TrkB-T1 receptors on Muller cells play critical role in brain-derived neurotrophic factor-mediated photoreceptor protection against phototoxicity.

PURPOSE: To determine how brain-derived neurotrophic factor (BDNF) protects photoreceptors against phototoxicity. METHODS: Iris pigment epithelial cells (IPE) that were transduced with different concentrations of adeno-associated virus (AAV) mediated BDNF (AAV-BDNF-IPE) were transplanted into the subretinal space of rats. We also injected small interfering RNAs (siRNAs) for TrkB, a BDNF receptor. The rats were exposed to continuous light to induce phototoxicity. We examined the expression of TrkB in the retina by Western blot and immunohistochemistry. RESULTS: Significant photoreceptor protection was detected when more than 1 x 10(7) capsids/ml AAV-BDNF was transplanted. An intravitreal injection of siRNAs showed that the photoreceptor protection by AAV-BDNF-IPE was reduced by injecting the siRNA of TrkB-T1, one of the TrkB isoforms. TrkB-T1 was slightly upregulated by Western blot, and one of the cells that upregulated TrkB-T1 was Muller cells by immunohistochemistry. CONCLUSION: We conclude that Muller cells are one of the cells responsible for the expression of TrkBs, and TrkB-T1 may play a role in the protection of photoreceptors against phototoxicity.

Topical doxycycline can induce expression of BDNF in transduced retinal pigment epithelial cells transplanted into the subretinal space.

PURPOSE: To determine whether topical doxycycline (DOX) induces the expression of brain-derived neurotrophic factor (BDNF) by BDNF-transduced retinal pigment epithelial (RPE) cells transplanted into the subretinal space of rats. METHODS: A rat RPE cell line that can express BDNF by exposure to DOX was created (Tet-BDNF-RPE). The expression of BDNF was examined by ELISA, Western blot analysis, and real-time PCR. The expression of BDNF was controlled by exposure to DOX in vitro. Tet-BDNF-RPE cells were transplanted into the subretinal space of rats, and the rats were exposed to constant light 1 day or 1 month after the transplantation. The rats were followed with or without topical DOX and examined electrophysiologically and histologically. RESULTS: The expression of BDNF was upregulated by exposure of Tet-BDNF-RPE cells to DOX in vitro. The optimal concentration for inducing BDNF expression was 0.5 to 1.0 microg/mL DOX. BDNF expression was also increased in vivo by topical DOX after subretinal transplantation of Tet-BDNF-RPE cells. Statistically significant protection of the electroretinogram amplitudes were found 3 days or 1 month after transplantation, and the outer nuclear layer was better preserved 7 days or 1 month after transplantation in the rats treated by 5 or 10 mg/mL/d topical DOX than rats treated by other conditions or sham-operation rats. CONCLUSIONS: The expression of BDNF can be significantly increased by topical DOX after Tet-BDNF-RPE subretinal transplantation. Better photoreceptor protection against phototoxicity was achieved by DOX eye drops after the cell transplantation.

Temporal and spatial differences in expression of TrkB isoforms in rat retina during constant light exposure.

Brain-derived neurotrophic factor (BDNF) has been reported to rescue neuroretinal cells under different toxic conditions. These cells include not only those expressing BDNF receptors (TrkB) but also those not expressing TrkB including photoreceptors. The purpose of this study was to determine the retinal sites at which BDNF and TrkB isoforms are expressed after different durations of continuous light exposure, and to compare these sites with those of TUNEL-positive cells in the same retina. Sprague-Dawley rats were exposed to continuous light for different durations. The expressions of BDNF and TrkB isoforms, TrkB-FL and TrkB-T1, were determined by Western blot analysis, real-time PCR, immunohistochemistry, and in situ hybridization before and after the light exposure. The number of TUNEL-positive cells reached a maximum at 48 to 72h after light exposure. The degree of up-regulation of the TrkB-T1 gene was significantly higher than that in normal control eyes at 24h by real-time PCR. Immunohistochemistry showed that TrkB-FL-positive cells were detected in all retinal layers except the outer nuclear layer (ONL), photoreceptor cells, and retinal pigment epithelium (RPE). The number of TrkB-FL-positive cells in the IPL was transiently decreased at 6h, and was increased on the processes of the Mueller cells in the ONL after 48h. TrkB-T1 was expressed in the INL, OPL, and RPE, and was up-regulated on the soma of Mueller cells after 24h. In situ hybridization showed that the expression of the TrkB-FL gene was up-regulated in the INL after 48h when the number of TUNEL-positive cells was at its peak. The TrkB-T1 gene was up-regulated before or just prior to the appearance of TUNEL-positive cells. These results suggest that BDNF transduces the signals using appropriate receptor isoforms that are expressed temporally and spatially differentially on Mueller cells during light-induced retinal degeneration.

Recombinant AAV-transduced iris pigment epithelial cell transplantation may transfer vector to native RPE but suppress systemic dissemination.

PURPOSE: To determine whether adenoassociated virus (AAV) vectors transduced into iris pigment epithelial (IPE) cells and transplanted into the subretinal space of rats will transfer the AAV genome to the host cells and whether the vectors are disseminated systemically. METHODS: Recombinant (r)AAV was transduced into rat IPE cells and transplanted into the subretinal space of rats. For the control, rAAVs alone were injected subretinally. The transplanted IPE cells were detected by LacZ staining. Immunohistochemistry, electron microscopy, electroretinography, and fluorescein-dextran angiography were performed. DNA was extracted from various organs and blood and examined for the AAV genome by polymerase chain reaction. RESULTS: No toxicity from rAAV transduction was observed in vitro. LacZ was expressed in the transplanted cells 1 and 2 weeks after transplantation. At 4 and 12 weeks, fewer transplanted cells were detected than at 1 week, and LacZ expression was occasionally detected at the level of host retinal pigment epithelial (RPE) cells. Expression was also detected in ciliary body epithelial cells. The electroretinograms and fluorescein-dextran angiography were only mildly altered. Significantly lower levels of AAV genome were detected in the organs and blood of rats receiving rAAV-IPE cell transplants than with direct intravenous injection of AAV vectors. CONCLUSIONS: AAV-mediated LacZ was expressed in the transplanted cells after subretinal transplantation, and the transplanted IPE cells may transfer the rAAV to host tissues, such as RPE cells, long after the transplantation. This method of gene delivery did not lead to systemic dissemination of the vectors.

Clinical course of macular edema in two cases of interferon-associated retinopathy observed by optical coherence tomography.

BACKGROUND: Interferon (IFN)-associated retinopathy is typically characterized by retinal hemorrhages and cotton wool spots at the posterior fundus, but visual function is usually maintained. With the use of optical coherence tomography (OCT), two patients with IFN-associated retinopathy who had developed macular edema and reduced visual acuity during the clinical course of IFN therapy were observed. CASES: A 37-year-old man with chronic hepatitis C and a 59-year-old man with chronic myeloid leukemia, both of whom had received IFN therapy, were referred to our outpatient clinic. The former patient had complained once that his visual acuity had decreased after the termination of IFN therapy, and the latter patient complained twice during IFN therapy that his visual acuity had decreased. OBSERVATIONS: In both patients, at the time of the visual disturbances, macular edema was clearly observed by OCT. Hypoalbuminemia and thrombocytopenia were observed at this time also. After the remission of the hypoalbuminemia and thrombocytopenia, the macular edema observed by OCT disappeared and visual acuity returned to normal. CONCLUSION: During and after IFN therapy, OCT is a useful examination technique for revealing macular edema in patients who have decreased vision. Ophthalmologists should be aware of the ocular side effects of IFN therapy and carefully monitor patients for the possible occurrence of hypoalbuminemia and thrombocytopenia. Jpn J Ophthalmol 2005;49:231-234 (c) Japanese Ophthalmological Society 2005.