized with peroxidase immunohistochemistry. Changes in the location of the hair cell lesion during the KM treatment were monitored by phalloidin immunofluorescence or scanning electron microscopy. Hair cell loss began at the base of the cochlea 6 days after the start of KM injections, whereas cell proliferation was first observed in the basal region between days 6 and 8 of the KM treatment. This indicates that the latency between cell loss and cell proliferation is less than 48 h. The region of cell proliferation shifted from the base toward the apex of the cochlea over a period of 6-8 days, but cell proliferation in a specific region of the cochlea only occurred for 2-4 days. The latency as well as the total duration of cell proliferation after KM ototoxicity was virtually equivalent to that observed after acoustic trauma (Hashino and Salvi, 1993), suggesting that similar cellular events are involved in triggering cell proliferation after mechanical destruction and metabolic destruction of avian hair cells. The spatio-temporal gradient of cell proliferation followed the pattern of hair cell loss, suggesting that some aspect of hair cell degeneration provides trigger signals for cell proliferation.

Developing vestibular ganglion neurons switch trophic sensitivity from BDNF to GDNF after target innervation

Recent evidence showing a distinctive cell loss in vestibular and cochlear ganglia of brain-derived neurotrophic factor (BDNF) versus neurotrophin-3 (NT-3) null mutant mice demonstrates that these neurotrophins play a critical role in inner ear development. In this study, biological functions of BDNF and NT-3 in the chick vestibular and cochlear ganglion development was assessed in vitro and compared to those of other neurotrophic factors. The embryonic day (E)8-12 vestibular ganglion neurons showed an extensive outgrowth in response to BDNF with less outgrowth to NT-3. In contrast, NT-3 had stronger neurotrophic effects on the E12 cochlear ganglion neurons compared to BDNF. These results support previous evidence that neurotrophins play important roles in the vestibular and cochlear ganglion neuron development. However, the responsiveness to the neurotrophins declined and became undetectable by E16. Unexpectedly, glial cell line-derived neurotrophic factor (GDNF) promoted neurite outgrowth from vestibular ganglia at E12-16, later than the stages at which BDNF had neurotrophic effects. The time of switching sensitivity of the vestibular ganglion neurons from BDNF to GDNF correlated with the time of completion of synaptogenesis on their peripheral and central targets. Furthermore, a factor released from E12 inner ears exerted neurotrophic effects on late-stage vestibular ganglion neurons that were not responsive to the E4 otocyst-derived factor. These results raise the possibility that the vestibular ganglion neurons become responsive to GDNF upon target innervation and that the changes in sensitivity are regulated by changes in available factors released from their peripheral targets, the inner ear epithelia.