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Gene therapy is a promising clinical solution to hearing loss. However suitable gene carriers for the auditory system are currently unavailable. Given the unique structure of the inner ear, the route of delivery and gene transfer efficiency are still not optimal at present. This study presented a non-viral delivery system of in vivo delivery of Atoh1 gene (a potentially therapeutic gene for hearing loss) to rat cochlea. We treated polyamidoamine (PAMAM) dendrimers by activating and modifying with Na-carboxymethyl-beta-cyclodextrins (cm-beta-CD) in sequence. A novel gene carrier (cm-beta-CD modified activated PAMAM dendrimers, CMAP) was then constructed. CMAP nanoparticles could bind pRK5-Atoh1-EGFP plasmids to form vector-DNA complexes (dendriplexes) with a mean particle size of 132 +/- 20 nm and zeta potential of 31 +/- 3 mV. These dendriplexes were locally applied on the round window membrane and delivered to the inner ear by passive gradient permeation. Results showed that the Atoh1 gene was successfully transferred into the cells as indicated by the green fluorescence detected in the inner ear. A relatively selective gene transfer with high efficiency was achieved in the auditory hair cells but not much in other cell types in the cochlea. Auditory brainstem response was determined seven days after inoculation, indicating good tolerance. This approach may provide a novel tool for inner ear gene therapy and initiate the applications of biomaterials to treat auditory disorders.

Background: The auditory complex of the chick, like that of humans, is made of intimate and highly ordered connections between the inner ear, the middle ear, and the outer ear. Unlike mammals, the middle ear of chick has only one ossicle, known as the columella. The independent lineages of the two suggest that some mechanism must exist that ensures the connectivity between the inner ear and the columella; however, the basis of integration is not known. Results: Using quail-chick chimeras, we demonstrate that columella development depends on signaling interactions. Specifically, both pharyngeal endoderm and cranial paraxial mesoderm can alter the morphology of the columella. Only a discrete region of pharyngeal endoderm exerts this patterning activity, and this region is specified by the overlying paraxial mesoderm. Conclusions: Paraxial mesoderm is also used in the induction of the inner ear, thus we propose that this overlapping source of signalling cues in both middle and inner ear development may underlie the integration of these structures. Developmental Dynamics 241:1104–1110, 2012. © 2012 Wiley Periodicals, Inc.

Bone marrow mesenchymal stem cells (MSCs) have the ability to differentiate into hair cells, and this method of culturing MSCs provides a useful tool for studies on mammalian cochlear hair cell regeneration.To investigate a method to induce bone marrow MSCs to differentiate into inner ear hair cells.Rat bone marrow MSCs were isolated from healthy rats and cultured in vitro. To make sure that the cultured cells were bone marrow MSCs, the expression of MSC markers such as SH2, CD31, CD34, and CD44 genes on the cultured cells was assessed by RT-PCR. Adipogenic cells and osteogenic cells were induced by the differentiation of the cultured cells, respectively, suggesting that the cultured cells have the characteristic of pluripotent differentiation. Then they were induced to differentiate into neural stem cells and hair cell progenitor cells. Immunohistochemistry experiments were carried out to detect the expression of molecular markers. Scanning electron microscope samples were prepared for observation of the morphology of the cells.Rat bone marrow MSCs were successfully isolated, purified, cultured, and identified in vitro. They were also successfully induced to differentiate into neural progenitor cells and then hair cell-like cells that expressed myosin VIIa.

Conclusion: Compared with traditional animal models, the miniature pig may be a better model for biomedical research because its morphology has many similarities with that of humans. Objective: To investigate the suitability of the miniature pig as an animal model for otological research as regards morphology. Methods: Microdissection of the temporal bone of 10 miniature pigs was performed and recorded on photographs. Results: The morphology and measurements of the external, middle, and inner ear, and the lateral recess of the miniature pigs were completed by microdissection. The temporal bone structures, including the external, middle, inner ear, and the lateral recess, were similar in the miniature pig and humans.

Gene therapy is a promising clinical solution to hearing loss. However suitable gene carriers for the auditory system are currently unavailable. Given the unique structure of the inner ear, the route of delivery and gene transfer efficiency are still not optimal at present. This study presented a non-viral delivery system of in vivo delivery of Atoh1 gene (a potentially therapeutic gene for hearing loss) to rat cochlea. We treated polyamidoamine (PAMAM) dendrimers by activating and modifying with Na-carboxymethyl-beta-cyclodextrins (CM-beta-CD) in sequence. A novel gene carrier (CM-beta-CD modified activated PAMAM dendrimers, CMAP) was then constructed. CMAP nanoparticles could bind pRK5-Atoh1-EGFP plasmids to form vector-DNA complexes (dendriplexes) with a mean particle size of 132 +/- 20 nm and zeta potential of 31 +/- 3 mV. These dendriplexes were locally applied on the round window membrane and delivered to the inner ear by passive gradient permeation. Results showed that the Atoh1 gene was successfully transferred into the cells as indicated by the green fluorescence detected in the inner ear. A relatively selective gene transfer with high efficiency was achieved in the auditory hair cells but not much in other cell types in the cochlea. Auditory brainstem response was determined seven days after inoculation, indicating good tolerance. This approach may provide a novel tool for inner ear gene therapy and initiate the applications of biomaterials to treat auditory disorders.

Mcl-1 is an anti-apoptotic member of the Bcl-2 family that modulates apoptosis-related signaling pathways and promotes cell survival. We have previously demonstrated a reduction of Mcl-1 expression in aging cochleae. To investigate whether restoring Mcl-1 expression would reduce aging-related cochlear degeneration, we developed a rat model of Mcl-1 overexpression. A plasmid encoding human Mcl-1/enhanced green fluorescent protein was applied to the round window of the cochlea. This in vivo treatment transfected both the sensory and supporting cells of the cochlear sensory epithelium and enhanced Mcl-1 expression at both the mRNA and the protein level. The upregulation of Mcl-1 expression reduced the progression of age-related cochlear dysfunction and sensory cell death. Furthermore, the transfection of Mcl-1 exerted its protective effect by suppressing cochlear apoptosis at the mitochondrial level. This study demonstrates that the genetic modulation of Mcl-1 expression reduces the progression of age-related cochlear degeneration.

Mouse embryonic stem cells (ESCs) transplanted into the scala tympani are able to migrate in the cochlea of rats deafened with aminoglycoside and partly restore the structure of sensory epithelia of the inner ear.To explore the migration and differentiation of enhanced green fluorescence protein (EGFP)-expressing ESCs by transplanting them into the scala tympani of rats with amikacin sulfate-induced hearing loss.Adult Sprague-Dawley (SD) rats were deafened with amikacin sulfate. Mouse ESCs expressing EGFP (EGFP-ESCs) were transplanted into the scala tympani. The migration and differentiation were observed at different time points.EGFP-ESCs transplanted into normal cochlea did not migrate, but those in the amikacin-damaged cochlea could survive and migrate into the scala media and the vestibular cisterna. For the first time, we observed that the EGFP-ESCs migrated into the scala media, took the place of the organ of Corti, and formed a structure just like the cochlear tunnel. Some grafted stem cells even expressed myosin VIIa, the molecular marker of hair cells. Some nerve fibers reached to the bottom of the hair cell-like cells. The ESCs migrated into the vestibule and restored the sensory epithelia of the ampullary crest. The number of the transplanted ESCs reduced over the 6 week period of the study.

To explore the changes of inferior collicular (IC) neurons after noise exposure cochlea injury in guinea pig to elucidate the encoding mechanism of pure tones, observe the changes of IC gamma-amino butyric acid (GABA) after cochlear damage by acoustic trauma and understand the possible mechanism of symptoms such as noise-induced tinnitus, hyperacusis and loudness recruitment.The responses of IC neurons to pure tone stimuli were observed in guinea pig at Day 1 and Days 11-21 after cochlear damage induced by noise exposure. And the IC neurons of normal guinea pig were assigned as the controls. Reverse transcription-polymerase chain reaction (RT-PCR) was used to measure the concentrations of GABA(A) and GABA(B) receptors.(1) The types of frequency reaction area (FRA) in the experiment group were the same as those in the control group (V-shape 84.8%, W-shape 8.9%, N-shape 6.3%). But the percentages of types were markedly different at Day 1 (V-shape 63.9%, W-shape 18.1%, N-shape, 18.1%) and Days 11-21 (V-shape 84.2%, W-shape 12.3%, N-shape 3.5%) after noise exposure. (2) After noise exposure, there was a marked fault in characteristic frequency (CF) and depth function map corresponding to 4 kHz (noise frequency). The rake ratio of CF and depth linear function map in the experiment group was lower than that of the control group. The control group, Day 1 and Days 11-21 after noise exposure, the rake ratios were 6.6, 5.8, 5.2 respectively. (3) GABA(A)/GABA(B) receptors decreased markedly at Days 1, 11 and 21 post-exposure compared to normal controls. And the values increased gradually with the prolonged time after exposure. The above findings conformed to the changes of electrophysiology of IC.After acoustic trauma, the responses of IC neurons to pure tone stimuli change with the elongation of time. It may be explained by the changes of IC GABA receptors after noise exposure.

The hallmark of mechanosensory hair cells is the stereocilia, where mechanical stimuli are converted into electrical signals. These delicate stereocilia are susceptible to acoustic trauma and ototoxic drugs. While hair cells in lower vertebrates and the mammalian vestibular system can spontaneously regenerate lost stereocilia, mammalian cochlear hair cells no longer retain this capability. We explored the possibility of regenerating stereocilia in the noise-deafened guinea pig cochlea by cochlear inoculation of a viral vector carrying Atoh1, a gene critical for hair cell differentiation. Exposure to simulated gunfire resulted in a 60–70 dB hearing loss and extensive damage and loss of stereocilia bundles of both inner and outer hair cells along the entire cochlear length. However, most injured hair cells remained in the organ of Corti for up to 10 days after the trauma. A viral vector carrying an EGFP-labeled Atoh1 gene was inoculated into the cochlea through the round window on the seventh day after noise exposure. Auditory brainstem response measured one month after inoculation showed that hearing thresholds were substantially improved. Scanning electron microscopy revealed that the damaged/lost stereocilia bundles were repaired or regenerated after Atoh1 treatment, suggesting that Atoh1 was able to induce repair/regeneration of the damaged or lost stereocilia. Therefore, our studies revealed a new role of Atoh1 as a gene critical for promoting repair/regeneration of stereocilia and maintaining injured hair cells in the adult mammal cochlea. Atoh1-based gene therapy, therefore, has the potential to treat noise-induced hearing loss if the treatment is carried out before hair cells die.