NGF stimulates extensive neurite outgrowth from implanted dorsal root ganglion neurons following transplantation into the adult rat inner ear.

Neuronal tissue transplantation is a potential way to replace degenerated spiral ganglion neurons (SGNs) since these cells cannot regenerate in adult mammals. To investigate whether nerve growth factor (NGF) can stimulate neurite outgrowth from implanted neurons, mouse embryonic dorsal root ganglion (DRG) cells expressing enhanced green fluorescent protein (EGFP) were transplanted into the scala tympani of adult rats with a supplement of NGF or artificial perilymph. DRG neurons were observed in the cochlea for up to 6 weeks postoperatively. A significant difference was identified in the number of DRG neurons between the NGF and non-NGF groups. In the NGF group, extensive neurite projections from DRGs were found penetrating the osseous modiolus towards the spiral ganglion. These results suggest the possibility that embryonic neuronal implants may become integrated within the adult auditory nervous system. In combination with a cochlear prosthesis, a neuronal implantation strategy may provide a possibility for further treatment of profoundly deaf patients.

Central migration of neuronal tissue and embryonic stem cells following transplantation along the adult auditory nerve.

The regeneration of the auditory nerve remains a challenge in restoring hearing. An interesting approach would be to use a cell replacement therapy with the potential to establish connections from the inner ear to the central auditory system. This hypothesis was tested by xenografted (mouse to rat) implantation of embryonic dorsal root ganglion (DRG) neurons and embryonic stem (ES) cells along the auditory nerve in the adult host. DRG neurons were obtained at embryonic day 13-14 in transgenic animals expressing enhanced green fluorescence protein (EGFP). For embryonic stem cells, a tau-GFP ES cell line was used as a donor. The fibers of the auditory nerve in the adult rat were transected through the modiolus at the first cochlear turn, and the biological implants were transplanted into the transection. The transplanted DRG neurons and ES cells survived for a postoperative survival time ranging from 3 to 9 weeks, verified by EGFP/GFP fluorescence, and neurofilament or TUJ1 immunostaining. At 9 weeks following implantation, the implanted DRG neurons were found to have migrated along the auditory nerve in the internal meatus. At the same postoperative time, the ES cells had migrated into the brain stem close to the ventral cochlear nucleus. The results demonstrate not only the survival and migration of xenografted DRG neurons and stem cells along the adult auditory nerve but also the feasibility of a cell replacement therapy in the degenerated auditory system.

Delayed neurotrophic treatment preserves nerve survival and electrophysiological responsiveness in neomycin-deafened guinea pigs.

Benefits of cochlear prostheses for the deaf are dependent on survival and excitability of the auditory nerve. Degeneration of deafferented auditory nerve fibers is prevented and excitability maintained by immediate replacement therapy with exogenous neurotrophic factors, in vivo. It is important to know whether such interventions are effective after a delay following deafness, typical for the human situation. This study evaluated the efficacy of brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor axokine-1 analogue (CNTF Ax1) application, 2 or 6 weeks postdeafening, in preventing further degeneration and a decrease in excitability. Guinea pigs were deafened and implanted with intracochlear stimulating electrodes, a scala tympani cannula-osmotic pump system, and auditory brainstem response (ABR) recording electrodes. Subjects received BDNF + CNTF Ax1 or artificial perilymph (AP) treatment for 27 days, beginning at 2 or 6 weeks following deafening. Electrical (E) ABR thresholds increased following deafening. After 1 week, in the 2-weeks-delayed neurotrophic factor treatment group, EABR thresholds decreased relative to AP controls, which were statistically significant at 2 weeks. In the 6-week delay group, a tendency to enhanced EABR sensitivity began at 2 weeks of treatment and increased thereafter, with a significant difference between neurotrophic factor- and AP-treated groups across the treatment period. A clear, statistically significant, enhanced survival of spiral ganglion cells was seen in both neurotrophic factor treatment groups relative to AP controls. These findings demonstrate that BDNF + CNTF Ax1 can act to delay or possibly even reverse degenerative and, likely apoptotic, processes well after they have been activated. These survival factors can rescue cells from death and enhance electrical excitability, even during the period of degeneration and cell loss when the spiral ganglion cell population is reduced by >50% (6 weeks). It is noteworthy that this same degree of ganglion cell loss, secondary to receptor damage, is typically observed after a period equivalent to some years of deafness in humans.

Survival of neuronal tissue following xenograft implantation into the adult rat inner ear.

The poor regenerative capacity of the spiral ganglion neurons of the mammalian inner ear has initiated research on how to assist the functional recovery of the injured auditory system. A possible treatment is to use a biological implant with a potential to establish central or peripheral synaptic contacts to develop into a functional auditory unit. The feasibility of this approach was tested by xenograft implantation of dorsal root ganglion (DRG) neurons from embryonic days 13 to 14, mouse expressing either LacZ or enhanced green fluorescent protein (EGFP) into the scala tympani of the adult rat inner ear. Transplanted DRG neurons survived in the scala tympani for a postoperative period ranging from 3 to 10 weeks, as verified by histochemical detection of LacZ, EGFP fluorescence and immunohistochemical labeling of the neuronal markers neurofilament and Thy 1.2. DRG neurons were found close to structures near the sensory epithelium (the organ of Corti) as well as adjacent to the spiral ganglion neurons with their peripheral dendrites. These results illustrate not only the survival of xenografted DRG neurons in the adult inner ear but also the feasibility of a neuronal transplantation strategy in the degenerated auditory system, thereby creating possibilities to replace spiral ganglion neurons.

Embryonic stem cells enhance the healing of tympanic membrane perforations.

OBJECTIVE: Tympanic membrane perforations may cause hearing impairment and otorhea. It is a common indication for ear surgery. The aim of the study was to test whether stem cells may enhance the healing of fresh tympanic membrane perforations. METHODS: In a first assay, the status of the tympanic membrane at 5 days after myringotomy was tested in five Mongolian gerbils that were treated on one side with embryonic stem cells and on the other side with control substance. In a second assay, nine gerbils were treated in the same way, except that fluorescent-labeled embryonic stem cells were used. The integration of the stem cells into the surface layer of the healing tympanic membrane was assessed with fluorescence microscopy, as well as the differentiation of these cells. RESULTS: In the first assay, all perforations in the treated ears were closed, whereas only two of the untreated ears were closed. The strength of the healed perforation was greater in the stem cell treated tympanic membranes (mean rupture pressure 120 daPa in three treated ears compared to 60 daPa in the one control ear). Two stem cell-treated tympanic membranes remained intact throughout the whole sequence of pressures, whereas only one control tympanic membrane remained intact. In three tympanic membranes in the second assay, a group of fluorescence-doped cells was detected in the region of the perforation. CONCLUSION: These findings indicate that stem cells enhance the healing of tympanic membrane perforations, possibly by differentiation and integration into the tympanic membrane tissue.

Neurotrophic factor intervention restores auditory function in deafened animals.

A primary cause of deafness is damage of receptor cells in the inner ear. Clinically, it has been demonstrated that effective functionality can be provided by electrical stimulation of the auditory nerve, thus bypassing damaged receptor cells. However, subsequent to sensory cell loss there is a secondary degeneration of the afferent nerve fibers, resulting in reduced effectiveness of such cochlear prostheses. The effects of neurotrophic factors were tested in a guinea pig cochlear prosthesis model. After chemical deafening to mimic the clinical situation, the neurotrophic factors brain-derived neurotrophic factor and an analogue of ciliary neurotrophic factor were infused directly into the cochlea of the inner ear for 26 days by using an osmotic pump system. An electrode introduced into the cochlea was used to elicit auditory responses just as in patients implanted with cochlear prostheses. Intervention with brain-derived neurotrophic factor and the ciliary neurotrophic factor analogue not only increased the survival of auditory spiral ganglion neurons, but significantly enhanced the functional responsiveness of the auditory system as measured by using electrically evoked auditory brainstem responses. This demonstration that neurotrophin intervention enhances threshold sensitivity within the auditory system will have great clinical importance for the treatment of deaf patients with cochlear prostheses. The findings have direct implications for the enhancement of responsiveness in deafferented peripheral nerves.