| Objective:To investigate the localization and expression of VEGF and nNOS in the retinas of the early diabetic rats, approach their effect on the pathogenesis of diabetic retinopathy, thus provide a new theoretical basis for the clinical prevention and cure of the early diabetic retinopathy.Methods:Forty-six male SD rats were randomly divided into the study group and the normal control group (NC). Rats of the study group were injected with large-dose (65mg/kg) of STZ by intraperitoneal injection to induce the type 1 diabetic rats model. Meanwhile the control group rats were given equal volume lemonacid buffer liquid. After model was succeeded, the diabetic rats were divided into the third week (DR3), the sixth week (DR6), the ninth week (DR9) diabetic group on average. Rats were anesthetized, their eyeballs were fixed, embedded in paraffin and sliced. Retina sections were stained by HE and the change of retina tissue structure were observed by the light microscope. Immunohistochemistry, computer image analysis and western blotting were used to investigate the distribution and expression of VEGF and nNOS protein on the retina of diabetic rats. Results:HE staining showed that there is no significant change in retinal structure of DR3 group. The pathological changes aggravate with the prolonged course of disease, including the hyalinization, thickening of retinal capillary wall and luminal stenosis. Immunohistochemistry showed that in NC group, VEGF-positive cells mainly distributed in the retinal ganglion cell layer, inner plexiform layer. With the progression of diabetes, the expression of VEGF extended to the retinal inner nuclear layer, outer plexiform layer and photoreceptor cell layer, VEGF protein levels increased gradually, there was significant difference in the positive expression of VEGF among DR3, DR6 and DR9 groups (P<0.05) nNOS mainly distributed in the retinal ganglion cell and the bipolar cells of inner plexiform layer. There was no difference in the positive expression of nNOS between NC group and DR3 group (P>0.05), but nNOS-positive cells were singnificantly higher in DR6 and DR9 groups than in NC group (P<0.05). There was significant difference in the positive expression of nNOS among DR3, DR6 and DR9 groups. The result of the western blotting showed that VEGF protein band was very weak in NC group. Moreover, the relative optical density of VEGF protein band were increased gradually in the DR3, DR6 and DR9 groups. The level of VEGF protein in diabetic retina were significantly higher than those in NC group (P<0.05). The nNOS protein bands were seen in both NC group and diabetic groups. There was no difference in the relative optical density of nNOS protein band between NC group and DR3 group (P>0.05), but the relative optical density of nNOS protein band were gradually higher in DR6 and DR9 groups than in NC group (P<0.05). There was significant difference in the expression of nNOS protein among DR3, DR6 and DR9 groups. Linear correlation analysis showed that there was a obvious positive correlation between the expression of VEGF and nNOS (P<0.01).Conclusions:1. The diabetic rat retinopathy model induced successfully by STZ may represent background stage of diabetic retinopathy in human.2. In the early diabetic retinopathy, the expression of VEGF and nNOS protein increased gradually with the progression of diabetes, we guessed it may be related to the disorder retinal level 3 neurons transmission function and nerve cell in experimental rats with diabetes.3. There was a significant positive correlation between the expression of VEGF and nNOS in the onset and the development of the early diabetic retinopathy.
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