Fukuichiro Iguchi

Novel strategy for treatment of inner ears using a biodegradable gel.

Objective: The present study aimed to evaluate the efficacy of a biodegradable hydrogel as a drug‐delivery medium for the inner ear. Brain‐derived neurotrophic factor (BDNF) was chosen as the agent to be administered.

Transplantation of bone marrow stromal cells into the cochlea of chinchillas.

This study aimed to evaluate the potential of bone marrow stromal cells for treatment of inner ear diseases. Autologous marrow cells labeled with DiI were implanted into the inner ear of five gentamicin-treated chinchillas. Histological analysis 3 weeks later revealed robust survival of grafted marrow cells in multiple regions within the cochlea. Marrow cells implanted in the basal turn of the cochlea migrated as far as the apical end or into the spiral ligament of the cochlea. Some grafted cells expressed a neuronal or glial cell marker, indicating their ability to differentiate into neuronal or glial cells. Survival, migrational mobility and differentiation of autologous marrow cells in damaged cochlea suggest their potential as transplants for treatment of various degenerative inner ear diseases.

Role of reactive radicals in degeneration of the auditory system of mice following cisplatin treatment.

Objective—It has been suggested that reactive radical species are involved in the mechanism of cisplatin-induced hearing loss. However, the nature of the free radicals involved is not fully understood. We examined the effects of two highly reactive species, hydroxyl radicals and peroxynitrite, on the auditory system of mice following cisplatin treatment. Material and Methods—Expression of 4-hydroxynonenal (HNE), a marker of lipid peroxidation by the hydroxyl radical, and nitrotyrosine (NT), a marker for protein peroxidation by peroxynitrite, was examined immunohistochemically in mouse cochleae injured by means of local application of cisplatin. Results—Loss of outer hair cells (OHCs) and spiral ganglia was found in cochleae affected by cisplatin. Both HNE and NT were detected in auditory epithelia and neurons damaged by cisplatin. Interestingly, auditory hair cells produced HNE, but not NT. Our findings indicate contributions by both HNE and NT to the degeneration of the auditory system due to cisplatin, and a crucial role of the hydroxyl radical in degeneration of OHCs. Conclusion—The hydroxyl radical may be a critical target for a strategy aimed at protecting auditory function from cisplatin toxicity.

Destiny of autologous bone marrow-derived stromal cells implanted in the vocal fold.

Objectives: The aim of this study was to investigate the destiny of implanted autologous bone marrow–derived stromal cells (BSCs) containing mesenchymal stem cells. We previously reported the successful regeneration of an injured vocal fold through implantation of BSCs in a canine model. However, the fate of the implanted BSCs was not examined. In this study, implanted BSCs were traced in order to determine the type of tissues resulting at the injected site of the vocal fold. Methods: After harvest of bone marrow from the femurs of green fluorescent transgenic mice, adherent cells were cultured and selectively amplified. By means of a fluorescence-activated cell sorter, it was confirmed that some cells were strongly positive for mesenchymal stem cell markers, including CD29, CD44, CD49e, and Sca-1. These cells were then injected into the injured vocal fold of a nude rat. Immunohistologic examination of the resected vocal folds was performed 8 weeks after treatment. Results: The implanted cells were alive in the host tissues and showed positive expression for keratin and desmin, markers for epithelial tissue and muscle, respectively. The implanted BSCs differentiated into more than one tissue type in vivo. Conclusions: Cell-based tissue engineering using BSCs may improve the quality of the healing process in vocal fold injuries.

Transplantation of neural stem cells into the modiolus of mouse cochleae injured by cisplatin.

This study aimed to examine the possibility of restoration of spiral ganglion neurons, which transmit sound stimulation to the brain, by transplantation of fetal neural stem cells (NSCs) into the modiolus of cochleae. Fetal mouse NSCs expressing green fluorescence were injected into the modiolus of cisplatin-treated cochleae of mice. The temporal bones were collected 14 days after transplantation, and provided histological examination. The cell fate of transplants was determined by immunohistochemistry for a neural or glial cell-marker. Histological analysis 2 weeks after transplantation revealed robust survival of transplant-derived cells in the modiolus of the cochlea. NSCs injected in the basal portion of cochleae migrated as far as the apical end of the modiolus Grafted NSCs expressing a neural cell marker were identified, but the majority of grafted NSCs differentiated into glial cells. These findings suggest the possible use of NSCs in cell therapy for restoration of spiral ganglion neurons. However, further treatments are required to increase the number of NSC-derived neurons in the modiolus to realize functional recovery.

Fates of mouse embryonic stem cells transplanted into the inner ears of adult mice and embryonic chickens.

The potential of embryonic stem (ES) cells to differentiate into inner ear hair cells was examined in this study. Undifferentiated mouse ES cells transplanted into neomycin-damaged mouse inner ears were evaluated by immunohistochemistry 4 weeks after transplantation. Some ES cells were positive for E-cadherin or NCAM, and most transplanted cells were positive for SSEA3 and Ki67. None were positive for Myosin VIIa or MF20. These results indicate that the damaged inner ear may have some activity inducing ES cells to develop into ectoderm cells, but the effect was insufficient to induce inner ear hair cells. Next, SDIA/BMP-treated ES cells were transplanted into embryonic chicken inner ear rudiments. Embryonic chickens were expected to share the same developmental systems as mice. SDIA/BMP treatment drove ES cells to the population including neural crest cells and probably placode cells ES colonies were found next to or in the otic vesicles but were not a part of vesicle walls, indicating that transplanted ES cells could not be expected to be the same kind of cells as chicken otic vesicle cells Some ES colonies were found at the vestibulo-cochlear ganglions. To induce inner ear hair cells in this system, the competency of ES cells and otic induction signals should be defined further.

A novel model for rapid induction of apoptosis in spiral ganglions of mice

Objectives/Hypothesis The survival of the spiral ganglion (SG) is a critical issue in preservation of hearing. Research on topics related to this issue requires a mouse experimental model because such a model has advantages including use of genetic information and knockout or “knockin” mice. Thus, the aim of the study was to establish a mouse model for induction of apoptosis of SG neurons with a definite time course.

Mechanisms of apoptosis induced by cisplatin in marginal cells in mouse stria vascularis.

Degeneration of the stria vascularis (SV) is amongst the major causes of cisplatin (CDDP)-induced hearing impairment. The pathways of apoptosis occurring in the SV due to CDDP were examined using a mouse experimental model. Temporal bones of adult C57BL/6 mice were collected on days 3, 7 and 14 after the local application of CDDP. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and immunostaining for apoptosis-related proteins or reactive radical species were employed for analysis. Local application of CDDP caused apoptotic cell death of marginal cells 3 days after CDDP treatment. Immunohistochemical analyses demonstrated activation of caspase-3 and -9, but not -8, and redistribution of cytochrome c in affected marginal cells, indicating a caspase-dependent, mitochondrion-mediated apoptotic pathway in marginal cells. Temporary expression of hydroxynonenal, nitrotyrosine and inducible nitric oxide synthase in the SV was observed at the induction of apoptosis in marginal cells. CDDP toxicity generates reactive radical species in the SV, which causes mitochondrial membrane permeabilization leading to apoptosis of marginal cells.

A novel technique for inducing local inner ear damage

With significant development of mouse genomics and the availability of transgenic and knockout mice, the mouse will be the preferred animal model for inner ear research. However, few studies have used mice as experimental animals for examination of hair cell degeneration, because of their relative resistance to ototoxic agents and difficulties in surgical treatment. This study presents a model for induction of apoptotic cell death in sensory epithelia of the mouse inner ear using injection of neomycin into the posterior semicircular canal. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay revealed that local application of neomycin produced sufficient induction of apoptotic cell death in both auditory and vestibular epithelia over a definite time course. Supplementation of the general caspase-inhibitor significantly reduced induction of TUNEL-positive cells, indicating caspase-dependency of apoptotic cell death observed in the present model. In addition, the approach to the posterior semicircular canal was an easy technique, and sham-operation induced no significant induction of TUNEL-positive cells. This model, hence, enables the use of various genetic tools in studies for mechanisms of hair cell apoptosis.

A new method for drug application to the inner ear

Inner ear sensory cells are very susceptible to injuries and recovery after damage is very difficult. Recently several drugs including neurotrophic factors have been reported to protect against inner ear injury. The purpose of this experimental study is to find new methods for applying drugs to the inner ear that effectively protect against inner ear damage. Biodegradable hydrogel was used as a carrier for application of brain-derived neurotrophic factor (BDNF) into the inner ear of guinea pigs through the round window membrane. After application of BDNF the number of surviving spiral ganglion neurons increased following injury of inner ear hair cells and spiral ganglion neurons by ototoxic treatment. This result indicates that BDNF provides effective protection against inner ear damage and that biodegradable hydrogel is useful for application of drugs to the inner ear.