Nobuhiro Hakuba

Basic fibroblast growth factor combined with atelocollagen for closing chronic tympanic membrane perforations in 87 patients.

Objective: To present the clinical results of closing chronic tympanic membrane (TM) perforations using basic fibroblast growth factor (bFGF) combined with an atelocollagen/silicone bilayer membrane patch. Study Design: Closure of TM perforations in 87 patients was attempted using bFGF, which is thought to promote the regeneration of TM tissues by facilitating the growth of fibroblasts and collagen fibers. Methods: Under an operating microscope, the margin of the perforation was trimmed, and a piece of an atelocollagen/silicone bilayer membrane was placed in the perforation with the silicon layer facing outward and then infiltrated with 0.1 ml of trafermin. Data obtained from patient records included patient age, perforation size, and duration of treatment, with a focus on hearing improvement and complete TM closure. Results: The mean perforation size before treatment was 14.4%. Complete closure of the TM perforation was achieved in 80 patients (92.0%), whereas pinholes remained in 5 patients (8.7%), and small perforations were observed in 2 patients (2.3%). In the patients with complete closure, the TM perforations closed after an average 1.8 treatments, and hearing improved by 13.6 dB. Conclusion: This study demonstrated that bFGF combined with atelocollagen is effective for the conservative treatment of TM perforation.

Efflux of glutamate into the perilymph of the cochlea following transient ischemia in the gerbil

Using a microdialysis technique followed by an enzyme cycling analysis, we measured changes in the glutamate levels in the perilymph of gerbil cochleae before, during and after transient ischemic insult. The basal glutamate level in perilymph was 0.35 +/- 0.22 pmol/microl. An almost immediate and continuous rise in the level of glutamate occurred after the ischemic insult, which advanced even further after recirculation; the average concentration was higher than 40 pmol/microl 55 min after recirculation. The compound action potentials (CAP) monitoring the auditory function totally disappeared after ischemic insult. However, CAP reappeared after recirculation; the threshold for acoustic stimulation was higher than that observed at the pre-ischemic state.

Insulin-like growth factor 1 treatment via hydrogels rescues cochlear hair cells from ischemic injury

This study was designed to investigate the protective effects of recombinant human insulin-like growth factor 1 (rhIGF1), applied locally via a hydrogel, against ischemic damage of the cochleae in gerbils. A hydrogel was immersed in rhIGF1 or saline and was applied on the round window membrane 30 min after the ischemia. Local rhIGF1 treatment significantly reduced the elevation of auditory brain responses thresholds at a frequency of 8 kHz on days 1, 4, and 7 after ischemia. A histological analysis revealed increased survival of inner hair cells in the animals treated with rhIGF1 via the hydrogel 7 days after ischemia. These findings showed that local rhIGF1 application using a hydrogel has the potential to protect the cochleae from ischemic injury.

Hearing loss and glutamate efflux in the perilymph following transient hindbrain ischemia in gerbils

The mechanism underlying ischemia‐induced hearing loss was studied in gerbils with transient hindbrain ischemia. Occlusion of the vertebral arteries caused an increase in the concentration of glutamate in the perilymph and elevated the compound action potential (CAP) threshold to 24.6 dB at 5 minutes. the CAP threshold subsequently recovered on reperfusion, gradually reaching 8.3 dB 120 minutes after reperfusion. Under electron microscopy, afferent dendrites of the cochlear nerve in contact with inner hair cells exhibited abnormal swelling 5 minutes after ischemia/reperfusion. These morphological changes were not observed in cochleas treated with an alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA)/kainate‐type glutamate receptor antagonist, 6‐7‐dinitroquinoxaline‐2,3‐dione (DNQX), before hindbrain ischemia; an N‐methyl‐D‐aspartate (NMDA)‐type receptor antagonist, D‐2‐amino‐5‐phosphonopentanoate (D‐AP5), was ineffective. Moreover, the histopathological alterations noted 5 minutes after reperfusion were spontaneously ameliorated 120 minutes after ischemia/reperfusion. These findings suggest that the ischemia‐induced increase in extracellular glutamate concentration with subsequent activation of AMPA/kainate receptors is responsible for neurite degeneration and hearing loss in the early stages following transient hindbrain ischemia. J. Comp. Neurol. 418:217–226, 2000.

Adenovirus-mediated overexpression of a gene prevents hearing loss and progressive inner hair cell loss after transient cochlear ischemia in gerbils

The use of adenoviral vectors has recently provided a novel strategy for direct gene transfer into the cochlea. In this study, we assessed the utility of an adenoviral vector expressing glial-cell-derived neurotrophic factor (GDNF) in ischemia–reperfusion injury of the gerbil cochlea. The vector was injected through the round window 4 days before ischemic insult. The distribution of a reporter transgene was confirmed throughout the cochlea from the basal to the apical turn and Western blot analysis indicated significant upregulation of GDNF protein 11 days following virus inoculation. Hearing ability was assessed by sequentially recording compound action potentials (CAP), and the degree of hair cell loss in the organ of Corti was evaluated in specimens stained with rhodamine-phalloidin and Hoechst 33342. On the seventh day of ischemia, the CAP threshold shift and inner hair cell loss were remarkably suppressed in the Ad-GDNF group compared with the control group. These results suggest that adenovirus-mediated overexpression of GDNF is useful for protection against hair cell damage, which otherwise eventually occurs after transient ischemia of the cochlea.

Ginsenoside Rb1 protects against damage to the spiral ganglion cells after cochlear ischemia.

The effects of transient cochlear ischemia on spiral ganglion cells (SGCs) were studied in Mongolian gerbils. Ischemic insult was induced by occluding the bilateral vertebral arteries of gerbils for 15min. Seven days after ischemia, the percentage of SGCs decreased to 67.5% from the preischemic baseline in the basal turn. Evaluation with immunohistochemical staining showed TUNEL-positive reactions in the SGCs with fragmented nuclei. In addition, we investigated the protective effects of ginsenoside Rb1 (gRb1) against ischemic injury to SGCs. Seven days after ischemia, the auditory brainstem response threshold shift was significantly reduced and the percentage of SGCs decreased to 90.2% from the preischemic baseline in the basal turn in the gRb1-treated group. These findings suggest that gRb1 prevented hearing loss caused by ischemic injury to SGCs in Mongolian gerbils.

Hypothermia prevents hearing loss and progressive hair cell loss after transient cochlear ischemia in gerbils

The effects of hypothermia on ischemia-reperfusion injury of the cochlea were studied in gerbils. Hearing was assessed by sequentially recording compound action potentials before, during and after the ischemia. The degree of hair cell loss in the organ of Corti was evaluated in specimens stained with rhodamine-phalloidin and the dye Hoechst 33342. Ischemic insult was applied to the animals by occluding the bilateral vertebral arteries for 15 min under normothermic or hypothermic (rectal temperature 32 degrees C) conditions. Interruption of the blood supply to the cochlea caused a tremendous increase in the compound action potential threshold, which usually recovered to some extent with reperfusion. In the ischemia/normothermic group, the threshold did not return to the pre-ischemic level. The average increase in the threshold seven days after ischemia was 20.0 dB. Histologically, the hair cell loss increased gradually until four days after the ischemic insult. On the seventh day, the mean loss of inner and outer hair cells at the basal turn was 31.1 % and 2.4 %, respectively. In the ischemia/hypothermic group, the threshold returned to the pre-ischemic level within 30 min after reperfusion and remained stable thereafter. The mean loss of inner and outer hair cells on the seventh day was 0.1 % and 0.2 %, respectively. These results indicate that hypothermia can prevent inner ear damage, which otherwise occurs after transient ischemia of the cochlea.

Ischemic damage increases nitric oxide production via inducible nitric oxide synthase in the cochlea

The present study was designed to elucidate the dynamic changes of nitric oxide (NO) production in the perilymph and to investigate the immunostaining for inducible nitric oxide synthase (iNOS) in the cochlea for 7 days after transient cochlear ischemia. Moreover, aminoguanidine, which is a selective iNOS inhibitor, was administrated immediately following ischemia and every 24h thereafter for 7 days to investigate whether the production of NO is dependent on the iNOS pathway. Significant increases in the oxidative NO metabolites, nitrite (NO(2)(-)) and nitrate (NO(3)(-)), were measured on day 1 using an in vivo microdialysis and on-line high performance liquid chromatography (HPLC) system. The immunostaining for iNOS was strongly expressed on days 1 and 4 and returned to normal on day 7 after the ischemia. The administration of aminoguanidine reduced the oxidative NO metabolites on day 1 and suppressed the expression of iNOS. These findings suggest that transient ischemia causes a remarkable increase in NO production in the perilymph, which might be attributable to the iNOS pathway.

Hypothermia reduces glutamate efflux in perilymph following transient cochlear ischemia.

The effect of hypothermia on ischemic injury of the cochlea in gerbils was studied with particular regard to glutamate efflux in the perilymph. Under normothermic conditions interruption of the blood supply to the cochlea for 15 min caused a remarkable elevation of the compound action potential (CAP) threshold, and an increase in perilymphatic glutamate. The CAP threshold recovered to some extent with reperfusion, but not to preischemic levels. CAP thresholds, under hypothermic condi- tions and with reperfusion, recovered promptly to near pre-ischemic levels, while glutamate concentration did not change. These results, together with electron microscopy studies, suggest that hypothermia prevents hearing loss primarily through reduction of glutamate efflux at the synopses between inner hair cells and primary afferent auditory neurons.

Hematopoietic stem cells prevent hair cell death after transient cochlear ischemia through paracrine effects.

Transplantation of hematopoietic stem cells (HSCs) is regarded to be a potential approach for promoting repair of damaged organs. Here, we investigated the influence of hematopoietic stem cells on progressive hair cell degeneration after transient cochlear ischemia in gerbils. Transient cochlear ischemia was produced by extracranial occlusion of the bilateral vertebral arteries just before their entry into the transverse foramen of the cervical vertebra. Intrascalar injection of HSCs prevented ischemia-induced hair cell degeneration and ameliorated hearing impairment. We also showed that the protein level of glial cell line-derived neurotrophic factor (GDNF) in the organ of Corti was upregulated after cochlear ischemia and that treatment with HSCs augmented this ischemia-induced upregulation of GDNF. A tracking study revealed that HSCs injected into the cochlea were retained in the perilymphatic space of the cochlea, although they neither transdifferentiated into cochlear cell types nor fused with the injured hair cells after ischemia, suggesting that HSCs had therapeutic potential possibly through paracrine effects. Thus, we propose HSCs as a potential new therapeutic strategy for hearing loss.