Transgenic BDNF induces nerve fiber regrowth into the auditory epithelium in deaf cochleae

Abstract

Sensory organs typically use receptor cells and afferent neurons to transduce environmental signals and transmit them to the CNS. When sensory cells are lost, nerves often regress from the sensory area. Therapeutic and regenerative approaches would benefit from the presence of nerve fibers in the tissue. In the hearing system, retraction of afferent innervation may accompany the degeneration of auditory hair cells that is associated with permanent hearing loss. The only therapy currently available for cases with severe or complete loss of hair cells is the cochlear implant auditory prosthesis. To enhance the therapeutic benefits of a cochlear implant, it is necessary to attract nerve fibers back into the cochlear epithelium. Here we show that forced expression of the neurotrophin gene BDNF in epithelial or mesothelial cells that remain in the deaf ear induces robust regrowth of nerve fibers towards the cells that secrete the neurotrophin, and results in re-innervation of the sensory area. The process of neurotrophin-induced neuronal regeneration is accompanied by significant preservation of the spiral ganglion cells. The ability to regrow nerve fibers into the basilar membrane area and protect the auditory nerve will enhance performance of cochlear implants and augment future cell replacement therapies such as stem cell implantation or induced transdifferentiation. This model also provides a general experimental stage for drawing nerve fibers into a tissue devoid of neurons, and studying the interaction between the nerve fibers and the tissue.

Figure 1

The auditory epithelium from normal cochleae (a–b) or cochleae analyzed one week after an insult with neomycin (c–d) and viewed by epi-fluorescence (a, c) or light microscopy of plastic sections (b, d). The left side of the plastic sections corresponds to the lower aspect of the whole-mounts. (a) Phalloidin labeled whole-mount shows the epithelial mosaic containing hair cells and supporting cells. The approximate site of the HP is depicted by a dotted line. (I, inner hair cell; P, pillar cells; 1, 2, 3, 1^st 2^nd and 3^rd rows of outer hair cells). (b) Sensory hair cells and non-sensory supporting cells and other cells and non-cellular components of the organ of Corti. The site of the HP is depicted with an arrow. (c) In the deaf cochlea, hair cells are absent and instead, a sheet of polymorphic cells with abundant actin in the intercellular junctions line the scala media and form the FE (FE). The dotted lines show the area of the HP. (d) The FE lines the scala media. The presumed site of the HP is depicted with an arrow. Scale bars are 20 µm and also apply to both images above.

Figure 2

Whole-mounts of the auditory epithelium stained for neurofilaments (red) and actin (green) and viewed with confocal microscopy, showing normal (a), neomycin-deafened (b–d), or Ad.BDNF treated ears 14 days after neomycin deafening (e–h). Dotted line depicts presumed area of HP in a–e, and h. Ad.BDNF was inoculated via scala media (e–g) or scala tympani (h) route. (a) The neurofilament positive nerve fibers exit the HP and reach inner (I) and outer hair cells (1–3), forming the inner (open arrowhead) and outer spiral bundle (arrow-head). (b) 7 days after neomycin deafening, nerve fibers are absent in the FE except for the area near the HP and a few loops (arrows) near the HP. (c–d) Fourteen (c) or 30 days (d) after Ad.Empty vector inoculation, no nerve fibers are seen in the epithelium beyond the HP area. (e) The diameter and orientation of nerve fiber extension into the FE varied. Narrower nerve fibers appeared further away from the HP (top of image) in a longitudinal (spiral) orientation. A nerve fiber that terminated not far from the HP (open arrow-head) exhibited an enlarged portion at the termination site. Most if not all nerve fibers weaved between the junctions of the FE cells (arrow-heads). (f) Enlarged neurofilament-positive areas appeared as spherical regions (dotted line) above and slightly medial to the HP, in a region corresponding to the inner sulcus or spiral limbus of a normal ear. (g) Nerve fibers in the interdental cell area projected while changing orientation (arrow). The surface of the nerve fibers in the interdental cell area appeared coarser than nerve fibers in the BMA (compare to e and h). (h) Following inoculation into the scala tympani, nerve fibers extended to the auditory epithelium weaving between FE cells. Scale bar=50 µm.

Figure 3

Cross-sections of the BMA observed with light (a) or TEM of ears treated with Ad.BDNF via the scala media and obtained for histology 14 days later (b–e). (a) Radial section of the FE after inoculation of Ad.Empty (top) or Ad.BDNF (bottom) stained for neurofilament with DAB (brown) and lightly counterstained with toluidine blue to reveal tissue structure. In Ad.Empty inoculated ears the nerve fibers rise near the surface of the HP (arrow) but do not extend further into the BMA. In Ad.BDNF inoculated ears, nerve fibers extend beyond the HP (arrow) and run in and under the FE, partially entering the ECM (arrow-heads). Scale barh 20 µm. (b) An un-myelinated nerve fiber (N) is seen beneath the cuboidal cells (CE) in the BMA and above the surface of the ECM. Tight junctions (arrow) and desmosomes (arrow-head) are observed between the cuboidal cells. An adherens junctional complex is seen between them. (c) A nerve fiber (N) is seen in contact with the ECM in an area devoid of basal lamina (area between black arrow-heads). The basal lamina can be observed adjacent to this area (open arrow-heads). (d) The area of contact between two flat epithelial cells above a nerve fiber (N) includes an irregular membrane structure (arrow-heads point to desmosomes). (e) Mitochondria (black arrow) are seen in the nerve fibers along with filaments and vesicles. Scale bars=500 nm.

Figure 4

Quantitative analysis of the nerve fibers projecting at least 50 µm into the BMA peripheral (lateral) to the HP. Comparison of Ad.Empty and Ad.BDNF shows a significant difference of average nerve fiber count in the 1^st and 2^nd turn at 14 (gray bars) and 30 (black bars) days after deafening. (*) indicates p < 0.01. Deafened animals with no inoculation had similar counts to Ad.Empty animals. Bars are standard deviation and statistical comparison was done by Student t-test.

Figure 5

Whole-mounts of ears treated with neomycin and then inoculated with AAV.GFP-BDNF via scala tympani (a–c) or scala media (d), observed with CFM (a and d) and epi-fluorescence (b and c). (a) Nerve fibers extend into the BMA and projections deviate toward the GFP-positive cells, and in some cases terminate near the cell (arrow-head). (b and c) Nerve fibers with large diameters are also seen to be attracted to GFP-positive cells in the residual Hensen cell area (H). (d) Nerve fibers deviate from outer spiral bundle area (dotted line) and move toward the GFP-positive cells. Scale bars=50 µm.

Figure 6

Light microscope micrographs of plastic sections through Rosenthal’s canal in the 1^st (a and b) and 2^nd (c and d) cochlear turns 14 days after inoculation of Ad.Empty (a and c), or Ad.BDNF (b and d), and quantification of SGNs (e). (a and c) SGNs are scarce. A presumed efferent nerve fiber bundle is present (arrow). (b and d) Rosenthal’s canal in Ad.BDNF inoculated ears exhibits a confluent population of neurons and no gliotic areas. In some of the Ad.BDNF inoculated ears, connective tissue could be observed in the scala tympani (arrow-head in b). Scale bars=100 µm. (e) Mean SGN density among the cochlear regions of basal and 2^nd turn of Ad.Empty and Ad.BDNF inoculated animals. Comparison of SGN density in Ad.Empty (gray bars) and Ad.BDNF-inoculated groups (black bars) in all regions showed significantly larger numbers of surviving SGN in the Ad.BDNF groups. Error bars are standard deviations. Statistical comparison was by one-way ANOVA. (*) indicates p < 0.01. (L=Lower and U=Upper).