Proteins were separated on a SuperSep Ace gel (Wako) and subsequently transferred onto a PVDF membrane (GE Healthcare). rat auditory system to transplant donor cells to gliotic auditory nerve and the primary antibodies used in this study. (and and and Fig. S1and Fig. S1and and Table S1). Latency analysis revealed that conduction velocity recovered in the 16-kHz FGH10019 region but not in the 4- and 8-kHz regions (Fig. 2and Table S1). This difference may be because the 16-kHz region was nearest to the transplantation site, allowing more time for myelination, so a similar recovery might have occurred at 4 and 8 kHz over a longer period. In the sham rats to which the medium-soaked gelatin sponge and fibrin glue were placed on the nerve (= 5), no significant changes in the ABRs were observed 3 mo later, indicating that spontaneous recovery did not occur 5 wk after auditory nerve compression. Open in a separate window Fig. 2. Recovery of ABRs after intraneural and surface transplantation of cells. (= 10). Open in a separate window Fig. S2. Neural generators of ABR and ABR waveform changes after surgical ablation of cochlear nucleus tissue and auditory nerve injury/subsequent glial scar formation. (and and and and and and and and is enlarged in and are enlarged in and indicates the site of compression. (and are approximately adjacent sections. (Scale bars, 200 m in and and and and and and and and is several sections apart from and roughly corresponds to upper part of and and and and is the approximate location in the posteroventral cochlear nucleus (PVCN) that is enlarged in the following panels. Dotted lines in and indicate the surface of the brainstem. (< 0.01. (Scale bars, 400 m in in and in in and and and are enlarged in each adjacent panel. The dotted line in is the fundus and wall of the IAC. (and and < 0.05, ***< 0.01, ****< 0.001. Cues for Inward Migration of Surface Cells. The cues that guide the migration of transplanted cells are unknown. However, in our model, the hair cells remain intact (10), and they should provide a source of neurotrophins, notably BDNF (brain-derived neurotrophic factor) and neurotrophin 3, which are indispensable for the maintenance and survival of auditory neurons (28). Correspondingly, auditory neurons express the relevant receptors, trkB and trkC, respectively (28). FGH10019 We found that BDNF concentrations were higher in ChABC-treated tissue (Fig. 8< 0.01. (is enlarged in and 20 m in and ?and3and Fig. S1= 4). In 1G2 and 2B6 antibodies, the fluorescence intensity in sham samples was manually reduced to approximately zero level, and at this level, the images of experimented specimens were photographed (= 4). Images were converted into grayscale images with Photoshop (CS3; Adobe) and transferred to National Institutes of Healths Image J, and positive pixel area (PPA) was analyzed applying the Erg same threshold. Western Blotting of GFAP. Samples were collected from experimental rats, 4 wk after compression (right side, = 19), and those from sham rats were used as control. After processing, proteins were separated on a SuperSep Ace gel (Wako) and transferred onto a PVDF membrane (GE Healthcare). Membranes were probed with GFAP antibody (1:15,000; DAKO; rabbit polyclonal) overnight, and HRP-linked anti-rabbit IgGs (1:10,000; GE Healthcare) were applied as the secondary antibody. GAPDH was used as a loading control. Detailed information is listed in at 85 dB SPL at 8 and 16 kHz and FGH10019 75 dB at 4 kHz to avoid large cochlear microphonics at 85 dB in this frequency. Statistical Analysis. An unpaired or paired Student two-tailed test was performed using Excel 2013 (Microsoft). For all statistical tests, < 0.05 was used as the criterion for statistical significance. In all figures, error bars indicate SD. SI Materials and Methods Immunohistochemistry. Temporal bones were decalcified with 10% (wt/vol) EDTA and HCl solution FGH10019 (pH 7.4) at 30 FGH10019 C for 5 wk using a microwave processor (MI-33; Azumaya Corporation). Serial 15-m frozen sections were made by a cryostat (Leica CM1850; Leica Biosystems) after embedding into OCT compound. After blocking in the mixture of 10% (vol/vol) normal goat serum (NGS) in PBS and 2% (wt/vol) BSA in PBS, sections were incubated in primary antibody diluted in 10% (vol/vol) NGS overnight at 4 C. The targets of the following primary antibodies are as follows.