Aims-To investigate the longevity and reproducibility of choroidal neovascularisation (CNV) induced by krypton laser photocoagulation in the rat. The presence of cell adhesion molecules (CAMs) and vascular endothelial growth factor (VEGF) during the development of CNV was also studied. Methods-67 pigmented rats underwent retinal photocoagulation by krypton laser. The eyes were examined by either single or serial fluorescein angiography at 3 days, 1, 2-3, 4-5, 7-8, and 12 weeks post photocoagulation. The expression of CAMs (ICAM-1, E-selectin, and CD44) and VEGF post photocoagulation was studied by immunohistochemistry. Results-CNV related fluorescein leakage appeared in 46.4% of 766 laser spots delivered to the 58 eyes that were tested at 2-3 weeks post treatment. The ratio of hyperfluorescent laser sites did not change significantly at 8 weeks post laser. The number of leaky spots was independent of the total number of lesions delivered to each eye (at 2-3 weeks post laser 10-15 spots/eye: 44% and 25-30 spots/eye: 49%; t=0.7673; p=0.3903). Nine eyes were followed by serial angiography between 2 and 12 weeks. The laser spots with fluorescein leakage at 2 weeks (51.5%) remained leaky at 12 weeks (51.5%). Histopathologically, macrophage accumulation peaked at 5 days and CNV was firstly observed at 1 week post photocoagulation. ICAM-1, E-selectin, CD44, and VEGF were maximally induced at 3-5 days post laser photocoagulation, and were localised to RPE, choroidal vascular endothelial, and inflammatory cells. VEGF was also detected in intravascular leucocytes at the sites of laser lesions. Conclusions-These studies demonstrated that krypton laser photocoagulation can be successfully used to produce lesions similar to those of human CNV. The response induced remained present for an extended period of time (12 weeks), thus oVering a potential model to screen candidate CNV inhibitory agents. In addition, it is proposed that the expression of ICAM-1, E-selectin, CD44, and VEGF before new vessel formation might be linked to the initiation of CNV.
BackgroundLeber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10–15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the Rpe65-/- knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function.MethodsrAAV.RPE65 was injected into the subretinal space of Rpe65-/- knockout mice and control mice. Histological and immunohistological analyses were performed to evaluate any rescue of photoreceptors and to determine longevity and pattern of transgene expression. Electron microscopy was used to examine ultrastructural changes, and electroretinography was used to measure changes in visual function following rAAV.RPE65 injection.ResultsrAAV-mediated RPE65 expression was detected for up to 18 months post injection. The delivery of rAAV.RPE65 to Rpe65-/- mouse retinas resulted in a transient improvement in the maximum b-wave amplitude under both scotopic and photopic conditions (76% and 59% increase above uninjected controls, respectively) but no changes were observed in a-wave amplitude. However, this increase in b-wave amplitude was not accompanied by any slow down in photoreceptor degeneration or apoptotic cell death. Delivery of rAAV.RPE65 also resulted in a decrease in retinyl ester lipid droplets and an increase in short wavelength cone opsin-positive cells, suggesting that the recovery of RPE65 expression has long-term benefits for retinal health.ConclusionThis work demonstrated the potential benefits of using the Rpe65-/- mice to study the effects and mechanism of rAAV.RPE65-mediated gene delivery into the retina. Although the functional recovery in this model was not as robust as in the dog model, these experiments provided important clues about the long-term physiological benefits of restoration of RPE65 expression in the retina.
The aim of this project was to determine if the subretinal delivery of a recombinant adenovirus encoding vascular endothelial growth factor (VEGF) was sufficient to induce changes resembling choroidal neovascularisation (CNV) in a rat model. A recombinant adenovirus was produced encoding vegf164 cDNA (Ad.RSV.VEGF). Transduction of cultured RPE cells confirmed VEGF expression and ensured the absence of Ad.RSV.VEGF-related toxicity. Following subretinal injection into rat eyes, fluorescein angiography indicated that the in vivo delivery of Ad.RSV.VEGF was associated with vascular leakage. Histological analysis demonstrated that changes resembling the early signs of CNV development were also present in the Ad.RSV.VEGF injected eyes. These results suggest that while a transient VEGF expression in the RPE layer is able to induce CNV-related changes, it may be insufficient for the development of a full neovascular membrane. This study demonstrates that virus-mediated gene delivery, in addition to its clinical applications, is a potentially efficient research tool for investigating gene expression-related physiological changes in vitro and in vivo.
There is accumulating evidence that an increased expression of vascular endothelial growth factor from retinal pigment epithelial cells may be important in choroidal neovascularization. In vivo studies have demonstrated that subretinal injection of recombinant adenovirus vectors produces long-term transgene expression specifically within retinal pigment epithelial cells. A recombinant adenovirus encoding of vascular endothelial growth factor (Ad.RSV.VEGF) was therefore produced and characterized in order to determine whether an upregulation of vascular endothelial growth factor expression is sufficient to induce choroidal neovascularization. Ad.RSV.VEGF was produced by homologous recombination and its identity confirmed by restriction enzyme analysis. Ad.RSV.VEGF was characterized in vitro by the transduction of cultured retinal pigment epithelial cells. The in vitro characterization confirmed vascular endothelial growth factor mRNA and protein expression from Ad.RSV.VEGF and demonstrated the biological activity of the vascular endothelial growth factor protein. A preliminary in vivo study suggested that the subretinal injection of Ad.RSV.VEGF induced vascular leakage.
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