Yayoi S. Kikkawa

Topical insulin-like growth factor 1 treatment using gelatin hydrogels for glucocorticoid- resistant sudden sensorineural hearing loss: a prospective clinical trial

BackgroundSudden sensorineural hearing loss (SSHL) is a common condition in which patients lose the hearing in one ear within 3 days. Systemic glucocorticoid treatments have been used as standard therapy for SSHL; however, about 20% of patients do not respond. We tested the safety and efficacy of topical insulin-like growth factor 1 (IGF1) application using gelatin hydrogels as a treatment for SSHL.MethodsPatients with SSHL that showed no recovery to systemic glucocorticoid administration were recruited. We applied gelatin hydrogels, impregnated with recombinant human IGF1, into the middle ear. The primary outcome measure was the proportion of patients showing hearing improvement 12 weeks after the test treatment. The secondary outcome measures were the proportion of patients showing improvement at 24 weeks and the incidence of adverse events. The null hypothesis was that 33% of patients would show hearing improvement, as was reported for a historical control after hyperbaric oxygen therapy.ResultsIn total, 25 patients received the test treatment at a median of 23 days (range 15-32) after the onset of SSHL, between 2007 and 2009. At 12 weeks after the test treatment, 48% (95% CI 28% to 69%; P = 0.086) of patients showed hearing improvement, and the proportion increased to 56% (95% CI 35% to 76%; P = 0.015) at 24 weeks. No serious adverse events were observed.ConclusionsTopical IGF1 application using gelatin hydrogels is well tolerated and may be efficacious for hearing recovery in patients with SSHL that is resistant to systemic glucocorticoids.

Local application of hepatocyte growth factor using gelatin hydrogels attenuates noise-induced hearing loss in guinea pigs

Conclusion: Local application of hepatocyte growth factor using biodegradable gelatin hydrogels attenuates noise-induced hearing loss in guinea pigs. Objectives: To develop an inner ear drug delivery system using gelatin hydrogels that is capable of a sustained delivery of growth factors to the cochlea. We examined the efficacy of the local application of gelatin hydrogels containing hepatocyte growth factor (HGF) in protecting cochlear hair cells from noise-induced damage. Materials and methods: A piece of gelatin hydrogel previously immersed in either HGF or saline was placed on the round window membrane of a guinea pig 1 h after noise exposure (4 kHz octave band noise at 120 dB sound pressure level for 3 h). Auditory function was monitored using auditory brainstem responses (ABRs), and the loss of hair cells was evaluated quantitatively. Results: Local HGF treatment significantly reduced the noise exposure-caused ABR threshold shifts and the loss of outer hair cells in the basal portion of the cochleae.

Hydrogen protects auditory hair cells from free radicals.

Reactive oxygen species (ROS) play a role in the degeneration of auditory hair cells because of aging, noise trauma, or ototoxic drugs. Hydrogenation is a fundamental reduction/deoxidation reaction in living organisms. This study thus examined the potential of hydrogen to protect auditory hair cells from ROS-induced damage. To generate ROS, we applied antimycin A to explant cultures of auditory epithelia, and examined the effect of hydrogen on the protection of hair cells against ROS. Incubation with a hydrogen-saturated medium significantly reduced ROS generation and subsequent lipid peroxidation in the auditory epithelia, leading to increased survival of the hair cells. These findings show the potential of hydrogen to protect auditory hair cells from ROS-induced damage.

Progression of Inner Ear Pathology in Ames Waltzer Mice and the Role of Protocadherin 15 in Hair Cell Development

The Ames waltzer (av) mouse mutant exhibits auditory and vestibular abnormalities resulting from mutation of protocadherin 15 (Pcdh15). Ames waltzer has been identified as an animal model for inner ear pathology associated with Usher syndrome type 1F. Studies correlating anatomical phenotype with severity of genetic defect in various av alleles are providing better understanding of the role played by Pcdh15 in inner ear development and of sensorineural abnormalities associated with alterations in Pcdh15 protein structure as a result of gene mutation. In this work we present new findings on inner ear pathology in four alleles of av mice with differing mutations of Pcdh15 as well as varying alterations in inner ear morphology. Two alleles with in-frame deletion mutations (Pcdh15av-J and Pcdh15av-2J) and two presumptive functional null alleles (Pcdh15av-3J and Pcdh15av-Tg) were studied. Light and electron microscopic observations demonstrated that the severity of cochlear and vestibular pathology in these animals correlates positively with the extent of mutation in Pcdh15 from embryonic day 18 (E18) up to 12 months. Electron microscopic analysis of immature ears indicated early abnormalities in the arrangement of stereocilia and the inner and outer hair cell cuticular plates, stereocilia rootlets, and the actin meshwork within the cuticular plate. In severe cases, displacement of the kinocilium and alterations in the shape of the cuticular plate was also observed. Mice harboring in-frame deletion mutations showed less disorganization of stereocilia and cuticular plates in the organ of Corti than the presumptive functional null alleles at P0–P10. A slower progression of pathology was also seen via light microscopy in older animals with in-frame deletions, compared to the presumptive functional null mutations. In summary, our results demonstrate that mutation in Pcdh15 affects the initial formation of stereocilia bundles with associated changes in the actin meshwork within the cuticular plate; these effects are more pronounced in the presumed null mutation compared to mutations that only affect the extracellular domain. The positive correlation of severity of effects with extent of mutation can be seen well into adulthood.

Hydrogen protects vestibular hair cells from free radicals

Abstract Conclusion: Hydrogen gas effectively protected against the morphological and functional vestibular hair cell damage by reactive oxygen species (ROS). Objective: ROS are generally produced by oxidative stress. In the inner ear, ROS levels increase as a result of noise trauma and ototoxic drugs and induce damage. It is thus important to control ROS levels in the inner ear. The protective effects of hydrogen gas in cochlear hair cells have been reported previously. Methods: This study examined the effects of hydrogen gas on mouse vestibular hair cell damage by ROS using antimycin A. Results: In the group *exposed to hydrogen gas, vestibular hair cells were morphologically well preserved and their mechano-electrical transduction activities were relatively well maintained when compared with controls. Hydroxyphenyl fluorescein (HPF) fluorescence in vestibular tissue was also reduced by hydrogen gas.

Growth factor-eluting cochlear implant electrode: impact on residual auditory function, insertional trauma, and fibrosis

BackgroundA cochlear implant (CI) is an artificial hearing device that can replace a damaged cochlea. The present study examined the use of growth factor-eluting gelatin hydrogel coatings on the electrodes to minimize inner ear trauma during electrode insertion. Insulin-like growth factor 1 (IGF1) and/or hepatocyte growth factor (HGF) were chosen as the agents to be administered.MethodsSilicone CI electrode analogs were prepared and coated with gelatin hydrogels. Adsorption/release profile of the hydrogel was measured using 125I-radiolabeled IGF. Hydrogel-coated electrodes were absorbed with IGF1, HGF, IGF1 plus HGF, or saline (control) and implanted into the basal turns of guinea pig cochleae (n = 5). Auditory sensitivity was determined pre-operatively, immediately after, and 3, 7, 14, 21, and 28 days post-operatively by using auditory brainstem response (ABR; 4–16 kHz). In addition, histological analysis was performed and auditory hair cell (HC) survival, spiral ganglion neuron (SGN) densities, and fibrous tissue thickness were measured.ResultsCompared to non-coated arrays, hydrogel-coated electrodes adsorbed significantly greater amounts of IGF1 and continuously released it for 48 h. Residual hearing measured by ABR thresholds after surgery were elevated by 50–70 dB in all of the electrode-implanted animals, and was maximal immediately after operation. Thresholds were less elevated after hydrogel treatment, and the hearing protection improved when IGF1 or HGF was applied. Histopathologically, hair cell survival, spiral ganglion cell survival, and fibrous tissue thickness were not different between the experimental groups. No serious adverse events were observed during the 4-week observation period.ConclusionsOur findings provide the first evidence that hydrogel-coated, growth factor-releasing CI electrodes could attenuate insertional trauma and promote recovery from it, suggesting that this combination might be a new drug delivery strategy not only in cochlear implantation but also in treating clinical conditions characterized by inner ear damage.

Inner ear drug delivery system from the clinical point of view

Abstract Conclusion: Three types of inner ear drug delivery systems (DDS) that were ready to be applied in clinics were developed. Objectives: To develop clinically applicable inner ear DDS for the treatment of inner ear disorders. Methods: Inner ear DDS using clinically applicable materials were developed and evaluated. Results: The systemic application of stealth-type nanoparticles encapsulating betamethasone provided superior therapeutic results for the treatment of noise-induced hearing loss compared with the systemic application of betamethasone in mice. Microparticles made of biodegradable polymer (poly (lactic/glycolic) acid, PLGA) encapsulating lidocaine were placed on the round window membrane of guinea pigs, and resulted in reasonable concentrations of lidocaine in the cochlea without serious adverse effects. The phase I/IIa clinical trial of the application of insulin-like growth factor-1 (IGF-1) in combination with gelatin hydrogel on the round window membrane was conducted, recruiting patients with acute sensorineural hearing loss after the failure of systemic application of steroids.

Selective vulnerability of adult cochlear nucleus neurons to de-afferentation by mechanical compression

It is well established that the cochlear nucleus (CN) of developing species is susceptible to loss of synaptic connections from the auditory periphery. Less information is known about how de-afferentation affects the adult auditory system. We investigated the effects of de-afferentation to the adult CN by mechanical compression. This experimental model is quantifiable and highly reproducible. Five weeks after mechanical compression to the axons of the auditory neurons, the total number of neurons in the CN was evaluated using un-biased stereological methods. A region-specific degeneration of neurons in the dorsal cochlear nucleus (DCN) and posteroventral cochlear nucleus (PVCN) by 50% was found. Degeneration of neurons in the anteroventral cochlear nucleus (AVCN) was not found. An imbalance between excitatory and inhibitory synaptic transmission after de-afferentation may have played a crucial role in the development of neuronal cell demise in the CN. The occurrence of a region-specific loss of adult CN neurons illustrates the importance of evaluating all regions of the CN to investigate the effects of de-afferentation. Thus, this experimental model may be promising to obtain not only the basic knowledge on auditory nerve/CN degeneration but also the information relevant to the application of cochlear or auditory brainstem implants.

A new spontaneous mutation in the mouse protocadherin 15 gene

We have characterized a new allele of the protocadherin 15 gene (designatedPcdh15(av-6J)) that arose as a spontaneous, recessive mutation in the C57BL/6J inbred strain at Jackson Laboratory. Analysis revealed an inframe deletion in Pcdh15, which is predicted to result in partial deletion of cadherin domain (domain 9) in Pcdh15. Morphologic study revealed normal to moderately defective cochlear hair cell stereocilia in Pcdh15(av-6J) mutants at postnatal day 2 (P2). Stereocilia abnormalities were consistently present at P5 and P10. Degenerative changes including loss of inner and outer hair cells were seen at P20, with severe sensory cell loss in all cochlear turns occurring by P40. The hair cell phenotype observed in the 6J allele between P0 and P20 is the least severe phenotype yet observed in Pcdh15 alleles. However, young Pcdh15(av-6J) mice are unresponsive to auditory stimulation and show circling behavior indicative of vestibular dysfunction. Since these animals show severe functional deficits but have relatively mild stereocilia defects at a young age they may provide an appropriate model to test for a direct role of Pcdh15 in mechanotransduction.

Surgical Invasiveness of Cell Transplantation into the Guinea Pig Cochlear Modiolus

Objective: Previous studies have demonstrated the potential of cell transplantation for regeneration of spiral ganglion neurons (SGNs). However, the effect of surgical invasion on host cochleae has yet to be evaluated. The present study investigated the efficiency and invasiveness of our surgical procedure using a fine glass pipette for injections into the cochlear modiolus. Methods: We examined the survival of transplanted embryonic stem cell-derived neurons in the cochlear modiolus of guinea pigs. Surgical invasiveness was assessed by measurements of electrically evoked auditory brainstem responses (eABRs) and SGN densities after an injection of 5 ml of saline into the cochlear modiolus. Results: All of the transplanted animals exhibited localization of transplanted cells in the cochlear modiolus. No significant alterations in the eABR thresholds or SGN densities were found following surgery. Conclusion: These findings indicate that our procedure is a viable method for testing the potential of transplants for SGN replacement.