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After three washes in TBST, membranes were incubated with peroxidase-conjugated secondary Abs (final dilution, 1:3000) in TBS for 1?h and subsequently washed as described earlier. Detection was performed by chemiluminescence using ECL-kit (Enhanced Chemiluminescence; Amersham Life Science, Braunschweig, Germany) and subsequent Multiple Gel-DOC system (Fujifilm, Tokyo, Japan). The following primary antibodies were used: anti-VEGF, anti-VEGF receptor, anti-erythropoietin, anti-erythropoietin receptor, anti-angiopoietin 1, anti-angiopoietin 2 antibodies as described earlier. Blood Fludarabine sugar level is elevated 2?days after injection of STZ into the abdominal cavity. After 1?week, blood sugar level was checked for diabetes induction, and the normal group had an average level of 138.1?��?12.6?mg/dL, the diabetes group had an average level of 557.8?��?51.4?mg/dL, and the lipoic acid-treated group had an average level of 484.2?��?61.4?mg/dL, which was over 300?mg/dL, so diabetes induction was confirmed. In the diabetic rat, blood glucose levels were not significantly altered by lipoic acid (P?>?0.05, diabetic vs. diabetic?+?lipoic acid). On weekly monitored glucose level, none of the animals showed a restoration of blood glucose level. Two months after the induction of diabetes, microscopic examination of H&E stained retinas showed that all of the retinal layers were normal, with no morphological differences among the groups (Fig.?1). The retinal thickness and each layer thickness were qualitatively similar among the groups. DHE is converted to ethidium bromide in the presence of superoxide. Ethidium bromide intercalates into DNA and has been used as an indirect measure of superoxide generation. Retinas from diabetic rats showed more fluorescence than samples from normal control. This increase was down-regulated in lipoic acid-treated rats (Fig.?2). It is known that NADPH oxidase contributes to total superoxide production in retinas of diabetic animals. Thus, we visualized NADPH oxidase with immunofluorescence staining. Significantly more fluorescence was present in the retina from lipoic acid-treated group than in the retina from normal group, but decreased immunofluorescence was seen in the retina from the diabetic group (Fig.?3). Four eyeballs from four rats in each three groups were chosen, respectively, and the retinas were detached and used for the following RT-PCR experiments. RT-PCR showed that VEGF and VEGF receptor mRNA levels were higher in retinas from the diabetic group than from the normal group, and VEGF mRNA levels were lower in the retinas from the lipoic acid-treated group than from the diabetic group (P?
