Deafferentiation-associated changes in afferent and efferent processes in the guinea pig cochlea and afferent regeneration with chronic intrascalar brain-derived neurotrophic factor and acidic fibroblast growth factor

Deafferentation of the auditory nerve from loss of sensory cells is associated with degeneration of nerve fibers and spiral ganglion neurons (SGN). SGN survival following deafferentation can be enhanced by application of neurotrophic factors (NTF), and NTF can induce the regrowth of SGN peripheral processes. Cochlear prostheses could provide targets for regrowth of afferent peripheral processes, enhancing neural integration of the implant, decreasing stimulation thresholds, and increasing specificity of stimulation. The present study analyzed distribution of afferent and efferent nerve fibers following deafness in guinea pigs using specific markers (parvalbumin for afferents, synaptophysin for efferent fibers) and the effect of brain derived neurotrophic factor (BDNF) in combination with acidic fibroblast growth factor (aFGF). Immediate treatment following deafness was compared with 3-week-delayed NTF treatment. Histology of the cochlea with immunohistochemical techniques allowed quantitative analysis of neuron and axonal changes. Effects of NTF were assessed at the light and electron microscopic levels. Chronic BDNF/aFGF resulted in a significantly increased number of afferent peripheral processes in both immediate- and delayed-treatment groups. Outgrowth of afferent nerve fibers into the scala tympani were observed, and SGN densities were found to be higher than in normal hearing animals. These new SGN might have developed from endogenous progenitor/stem cells, recently reported in human and mouse cochlea, under these experimental conditions of deafferentation-induced stress and NTF treatment. NTF treatment provided no enhanced maintenance of efferent fibers, although some synaptophysin-positive fibers were detected at atypical sites, suggesting some sprouting of efferent fibers. J. Comp. Neurol. 507:1602–1621, 2008. © 2008 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/58023/1/21619_ftp.pd

Glial cell line-derived neurotrophic factor and chronic electrical stimulation prevent VIII cranial nerve degeneration following denervation

As with other cranial nerves and many CNS neurons, primary auditory neurons degenerate as a consequence of loss of input from their target cells, the inner hair cells (IHCs). Electrical stimulation (ES) of spiral ganglion cells (SGCs) has been shown to enhance their survival. Glial cell line-derived neurotrophic factor (GDNF) has also been shown to increase survival of SGCs following IHC loss. In this study, the combined effects of the GDNF transgene delivered by adenoviral vectors (Ad- GDNF ) and ES were tested on SGCs after first eliminating the IHCs. Animal groups received Ad- GDNF or ES or both. Ad- GDNF was inoculated into the cochlea of guinea pigs after deafening, to overexpress human GDNF . ES-treated animals were implanted with a cochlear implant electrode and chronically stimulated. A third group of animals received both Ad- GDNF and ES (GDNF/ES). Electrically evoked auditory brainstem responses were recorded from ES-treated animals at the start and end of the stimulation period. Animals were sacrificed 43 days after deafening and their ears prepared for evaluation of IHC survival and SGC counts. Treated ears exhibited significantly greater SGC survival than nontreated ears. The GDNF/ES combination provided significantly better preservation of SGC density than either treatment alone. Insofar as ES parameters were optimized for maximal protection (saturated effect), the further augmentation of the protection by GDNF suggests that the mechanisms of GDNF- and ES-mediated SGC protection are, at least in part, independent. We suggest that GDNF/ES combined treatment in cochlear implant recipients will improve auditory perception. These findings may have implications for the prevention and treatment of other neurodegenerative processes. J. Comp. Neurol. 454:350–360, 2002. © 2002 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34465/1/10480_ftp.pd

Viral-mediated Ntf3 overexpression disrupts innervation and hearing in nondeafened guinea pig cochleae

Synaptopathy in the cochlea occurs when the connection between inner hair cells and the auditory nerve is disrupted, leading to impaired hearing and nerve degeneration. Experiments using transgenic mice have shown that overexpression of NT3 by supporting cells repairs synaptopathy caused by overstimulation. To accomplish such therapy in the clinical setting, it would be necessary to activate the neurotrophin receptor on auditory neurons by other means. Here we test the outcome of NT3 overexpression using viral-mediated gene transfer into the perilymph versus the endolymph of the normal guinea pig cochlea. We inoculated two different Ntf3 viral vectors, adenovirus (Adv) or adeno-associated virus (AAV) into the perilymph, to facilitate transgene expression in the mesothelial cells and cochlear duct epithelium, respectively. We assessed outcomes by comparing Auditory brainstem response (ABR) thresholds prior to that at baseline to thresholds at 1 and 3 weeks after inoculation, and then performed histologic evaluation of hair cells, nerve endings, and synaptic ribbons. We observed hearing threshold shifts as well as disorganization of peripheral nerve endings and disruption of synaptic connections between inner hair cells and peripheral nerve endings with both vectors. The data suggest that elevation of NT3 levels in the cochlear fluids can disrupt innervation and degrade hearing

Nanofibrous scaffolds for the guidance of stem cell-derived neurons for auditory nerve regeneration

<div><p>Impairment of spiral ganglion neurons (SGNs) of the auditory nerve is a major cause for hearing loss occurring independently or in addition to sensory hair cell damage. Unfortunately, mammalian SGNs lack the potential for autonomous regeneration. Stem cell based therapy is a promising approach for auditory nerve regeneration, but proper integration of exogenous cells into the auditory circuit remains a fundamental challenge. Here, we present novel nanofibrous scaffolds designed to guide the integration of human stem cell-derived neurons in the internal auditory meatus (IAM), the foramen allowing passage of the spiral ganglion to the auditory brainstem. Human embryonic stem cells (hESC) were differentiated into neural precursor cells (NPCs) and seeded onto aligned nanofiber mats. The NPCs terminally differentiated into glutamatergic neurons with high efficiency, and neurite projections aligned with nanofibers <i>in vitro</i>. Scaffolds were assembled by seeding GFP-labeled NPCs on nanofibers integrated in a polymer sheath. Biocompatibility and functionality of the NPC-seeded scaffolds were evaluated <i>in vivo</i> in deafened guinea pigs (<i>Cavia porcellus</i>). To this end, we established an ouabain-based deafening procedure that depleted an average 72% of SGNs from apex to base of the cochleae and caused profound hearing loss. Further, we developed a surgical procedure to implant seeded scaffolds directly into the guinea pig IAM. No evidence of an inflammatory response was observed, but post-surgery tissue repair appeared to be facilitated by infiltrating Schwann cells. While NPC survival was found to be poor, both subjects implanted with NPC-seeded and cell-free control scaffolds showed partial recovery of electrically-evoked auditory brainstem thresholds. Thus, while future studies must address cell survival, nanofibrous scaffolds pose a promising strategy for auditory nerve regeneration.</p></div

Surgical approach and histological assessment of placement and tissue response.

<p>(A–C) Guinea pig temporal bone showing the access to and the scaffold placement in the IAM. (A) The scaffold was advanced into the IAM through a cochleostomy in the base of the cochlea. (B) Positioning of the electrode co-implanted for eABR recordings. (C) View on the positioned scaffold from the brain side. (D and E) Plastic cross-sections of the IAM with implanted cell-free scaffold 15 days post-implantation. (D) Cross section showing the IAM with its bony wall and the scaffold (arrowhead; sheath shaded in blue) embedded in host tissue. Host tissue was found to infiltrate the full length of the scaffold. (E) Higher magnification of the scaffold’s interior with host tissue embedding the nanofibers (shaded in green), which adopt a chain-like arrangement. (F) IAM immunohistochemistry for the hematopoietic lineage marker CD45 shows few immune cells within the IAM 4 days after implantation with an NPC-seeded scaffold. (G) Additional samples 1 month post-implantation were examined for CD45 labeling, including sham, cell-free scaffold, and NPC scaffold animal groups, as well as untreated controls. Few immune-positive cells were identified in these sections indicating that the surgery, scaffolds, and NPCs did not trigger a significant immune response. (H) Immunohistochemistry for TUJ1 (neurons), GFAP (glia) and vimentin (Schwann cells/fibroblasts) in control, sham and cell-free scaffold (1-month post implant) shows involvement of vimentin and GFAP positive cells in tissue repair. Scale bars represent 1 mm in A—C, 100 μm in D—H.</p

Neurite alignment on nanofiber mats <i>in vitro</i>.

<p>(A–C) Representative images of TUJ1 stained terminally differentiated H7 hESC derived neurons (green) on PS coverslips (A) and unaligned (B) and aligned (C) nanofiber mats. Insets in B’ and C’ show light microscopic images of unaligned and aligned nanofiber mats. (D–F) Plot of image intensity as a function of angle with corresponding FWHM values after Fourier transformation of fluorescence images of A, B and C illustrating the extent of alignment. A low FWHM indicates high alignment. (G) Quantification of FWHM values of coverslips (n = 8) and unaligned (n = 7) and aligned (n = 6) fibers terminally differentiated for 2–6 weeks. For random samples, only one out of 7 samples allowed FWHM calculation. (H) Immunohistochemistry for TUJ1 (green) and VGLUT1 (red) shows that neurons maintain a glutamatergic fate on fiber mats. Scale bars represent 100 μm in A, B, C and H and 50 μm in B’ and C’.</p

Neuronal differentiation protocol and phenotype analysis.

<p>(A) Illustration of the neuronal differentiation protocol. (B–D) Representative images of H7 hESC derived precursors after formation of EBs (B), neuronal rosettes (C) and NPCs (D). (E–G) qPCR analysis of gene expression during EB (green, n = 3), neuronal rosette (blue, n = 4) and NPC (grey, n = 3) stages compared to undifferentiated hESC (red, n = 3). Error bars indicate standard error of the mean. (H–J) Representative images of protein expression 4 weeks after terminal differentiation. Nuclei are counterstained with Hoechst. (H) Differentiated cells express the neuronal marker TUJ1 and the vesicular glutamate transporter VGLUT1. (I) MAP-2 expression indicates maturation of derived neurons. (J) Neurons stained positive for the synaptic protein synaptophysin (SYP, counterstained with TUJ1). Scale bars represent 200 μm in B, 50 μm C and J, and 100 μm in D, H, and I.</p

Nanofibrous scaffold assembly, NPC seeding and pre-implantation adhesion.

<p>(A and B) High resolution images of single (A, SEM image) and assorted (B, light microscopic image) PLLA:PCL nanofibers. (C and D) SEM images of assembled scaffolds consisting of nanofiber bundle and PCL sheath. (E) Illustration of procedures for seeding NPCs on nanofibers for implantation. A long nanofiber bundle with sheath was attached to a coverslip. (1) Concentrated cell suspension (10,000 cells/μl) was deposited on a nanofiber portion outside the sheath and the cells allowed to settle. (2) The sheath was shifted and positioned over the nanofiber area with attached cells. (3) The excess length of nanofibers was cut, releasing the scaffold for implantation. (F) Nuclear Hoechst stain of NPCs one hour after implantation. (G) TUJ1 stain (green, counterstained with Hoechst) shows fiber extension and alignment on Nanofiber bundles as early as 24h after seeding. (H) Quantification of Hoechst stain based cell counts on coverslips and bundles of parallel cultures. Error bars represent standard error of the mean. (I) Neurite extension aligned with nanofiber orientation 6 weeks after seeding (green: TUJ1, red: Nestin, blue: Hoechst). Scale bars represent 1 μm in A, 100 μm in B, D, F, G and I and 1 mm in C.</p

Post-implant physiology and histological assessment of H9-GFP derived NPC survival.

<p>(A) Representative examples of in vivo threshold eABR waveforms of cell-free and NPC scaffolds at post implantation days indicated. In both groups, waveforms are flattened after deafening and implantation. Profiles of distinct waves partially recover over the one month observation period. All responses were evoked with a 300 μA stimulus, except for the day-9 time point of the cell-free scaffold subject (indicated by *, 300 μA was below threshold and thus a 370 μA response is shown for illustration). (B) Course of post implantation eABR thresholds for individual animals implanted with cell-free scaffolds (n = 4) and NPC scaffolds (n = 9) is shown alongside the mean thresholds for NPC-only injections (n = 3). Gray triangle symbols indicate two animals implanted with dye-labeled NPC-scaffolds; the elevated thresholds in these animals may indicate a negative impact from the FluoroRuby dye on NPC health. Data points are slightly shifted in time for clarity. (C) hrGFP immunohistochemistry for the detection of H9-GFP derived NPCs in sections of control and implanted specimen collected 4 days post implantation. The excerpt shows a magnification of the interior of the scaffold with hrGFP expression in two Hoechst-positive cells (blue channel enhanced for illustration). (D) There was no hrGFP signal detected in 1 month implantations. TUJ1 stain shows interspersed fibers both in cell-free and NPC-seeded conditions. Scale bars represent 100 μm in C and D and 50 μm in the excerpt of C.</p