Combinatorial Atoh1 and Gfi1 induction enhances hair cell regeneration in the adult cochlea.

Mature mammalian cochlear hair cells (HCs) do not spontaneously regenerate once lost, leading to life-long hearing deficits. Attempts to induce HC regeneration in adult mammals have used over-expression of the HC-specific transcription factor Atoh1, but to date this approach has yielded low and variable efficiency of HC production. Gfi1 is a transcription factor important for HC development and survival. We evaluated the combinatorial effects of Atoh1 and Gfi1 over-expression on HC regeneration using gene transfer methods in neonatal cochlear explants, and in vivo in adult mice. Adenoviral over-expression of Atoh1 and Gfi1 in cultured neonatal cochlear explants resulted in numerous ectopic HC-like cells (HCLCs), with significantly more cells in Atoh1 + Gfi1 cultures than Atoh1 alone. In vitro, ectopic HCLCs emerged in regions medial to inner HCs as well as in the stria vascularis. In vivo experiments were performed in mature Pou4f3DTR mice in which HCs were completely and specifically ablated by administration of diphtheria toxin. Adenoviral expression of Atoh1 or Atoh1 + Gfi1 in cochlear supporting cells induced appearance of HCLCs, with Atoh1 + Gfi1 expression leading to 6.2-fold increase of new HCLCs after 4 weeks compared to Atoh1 alone. New HCLCs were detected throughout the cochlea, exhibited immature stereocilia and survived for at least 8 weeks. Combinatorial Atoh1 and Gfi1 induction is thus a promising strategy to promote HC regeneration in the mature mammalian cochlea.

Four-point impedance as a biomarker for bleeding during cochlear implantation.

Cochlear implantation has successfully restored the perception of hearing for nearly 200 thousand profoundly deaf adults and children. More recently, implant candidature has expanded to include those with considerable natural hearing which, when preserved, provides an improved hearing experience in noisy environments. But more than half of these patients lose this natural hearing soon after implantation. To reduce this burden, biosensing technologies are emerging that provide feedback on the quality of surgery. Here we report clinical findings on a new intra-operative measurement of electrical impedance (4-point impedance) which, when elevated, is associated with high rates of post-operative hearing loss and vestibular dysfunction. In vivo and in vitro data presented suggest that elevated 4-point impedance is likely due to the presence of blood within the cochlea rather than its geometry. Four-point impedance is a new marker for the detection of cochlear injury causing bleeding, that may be incorporated into intraoperative monitoring protocols during CI surgery.

"Emergency" Cochlear Implantation in Labyrinthitis Ossificans Secondary to Polyarteritis Nodosa: How to Face a Rare Entity.

Polyarteritis nodosa (PAN) is a systemic vasculitis affecting the small- and medium-sized arteries that may present with hearing impairment. In rare cases, PAN may be associated with progressive labyrinthitis ossificans (LO), an otologic emergency requiring early cochlear implantation (CI) to restore hearing before the complete, irreversible cochlear ossification. We report the first case in the literature of a patient affected by PAN with bilateral sudden sensorineural hearing loss and rapid LO who underwent "emergency" bilateral simultaneous CI. This case report emphasizes the importance of an early audiological evaluation in patients with PAN when LO is suspected. Multidisciplinary approach is mandatory when facing organ-specific manifestations in patients with PAN. Detailed discussion is provided with particular regard to clinical and radiological presentation as well as CI outcomes in such a rare and challenging case.

Macrophages in the Human Cochlea: Saviors or Predators-A Study Using Super-Resolution Immunohistochemistry.

The human inner ear, which is segregated by a blood/labyrinth barrier, contains resident macrophages [CD163, ionized calcium-binding adaptor molecule 1 (IBA1)-, and CD68-positive cells] within the connective tissue, neurons, and supporting cells. In the lateral wall of the cochlea, these cells frequently lie close to blood vessels as perivascular macrophages. Macrophages are also shown to be recruited from blood-borne monocytes to damaged and dying hair cells induced by noise, ototoxic drugs, aging, and diphtheria toxin-induced hair cell degeneration. Precise monitoring may be crucial to avoid self-targeting. Macrophage biology has recently shown that populations of resident tissue macrophages may be fundamentally different from circulating macrophages. We removed uniquely preserved human cochleae during surgery for treating petroclival meningioma compressing the brain stem, after ethical consent. Molecular and cellular characterization using immunofluorescence with antibodies against IBA1, TUJ1, CX3CL1, and type IV collagen, and super-resolution structured illumination microscopy (SR-SIM) were made together with transmission electron microscopy. The super-resolution microscopy disclosed remarkable phenotypic variants of IBA1 cells closely associated with the spiral ganglion cells. Monitoring cells adhered to neurons with "synapse-like" specializations and protrusions. Active macrophages migrated occasionally nearby damaged hair cells. Results suggest that the human auditory nerve is under the surveillance and possible neurotrophic stimulation of a well-developed resident macrophage system. It may be alleviated by the non-myelinated nerve soma partly explaining why, in contrary to most mammals, the human's auditory nerve is conserved following deafferentiation. It makes cochlear implantation possible, for the advantage of the profoundly deaf. The IBA1 cells may serve additional purposes such as immune modulation, waste disposal, and nerve regeneration. Their role in future stem cell-based therapy needs further exploration.

Delivery of Adeno-Associated Virus Vectors in Adult Mammalian Inner-Ear Cell Subtypes Without Auditory Dysfunction.

Hearing loss, including genetic hearing loss, is one of the most common forms of sensory deficits in humans with limited options of treatment. Adeno-associated virus (AAV)-mediated gene transfer has been shown to recover auditory functions effectively in mouse models of genetic deafness when delivered at neonatal stages. However, the mouse cochlea is still developing at those time points, whereas in humans, the newborn inner ears are already fully mature. For effective gene therapy to treat genetic deafness, it is necessary to determine whether AAV-mediated therapy can be equally effective in the fully mature mouse inner ear without causing damage to the inner ear. This study tested several AAV serotypes by canalostomy in adult mice. It is shown that most AAVs transduce the sensory inner hair cells efficiently, but are less efficient at transducing outer hair cells. A subset of AAVs also transduces non-sensory cochlear cell types. Neither the surgical procedure of canalostomy nor the AAV serotypes damage hair cells or impair normal hearing. The studies indicate that canalostomy can be a viable route for safe and efficient gene delivery, and they expand the repertoire of AAVs to target diverse cell types in the adult inner ear.

Molecular pathways involved in apoptotic cell death in the injured cochlea: cues to novel therapeutic strategies.

Most hearing loss results from lesions of the sensory cells and/or neurons of the auditory portion of the inner ear. To date, only the cochlear implantation offers long-term hearing-aid benefit, but still with limited performance and expensive cost. While the underlying causes of deafness are not clear, the death or hair cells and/or neurons and the loss of neuronal contacts are key pathological features. Pinpointing molecular events that control cell death in the cochlea is critical for the development of new strategies to prevent and treat deafness, whether in combination or not with cochlear implant therapy.

Passive transfer of experimental autoimmune labyrinthitis.

The aim of the present study was to establish an animal model of autoimmune labyrinthitis using heterologous inner ear antigen (IEAg) and to elucidate whether the experimentally induced labyrinthitis could be passively transferred. Cochlear and vestibular membranous labyrinthine tissues from bovine temporal bones were used as IEAg. Donor mice were inoculated intracutaneously at multiple sites with an emulsion consisting of equal parts of IEAg and complete Freund's adjuvant. After 10 days, mononuclear cells were collected from lymph nodes, spleen and blood of the donor mice and injected intravenously into naive recipient mice. Cellular infiltration was observed in the perilymphatic space of the cochlea of all donor and recipient mice. Endolymphatic hydrops was also observed in 63% of donor and 42% of recipient mice. These findings suggest that the experimentally induced labyrinthitis observed in this animal model was probably due to an autoimmune reaction to the IEAg and was passively transferred by a cell-mediated immune reaction.

[Transplantation of fetal tissues in otorhinolaryngology: current status and prospects for the future]. / Ispol'zovanie transplantatsii fetal'nykh tkanei v otorinolaringololgii. Analiz sostoianiia problemy i perspektivy razvitiia.

Insufficient efficacy of treatment of neurosensory hypoacusis makes investigators search for new methods. Transplantation of fetal tissue taken from healthy embryos of gestation stage II tried in various fields of medicine, such as neurology, endocrinology, surgery, etc., was also tested in management of various forms of neurosensory hypoacusis. Endonasal, endaural, endolumbal and intracochlear methods of the transplantation are detailed. Results of combined dynamic audiological control of these patients including tonal threshold audiometry in extended frequency range are presented. A 1-year follow-up provided evidence in favour of endolumbal and intracochlear transplantation. Further investigations are needed.

Stage dependent development of intraocular cochlear grafts.

The intraocular grafting technique was employed to test whether the peripheral hearing organ, the cochlea, is capable of survival and an organized development in total isolation from the temporal bone. Rat cochleae obtained from gestation day 16, postnatal day 1 and 7 were chosen for transplantation into the anterior chamber of the eye of adult Sprague-Dawley rats. The grafts were maintained in the anterior chamber for 6, 10, or 15 weeks survival time. The salient features of this study is that 1) cochlear structures survive and, 2) the cochlear structures develop beyond their pre-grafted stage as determined from light and electron micrographs. In the present study, the grafts obtained at gestation day 16 (GD 16) and postnatal day 1 gave a much higher rate of survival and development than the postnatal day 7 grafts. In addition, grafts maintained for either 6 or 10 weeks had a better survival rate than those grafts left for 15 weeks. It is estimated from light and electron micrographs that the gestation day 16 otocysts that were maintained for 10 weeks, developed to the equivalent of a postnatal day 10 cochlea. The grafts obtained from postnatal day one rats developed to the equivalent of approximately 14 days after birth. Interestingly, in the absence of synaptic contact, the inner and outer hair cells were capable of survival, differentiation and maturation. It remains to be determined if the spiral ganglion cells require additional neurotrophic factors for survival in the anterior chamber of the eye.

Development of location-specific hair cell stereocilia in denervated embryonic ears.

The developmental mechanisms that allow physiological coding of acoustic pitch have remained unexplained. Cochlear hair cells that have different structures respond to different sound frequencies and synapse with neurons that project to different locations in the brain. How do these hair cells develop appropriate structures, and how are the connections between specific hair cells and the neurons that code for their pitch sensitivities matched? We have investigated one aspect of this by denervating embryonic chicken ears, before the time of hair cell production, and then transplanting them to the aneural chorioallantoic membrane of host embryos where they have continued to develop. We report that vestibular and auditory hair cell phenotypes differentiate appropriately and that correct gradients of hair cell structural phenotypes, as expressed in stereocilia bundles, develop in the cochleae of these denervated ears. Therefore, the normal development of gradients in hair cell stereocilia properties must be controlled by location-specific cues originating in the ear itself. Neuronally directed modification of target cell phenotypes is not required for the quite specific phenotype development represented by the stereocilia bundles of individual hair cells and the connectional matching in the numerous distinct peripheral information lines of the auditory system.

Ganglion cell populations in normal and pathological human cochleae. Implications for cochlear implantation.

A histological study of 100 hearing ears showed that some capability for speech discrimination requires at least 10,000 spiral ganglion cells. The spiral ganglion cell populations were then estimated in another group of 62 ears which were profoundly deaf for a variety of causes and it was found that 45% of these met the criterion of having 10,000 ganglion cells. The ganglion cell populations were largest in ears deafened by sudden deafness, Meniere's Disease, and ototoxic drugs; they were somewhat less for those with vascular occlusion, temporal bone fracture, otosclerosis, and cochlear dysplasias; they were least in those with measles, bacterial labyrinthitis and congenital syphilis. The data is of relevance in the selection of patients for cochlear implantation.