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The ISIN code system is an international standard on many financial markets including the Euronext markets, and facilitates the identification of securities, particularly in cross-border transactions, since ISIN assigns each security a single number used in all countries. Retinal protein was measured according to Lowry et al. Panendothelial cell marker CD31 was used to determine vascular changes as described elsewhere.
Immunohistochemical staining was performed on formalin-fixed, parafin-embedded tissue by using the streptavidin-biotin method and 4—5 sections were obtained for evaluation from each eye. Tissue sections were subject to antigen retrieval by microwave treatment in 0. Slides were counterstained with Mayer's hematoxylin. The isotype controls were also done for immunohistochemistry. The immunstained specimens were evaluated by using light microscopes and pictures were taken with an digital camera attached to the microscope.
Blood biochemical parameters including complete blood count, glucose, HbA1c, blood urea nitrogen, creatinine, and albumin levels were measured. All parameters were calculated according to own SD. The post hoc multiple comparisons among the groups were done with Tukey's honestly significant differences test after the group variances were shown to be equal by Levene's test. At the time of killing, the mean weights of the rats were IP streptozotocin caused a significant rise in blood sugar.
There were no significant differences with regard to blood glucose and HbA1c levels between diabetic animals. Retinal nitrotyrosine levels between the groups. Briefly, aliquots of homogenates were centrifuged and the supernatants were analyzed by ELISA method for retinal nitrotyrosine. Error bars represent SD. Retinal lipid peroxidation levels in diabetic and control rats.
The retina lipid peroxidation total MDA were determined according to Ohkawa et al 11 with modifications. Briefly, aliquots of homogenates were centrifuged and the supernatants were analyzed by ELISA method. Immunohistochemistry revealed vascular engorgement and dilatation in diabetic animals Figure 5.
CD31 staining was more denser in diabetic retinas. The increased luminal space is also shown. With melatonin use, vascular derangement disappeared and vascular distortion was lost. Melatonin treatment resulted in a better retinal outcome with respect to the vascular structure. Immunoperoxidase staining of retinal sections of rats with anti-CD Middle left Diabetic rat retina diabetic group.
Large diameter vessels are also seen. Small pale pink-stained vessels are seen especially in the nerve fiber layer. Intensely stained and large size vessels are prominent in the nerve fiber layer of the retina. Streptozotocin is an antibiotic commonly used in pancreatic islet cell carcinoma. When uptaken, it causes rapid degeneration and necrosis of these cell, resulting in a condition similar to type 1 diabetes mellitus.
In our study, the retinal images showed vascular engorgement in the diabetic group. We also confirmed this morphological changes biochemically by measuring tissue VEGF levels. Abnormal vascular changes such as dilatation, microaneurysm, and tortuosity are prominent features of diabetic retinopathy. Preponderant CD staining with marked vascular dilatations were reported in diabetic patients elsewhere, mainly created by high levels of VEGF.
Although we did not measure retinal edema marker such as PAL-E antigen or fibrinogen, they are mostly to accompany such kind of vascular deformation. Melatonin supplementation was successful to reduce diabetic microvascular deterioration in this study. Subcutaneous implantation of melatonin therapy prevented retinal VEGF staining in diabetic rats.
How melatonin decreases VEGF may be a concern of future investigations. As showed elsewhere, 14 diabetic retinopathy was associated with oxidative and nitrosative stress in our experiment. Another study established the elevated nitrotyrosine levels in nerve fiber layer and endothelial cells of diabetic retina. Our experiment revealed a high nitrotyrosine level in diabetic group, confirming nitrative damage.
Malondialdehyde MDA is one of several low-molecular-weight end products formed via the decomposition of lipid peroxidation. Malondialdehyde and other thiobarbituric acid reactive substances TBARS condense with thiobarbituric acid to give a fluorescent red derivative that can be assayed spectrophotometrically. TBARS are widely known indicator of oxygen radicals attack.
In our experiment, diabetic group had higher MDA levels compared with control. Hyperglycemia oxidized retina and increased MDA content more than twofold. Studies showed that hyperglycemia causes oxidation via induction of several pathways, including protein kinase C, sorbitol, and phoshoinositide 3-kinase pathways. Free radicals impair cellular membranes and may disrupt endothelial cell integrity, creating both ischemic and edematous areas. Melatonin 5-methoxy-N-acetyltryptamine is a neurohormone mainly secreted by pineal gland.
Apart from pineal gland, it is also synthesized in the retina. Melatonin is secreted from photoreceptor cells in the retina and activates dark induced changes. Retinal diurnal rhytmicity are heavily related to the melatonin and light interaction.
During the night, retinal melatonin synthesis increases, and during the daytime vice versa occurs. Once secreted, it can easily diffuse throughout the retina because of its lipophilic and hydrophilic nature and exerts its functons through MT1 and MT2 receptors, both of which exist within the eye. Melatonin can be dissolved in dimethylsulfoxide or ethanol succesfully. Previous studies showed that melatonin membrane receptors were distributed throughout the retina 23 and melatonin administered systemically could reach to the eye, increasing retinal methoxyindole levels.
We found that melatonin decreased nitrotyrosine in retinal homogenates obtained from diabetic rats, whereas vehicle administration had no effect on glucose-induced damage. It is reported that melatonin suppresses the activity of NO synthase. Another contribution by melatonin is its free radical scavenging property. Therefore, it has been referred to as a terminal or suicidal antioxidant.
This aspect might be related to the diminished damage in this study. A posivite impact on the activity of antioxidant enzymes by melatonin is also possible inasmuch as superoxide dismutase, glutathione peroxidase, and calatalase levels are favored with melatonin therapy. Although we did not measure retinal antioxidant enzyme levels, our possible results may be linked to this situation.
Furthermore, VEGF, a cytoprotective factor, can be activated upon various insults including hyperglycemia and oxidative stress may be a key factor for stimulation. From another point of view, it has been reported that raised level of VEGF may be a precipitating event in diabetic retinopathy. A direct suppressive effect of melatonin on vascular endothelial growth factor is reported at Mueller cell level.
Melatonin's effect on angiomodulator agents may be a promisory approach in microvasculopathies. PEDF is a glycoprotein that was firstly identified from fetal human retinal pigment epithelial cell culture. We detected higher levels of PEDF in the diabetic group as compared with the control animals. Although several authors found that hyperglycemia depressed the level of PEDF in cell cultures, 30 controversy still exists.
Elevated levels of PEDF in diabetic rats were demonstrated by some authors in accordance with our results. With a chronic ongoing damage, PEDF depletion may be a possibility. PEDF may have been reduced because of diminished oxidative stress by melatonin. In addition, a direct inhibitory effect with melatonin on PEDF synthesis should be investigated as well. We detected that glucose was increased more than fivefold and HbA1c roughly twofold in diabetic animals.
Although melatonin is reported to have decreased blood sugar, 8 this is not the case in our study. Other researchers reported no effect of melatonin on blood glucose levels like us. Melatonin treatment did not cause any disorder on blood biochemical findings.
As a result, we were successful in reducing diabetic retinal damage with melatonin supplementation. Being an ideal antioxidant because of its unique features, melatonin may have the potential for therapeutic applications. Further analyses are required to elucidate the effect of melatonin on diabetic complications.
National Center for Biotechnology Information , U. Journal List Eye Lond v. Eye Lond. Published online Jun Author information Article notes Copyright and License information Disclaimer. E-mail: moc. Received Aug 28; Accepted Apr This article has been cited by other articles in PMC. Abstract Purpose To evaluate the role of melatonin, an antioxidant agent, in diabetic oxidative stress and vascular damage. Methods Diabetes was induced in 21 male Wistar rats by intraperitoneal IP administration of streptozotocin and then the rats were equally and randomly allocated to diabetic, melatonin, and vehicle groups.
Results Hyperglycemia increased retinal oxidation as measured through levels of nitrotyrosine and malondialdehyde. Conclusion Melatonin may show some advantage on diabetic vascular changes through decreasing oxidative stress and vessel-related cytokines. Introduction Diabetic retinopathy is a common disorder and a leading cause of blindness worldwide. Retinal immunohistochemistry for CD31 Panendothelial cell marker CD31 was used to determine vascular changes as described elsewhere.
Results At the time of killing, the mean weights of the rats were Open in a separate window.
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