Experimental verification of subthreshold laser therapy using conventional pattern scan laser.

PURPOSE: Leading-edge therapeutic laser technologies are not available at every medical facility; therefore, alternative approaches incorporating novel advances in digital and laser technology into more readily available conventional methods have generated significant research interest. Using a rabbit model, this study investigated whether the algorithm used in the Endpoint Management (EM) software system of the latest devices could enable subthreshold laser treatment in conventional retinal tissue laser therapy systems. MATERIALS AND METHODS: Two types of devices were used, the PASCAL Streamline 577 and the MC 500-Vixi™, and the laser method was classified into three categories: EM; single-shot using PASCAL with arbitrary energy settings (PSS-SDM); and MC500-VixiTM (VX-SDM), which were performed in eight eyes from four Dutch-Belted rabbits. In EM, 100 mW (100%) was set as a landmark, and the laser energy parameters were gradually decreased to 80%, 60%, 50%, 40%, 30%, 20%, and 10%, using a 2 × 3 square pattern. In PSS-SDM and VX-SDM, as control, the laser energy was gradually decreased to 100, 80, 60, 50, 40, 30, 20, and 10 mW. The laser settings were fixed at 200 µm, 20 ms, and a wavelength of 577 µm. To identify and compare the extent of tissue damage at each spot size, optical coherence tomography (OCT) and histological findings were used to construct a three-dimensional histopathology image using a confocal laser scanning fluorescence microscope. RESULTS: The spot size at 50% setting on EM was 7183 µm2; PSS-SDM required 50 mW (5503 µm2) to 60 mW (10279 µm2) and VX-SDM required 50 mW (7423 µm2) to create the approximate spot size. Furthermore, at 50 mW of PSS-SDM and VX-SDM, the extent of tissue damage in all three methods was generally in accord with the outer nuclear layer by OCT and inner nuclear layer by histopathological imaging. CONCLUSION: These findings suggest that it may be possible to perform subthreshold laser therapy using approximations from the EM algorithm.

Periocular injection of in situ hydrogels containing Leu-Ile, an inducer for neurotrophic factors, promotes retinal ganglion cell survival after optic nerve injury.

Intraocular administration of neurotrophic factors has been shown to delay irreversible degeneration of retinal ganglion cells (RGCs). It would be beneficial for the treatment of optic nerve (ON) injury if such neurotrophic factors could be delivered in a less-invasive manner. The dipeptide leucine-isoleucine (Leu-Ile) appears to induce the production of neurotrophic factors, including brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF), in the brain. We therefore administered Leu-Ile via periocular depot injection in rats and investigated the dipeptide's ability to induce BDNF and GDNF in the retina and to delay RGC loss in an ON injury model. Poloxamer-alginate hydrogels containing Leu-Ile were injected into the subconjunctival space of intact or ON-injured rats. BDNF and GDNF levels in the retina were determined by an enzyme immunoassay. Survival of RGCs was assessed in retinal flatmounts. Activation of extracellular signal-regulated kinases (ERK) and cAMP response element binding protein (CREB) in the retina was examined by Western blotting. At 2 h after injection of fluorescein isothiocyanate-conjugated Leu-Ile, the fluorescence intensities in the retina were 4.3-fold higher than those in the saline control. Treatment with Leu-Ile significantly increased the retinal levels of BDNF at 6 h and GDNF at 6-72 h after injection. Treatment with Leu-Ile significantly increased RGC survival to 14 days after ON injury and enhanced the activation of ERK at 72 h and CREB at 48 h after injection in the ON-injured retina. These results suggest that periocular delivery of Leu-Ile induces BDNF and GDNF production in the retina, which may eventually enhance RGC survival after ON injury.

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.