Search

Purpose To investigate the effect of thermal stimulation of the retina (TS-R) on Bruch's membrane (BrM) thickness in age-related macular degeneration (AMD) mouse models as a novel concept for the prophylaxis and treatment of dry AMD. Methods Two knockout AMD mouse models, B6.129P2-Apoetm1Unc/J (ApoE-/-) and B6.129X1-Nfe2I2tm1Ywk/J (NRF2-/-), were chosen. One randomized eye of each mouse in four different groups (two of different age, two of different genotype) of five mice was treated by TS-R (532 nm, 10-ms duration, 50-μm spot size), the fellow eye served as control. Laser power was titrated to barely visible laser burns, then reduced by 70% to guarantee for thermal elevation without damage to the neuroretina, then applied uniformly to the murine retina. Fundus, optical coherence tomography (OCT), and fluorescein angiography (FLA) images were obtained at the day of treatment and 1 month after treatment. Eyes were enucleated thereafter to analyze BrM thickness by transmission electron microscopy (TEM) in a standardized blinded manner. Results Fundus images revealed that all ApoE-/- and NRF2-/- mice had AMD associated retinal alterations. BrM thickness was increased in untreated controls of both mouse models. Subvisible TS-R laser spots were not detectable by fundus imaging, OCT, or FLA 2 hours or 1 month after laser treatment. TEM revealed a significant reduction of BrM thickness in laser-treated eyes of all four groups compared to their fellow control eyes. Conclusions TS-R reduces BrM thickness in AMD mouse models ApoE-/- and NRF2-/- without damage to the neuroretina. It may become a prophylactic or even therapeutic treatment option for dry AMD. Translational relevance TS-R may become a prophylactic or even therapeutic treatment option for dry AMD.

PURPOSE. To investigate the influence of VEGF antagonists on the barrier function of the retinal pigment epithelium and underlying mechanisms. METHODS. Porcine RPE cells were cultured on six-well membrane inserts. The cells were exposed to bevacizumab (62.5 g/mL) or ranibizumab (25 g/mL) for 24 hours (short term) or 9 days (long term). Transepithelial flux of FITC-dextran and intracellular levels of reduced glutathione (GSH) at normal and low-glucose conditions were investigated at different points in time. The influence of the addition of triamcinolone acetonide (TA) was investigated. The effect of GSH depletion on RPE permeability was examined using L-buthionine sulfoximine (BSO), a -glutamylcysteine synthethase inhibitor. RESULTS. After short-term exposure, VEGF antagonists increased the transepithelial flux of FITC-dextran significantly on day 2. Bevacizumab, but not ranibizumab, increased permeability up to 9 days. Under long-term exposure, both drugs enhanced permeability for 7 days; bevacizumab had the stronger effect. The addition of TA inhibited this increase. At the ninth day of short- and long-term exposure, bevacizumab-exposed cells, but not ranibizumab-exposed cells, exhibited a significantly lower GSH level. In the low-glucose condition, both drugs accelerated the decrease of intracellular GSH for the first 48 hours. GSH depletion increased the permeability of retinal pigment epithelium. TA had no effect on BSO-induced GSH depletion. CONCLUSIONS. The results suggest that bevacizumab and ranibizumab may decrease RPE barrier function, with bevacizumab exhibiting a prolonged and more profound effect. Combination with TA is thought to be beneficial because of its protective effect on stabilizing RPE junctional integrity. (Invest Ophthalmol Vis Sci. 2010;51:4848‐4855) DOI:10.1167/iovs.09-4699

PURPOSE. To investigate the effect of selective retina therapy (SRT) on the release of AMD-relevant cell mediators, such as matrix metalloproteinases (MMPs), VEGF, and pigment epithelium derived factor (PEDF) using different laser spot sizes and densities. METHODS. Porcine RPE-choroid explants were treated with a pulsed 532 nm Nd:YAG laser using (1) large spot sizes, (2) small spot sizes with a high-density (hd) treatment, and (3) small spot sizes with a low-density (1d) treatment. Explains were cultivated in modified Ussing chambers. RPE regeneration and RPE cell death were investigated by calcein-AM staining and immunofluorescence. The MMP release was examined via zymography and immunofluorescence. VEGF and PEDF secretion was analyzed by ELISA. RESULTS. During pigment epithelium regeneration (PER), mitosis and RPE cell migration were observed. Four days after SRT (large spot size) the content of active MMP2 increased significantly (P < 0.01). Hd treatment with small spot sizes resulted also in an increase of active MMP2 (P < 0.05). In immunofluorescence explants showed a localized expression of MMP2 within the healing lesions after irradiation. The PEDF level increased significantly (P = 0.01) after SRT with large spot sizes. VEGF secretion decreased significantly (P < 0.05) following SRT with large spot sizes and with hd treatment of small spot sizes. CONCLUSIONS. SRT induces a cytokine profile, which may improve the flux across Brach's membrane, slows down progression of early AMD by RPE regeneration, and inhibits the formation of choroidal neovascularization. The cytokine release depends on the size and density of applied laser spots.

PURPOSE In several retinal complications, such as age-dependent macular degeneration (AMD), oxidative stress is increased and cytokine level is elevated. These are shown to alter the activation and expression of matrix metalloproteinase (MMP) both in human primary and immortalized retinal pigment epithelial (RPE) cells. However, the effects on human embryonic stem cell (hESC)-derived RPE cells remain to be elucidated. METHODS The mature hESC-RPE cells were exposed to inflammatory cytokines (IFN-γ or TNF-α) for 24 hours or oxidative stress (H2O2) for 1 hour. Effects on barrier properties were analyzed with transepithelial electrical resistance (TEER), the expression of MMP-1, MMP-2, MMP-3, MMP-9, collagen I, and collagen IV genes with quantitative RT-PCR, and the expression of MMP-1 and MMP-3 proteins with Western blot or ELISA, respectively. Also, activation and secretion of MMP-2 and -9 proteins were analyzed with zymography. RESULTS In normal state, mature hESC-RPE cells expressed MMP-1, -2, -3, and -9 genes in low levels, respectively. Tumor necrosis factor-α increased MMP-1 and -2 gene expression, and H2O2 increased MMP-3 and -9 gene expression. Zymography revealed IFN-γ- and TNF-α-induced secretion of MMP-2 and high-molecular-weight species of MMP (HMW MMP), but H2O2 decreased their secretion. Furthermore, TNF-α and H2O2 significantly decreased barrier properties. CONCLUSIONS Here, cytokines induced the MMP-1 and -2 gene and protein expression. Also, H2O2 induced MMP-3 and -9 gene expression, but not their protein secretion. These data propose that under oxidative stress and cytokine stimuli, mature hESC-RPE cells resemble their native counterpart in the human eye in regard to MMP secretion and expression and could be used to model retinal disorders involving alterations in MMP activity such as AMD, diabetic retinopathy, or proliferative vitreoretinopathy in vitro.