Fig 1: Expression of BDNF and NT-3 in neomycin-treated utricle cultures over time. Adult mouse utricles were treated with neomycin for 48 h and maintained in vitro for 1 month. Controls were maintained in defined medium, and experimental groups were treated with an Ad28 vector delivering the BDNF or NTF3 genes driven either with the herpes latency promoter or the human CMV promoter. Medium was exchanged every 72 h, and BDNF or NT-3 levels were determined by ELISA. Error bars represent standard deviation. Controls and the lat promoter groups showed a low production of BDNF at the limits of the ELISA sensitivity. Utricles treated with the CMV-driven BDNF showed steady growth of BDNF expression over the first 10 days in vitro that was maintained at high levels for the duration of the experiment. NT-3 was barely detectable in control utricle explants. NTF3 driven by the lat promoter produced a low level of NT-3, whereas use of a CMV promoter resulted in higher expression levels of NT-3.
Fig 2: Effect of neurotrophin expression in the normal hearing ear. Expression of BDNF in the cochlea appeared to induce a mild change in hearing when expressed at high levels using the CMV promoter (A). This was not significant compared with expression driven by the lat promoter or overexpression of GFP driven by the CMV promoter. Overexpression of NTF3 at high levels caused significant elevation of thresholds at 8 k, 16 k, and 32 kHz, compared with lat-driven NTF3 or control inner ears (B). Evaluation of spiral ganglion neurons by immunofluorescence demonstrated abnormal migration of spiral ganglion neurites after delivering the NTF3 gene with a strong promoter [(C), Arrow]. Error bars represent standard deviation (n = 5). Scale bar = 60 µm.
Fig 3: The graphical abstract depicts that Vav3–/– astrocytes promote the branching and the elongation of dendrites on wildtype neurons in an indirect co-culture system. Knockout astrocytes show a higher secretion of the chemokine CCL5 which has a neurotropic potential. Contrary, the pro-inflammatory cytokine IL-6 is released in lower amounts. In contrast to wildtype astrocytes, Vav3–/– astrocytes do not release CXCL11. On mRNA level Vav3–/– astrocytes express more NT-3 and TSP-1.
Fig 4: Neuronal survival after low-level neurotrophin gene therapy using the lat promoter (A). Section through Rosenthal’s canal in the basal turn of an adult hearing mouse shows spiral ganglion neurons immunostained for neurofilament (red). Cell nuclei are stained in blue using DAPI injection of neomycin into the round window that resulted in loss of greater than 50% of spiral ganglion cells (B) Example of missing neurons are shown with arrow (B) Neomycin treatment followed by delivery of Ad28.lat.bdnf into the posterior semicircular canal resulted in improved survival of spiral ganglion cells (C). Animals treated with Ad28.lat.ntf3 after neomycin treatment resulted in optimal neuronal survival and morphology similar to controls (D). Scale bar = 10 µm.
Fig 5: Neuronal survival after neomycin treatment and after neomycin treatment followed by delivery of Ad28.lat.bdnf or Ad28. lat.ntf3. Delivery of BDNF resulted in statistically significant improvement of spiral ganglion survival in the basal turn. NT-3 delivery significantly improved spiral ganglion survival in both the basal and apical turns. Error bars represent standard deviation (n = 3).
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