DOI 10.1007/s10517-014-2723-6
206
Bulletin of Experimental Biology and Medicine, Vol. 158, No. 2, December, 2014 GENERAL PATHOLOGY AND PATHOPHYSIOLOGY
Superoxide Dismutase and Catalase Activities in the Retina during Experimental Diabetes and Electric Stimulation of the Paleocerebellar Cortex N. V. Kresyun and L. S. Godlevskii Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 158, No. 8, pp. 168-170, August, 2014 Original article submitted April 3, 2013 In 1.5 months after modeling of streptozotocin-induced diabetes mellitus, superoxide dismutase and catalase activities decreased by 40.7 and 32.0%, respectively, in comparison with the corresponding values in Wistar rats without diabetes. Electric stimulations (100 Hz, 0.25 msec, 50-100 μA, 2.5 sec) of the paleocerebellar cortex (V-VII lobules) were conducted 3 times a day over 1 month. These stimulations prevented the decrease in antioxidant enzyme activity in rats with experimental diabetes. Key Words: streptozotocin; diabetic retinopathy; superoxide dismutase; catalase; electric stimulation of the cerebellum
Mechanisms of free radical generation play an important role in the pathogenesis of diabetic retinopathy, while treatment with antioxidants is a pathogenically substantiated therapy in this pathology [1,7]. It is shown that electric stimulation (ES) of the cerebellar fastigial nucleus produces a neuroprotective effect during ischemia-induced damages of rat retina [6], which is probably mediated by the antioxidant mechanism [2]. Here we studied activities of antioxidant enzymes superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6) in rats during streptozotocin-induced diabetes and the dynamics of their changes during ES of the paleocerebellar cortex. We also studied the dynamics of changes in body weight and blood glucose level in rats during ES of the cerebellum.
MATERIALS AND METHODS Chronic experiments were performed on male Wistar rats weighting 170-240 g kept under standard vivarium conditions. The studied were conducted in accordance to the Rules of GLP and Bioethics Commission of the Odessa National Medical University, Ukraine. Address for correspondence: [email protected]
Odessa National Medical University (protocol No. 84, 10 October 2008). Bipolar nichrome electrodes were implanted into the lobuli V-VII of the paleocerebellar cortex under ketamine (100 mg/kg, intraperitoneally) anesthesia; the distance between the electrodes 0.25-0.30 mm. The electrodes were fixed to the scull with quick setting dental plastic paste Noracryl. The animals were observed starting from days 7-10 after surgery. Experimental diabetes mellitus was modeled by intraperitoneal injection of 50.0 mg/kg streptozotocin dissolved in sodium citrate buffer (pH 4.5) before meal (Sigma-Aldrich) [1,7]. In 1 and 2 weeks, venous blood was taken from the caudal vein for glucose measurement; the animals with glucose concentration >300 mg/liter were used in further experiments [7]. Glucose level was estimated at 09.00; animals were allowed access to food during the night. The rats received insulin (0-2 ME subcutaneously, 2-5 times a week) throughout the experiment [7]. The animals were randomized into 6 groups: 1) sham-operated rats (control, n=11); 2) rats subjected to daily single ES of the paleocerebellar cortex (n=7); 3) rats subjected to daily ES of the paleocerebellar cortex three times a day (n=7); 4) non-treated rats with diabetes mellitus (n=11); 5) rats with diabetes sub-
0007-4888/14/15820206 © 2014 Springer Science+Business Media New York
N. V. Kresyun and L. S. Godlevskii
jected to daily single ES of the paleocerebellar cortex (n=10); 6) rats with diabetes subjected to daily ES of the paleocerebellar cortex three times a day (n=10). On day 14 after streptozotocin injection and over the subsequent 4 weeks, ES of the paleocerebellar cortex was performed (rectangular pulses, 50-100 μA, 100 Hz, 2.5 sec). The target structures and regimens of ES were chosen based on previous data on strengthening of the antioxidant potential of the brain tissue after these exposures [2]. Two regimens of ES were used: daily single exposure (9 am) and daily triple exposures (at 09.00, 14.00, and 19.00). The animals were weighted every 3 days. The animals were decapitated at the end of experiments; tissue samples were frozen and stored in liquid nitrogen. Isolated retina samples were washed with PBS for removal of blood and homogenized in 0.1 M of phosphate buffer (pH 7.0; 1:10 w/v). Homogenized samples were centrifuged at 13,000g and 4ºC for 15 min. SOD activity was measured [4] and expressed as percent of blockage of NBT reduction to nitroblue formazan [1]. CAT activity (H2O2/min/mg of protein, nM) was measured as described elsewhere [3]. Protein concentration was measured the method of Lowry. Experimental data were analyzed by ANOVA and Kruskal–Wallis test. The distribution of the experimental data complied with normal law.
207 It should be emphasized that at the end of experiments, the body weight of control group animals increased by 36.2% in comparison with the initial value (297.0±18.3 g). Body weight of rats exposed to daily single and triple ES increased by 29.3 and 27.0%, respectively, in comparison with this parameter before ES (p
206
Bulletin of Experimental Biology and Medicine, Vol. 158, No. 2, December, 2014 GENERAL PATHOLOGY AND PATHOPHYSIOLOGY
Superoxide Dismutase and Catalase Activities in the Retina during Experimental Diabetes and Electric Stimulation of the Paleocerebellar Cortex N. V. Kresyun and L. S. Godlevskii Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 158, No. 8, pp. 168-170, August, 2014 Original article submitted April 3, 2013 In 1.5 months after modeling of streptozotocin-induced diabetes mellitus, superoxide dismutase and catalase activities decreased by 40.7 and 32.0%, respectively, in comparison with the corresponding values in Wistar rats without diabetes. Electric stimulations (100 Hz, 0.25 msec, 50-100 μA, 2.5 sec) of the paleocerebellar cortex (V-VII lobules) were conducted 3 times a day over 1 month. These stimulations prevented the decrease in antioxidant enzyme activity in rats with experimental diabetes. Key Words: streptozotocin; diabetic retinopathy; superoxide dismutase; catalase; electric stimulation of the cerebellum
Mechanisms of free radical generation play an important role in the pathogenesis of diabetic retinopathy, while treatment with antioxidants is a pathogenically substantiated therapy in this pathology [1,7]. It is shown that electric stimulation (ES) of the cerebellar fastigial nucleus produces a neuroprotective effect during ischemia-induced damages of rat retina [6], which is probably mediated by the antioxidant mechanism [2]. Here we studied activities of antioxidant enzymes superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6) in rats during streptozotocin-induced diabetes and the dynamics of their changes during ES of the paleocerebellar cortex. We also studied the dynamics of changes in body weight and blood glucose level in rats during ES of the cerebellum.
MATERIALS AND METHODS Chronic experiments were performed on male Wistar rats weighting 170-240 g kept under standard vivarium conditions. The studied were conducted in accordance to the Rules of GLP and Bioethics Commission of the Odessa National Medical University, Ukraine. Address for correspondence: [email protected]
Odessa National Medical University (protocol No. 84, 10 October 2008). Bipolar nichrome electrodes were implanted into the lobuli V-VII of the paleocerebellar cortex under ketamine (100 mg/kg, intraperitoneally) anesthesia; the distance between the electrodes 0.25-0.30 mm. The electrodes were fixed to the scull with quick setting dental plastic paste Noracryl. The animals were observed starting from days 7-10 after surgery. Experimental diabetes mellitus was modeled by intraperitoneal injection of 50.0 mg/kg streptozotocin dissolved in sodium citrate buffer (pH 4.5) before meal (Sigma-Aldrich) [1,7]. In 1 and 2 weeks, venous blood was taken from the caudal vein for glucose measurement; the animals with glucose concentration >300 mg/liter were used in further experiments [7]. Glucose level was estimated at 09.00; animals were allowed access to food during the night. The rats received insulin (0-2 ME subcutaneously, 2-5 times a week) throughout the experiment [7]. The animals were randomized into 6 groups: 1) sham-operated rats (control, n=11); 2) rats subjected to daily single ES of the paleocerebellar cortex (n=7); 3) rats subjected to daily ES of the paleocerebellar cortex three times a day (n=7); 4) non-treated rats with diabetes mellitus (n=11); 5) rats with diabetes sub-
0007-4888/14/15820206 © 2014 Springer Science+Business Media New York
N. V. Kresyun and L. S. Godlevskii
jected to daily single ES of the paleocerebellar cortex (n=10); 6) rats with diabetes subjected to daily ES of the paleocerebellar cortex three times a day (n=10). On day 14 after streptozotocin injection and over the subsequent 4 weeks, ES of the paleocerebellar cortex was performed (rectangular pulses, 50-100 μA, 100 Hz, 2.5 sec). The target structures and regimens of ES were chosen based on previous data on strengthening of the antioxidant potential of the brain tissue after these exposures [2]. Two regimens of ES were used: daily single exposure (9 am) and daily triple exposures (at 09.00, 14.00, and 19.00). The animals were weighted every 3 days. The animals were decapitated at the end of experiments; tissue samples were frozen and stored in liquid nitrogen. Isolated retina samples were washed with PBS for removal of blood and homogenized in 0.1 M of phosphate buffer (pH 7.0; 1:10 w/v). Homogenized samples were centrifuged at 13,000g and 4ºC for 15 min. SOD activity was measured [4] and expressed as percent of blockage of NBT reduction to nitroblue formazan [1]. CAT activity (H2O2/min/mg of protein, nM) was measured as described elsewhere [3]. Protein concentration was measured the method of Lowry. Experimental data were analyzed by ANOVA and Kruskal–Wallis test. The distribution of the experimental data complied with normal law.
207 It should be emphasized that at the end of experiments, the body weight of control group animals increased by 36.2% in comparison with the initial value (297.0±18.3 g). Body weight of rats exposed to daily single and triple ES increased by 29.3 and 27.0%, respectively, in comparison with this parameter before ES (p
