The Role Of Retinal Müller Cell-Derived VEGF In Diabetes-Induced Retinal Inflammation And Vascular Lesions

Objective To generate retinal Muller cell-derived VEGF knockout mice and provide the basis for investigating the role of retinal Muller cell-derived VEGF under normal and pathological conditions.Methods Muller cell-derived VEGF knockout mice were generated by mating the Muller cell-specific Cre mice with floxed VEGF mice. Doxycycline was administrated through the drinking water of pregnant mothers at a concentration of 0.5mg/ml in 5% sucrose from E15 to P1. Genotyping of tail DNA was performed with PCR analysis for Cre and VEGF at P15. Mice were divided into KO group and WT group according to PCR results. At P21, Western blot analysis and immunofluorescence were used to detect the expression and location of VEGF in retinas, FITC-dextran angiography was performed to observe the morphology of retinal vessels.8 months after birth, histological analysis was performed to detect retinal morphology.Results At P21, immunoblotting demonstrated that VEGF expression was significantly reduced (43.2%) in KO mice compared with that of WT mice. Immunofluorescence showed that the decreased VEGF was from inner nuclear layer and the signal in other layers was similar between KO and WT mice. There was no significant difference in the density and pattern of the retinal vessels between KO and WT mice.8 months after birth, no significant difference in thickness of total retina, ONL and INL was observed between KO and WT mice.Conclusion Retinal Muller cell-derived VEGF knockout mice were generated sucessfully, and it is an ideal model for investigating the role of Muller cell-derived VEGF under normal and pathological conditions. Objective To investigate the role of Muller cell-derived VEGF in diabetes-induced inflammation.Methods KO and WT mice were randomly divided into diabetic group and non-diabetic group. Diabetes was induced by intraperitoneal injection of streptozotocin for 5 days. Body weight and blood glucose level were measured every two weeks. Mice with blood glucose levels greater than 300mg/dL were deemed diabetic. Two months after diabetes, Western blot analysis was performed to detect retinal expression of VEGF, HIF-1α, ICAM-1, TNF-αand phosphorylated NF-κB (p-p65). Immunofluorescence was performed to detect the expression and location of retinal VEGF. Leukostasis was determined by counting the number of fluorescently labeled leukocytes inside retinal vasculature.Results There was a significant elevation of blood glucose level and body weight loss in diabetic groups, compared with non-diabetic controls (p<0.001),2 months after induction of diabetes. However, there was no significant change in blood glucose levels or body weights between KO and WT mice under normal or diabetic conditions. Under diabetic stress, disruption of Muller cell-derived VEGF resulted in a 51.4% reduction in VEGF overexpression. This result was supported by immunohistochemical analysis. No signifcant difference was observed in HIF-la levels between diabetic KO and WT mice. Under diabetes, compared with WT mice, KO mice demonstrated a 74.5% reduction of adherent leukocytes in retinal vessels, and showed 62.3%,52.9% and 48% reduction of ICAM-1, TNF-αand p-p65 respectively in retinas.Conclusion Our study demonstrates that disruption of Muller cell-derived VEGF significantly inhibits diabetes-induced retinal inflammation. Objective To investigate the role of Miiller cell-derived VEGF in diabetes-induced vascular lesions.Methods Diabetes was induced by intraperitoneal injection of streptozotocin as described in chapter 2. Six months after diabetes, Western blot analysis and FITC-BSA angiography were performed to detect the extravascular albumin content in the retina and vitreous. The expression and location of retinal tight junction proteins, occludin and ZO-1 were detected by Western blot analysis and immunolocalization assay. Trypsin digestion assay was performed to measure the number of acellular capillaries and pericytes.Results Six months after diabetes, the difference in the body weights and blood glucose levels among each groups was similar to that in two months after diabetes. Compared with diabetic WT mice, there was a 58.9% reduction of albumin leakage in diabetic KO mice, and KO mice demonstrated 59.7% and 130.3% increases of occludin and ZO-1 in diabetic retinas. ZO-1 immunoreactivity was intense in the main arteriols and capillaries of the outer retina in normal mice. ZO-1 immunoreactivity was elevated in retinas from diabetic KO mice compared to diabetic WT mice. Compared with WT mice, KO mice demonstrated a 62.5% increase of pericytes and a 43.4% reduction of acellular capillaries under diabetic condition.Conclusion Our study demonstrates that disruption of Muller cell-derived VEGF significantly inhibits diabetes-induced retinal vascular leakage and capillary degeneration.