Increased nitric oxide level in diabetic rats from vitamin C supplementation: an in vivo detection using diaminofluorescein

Abstract

Background: Vitamin C is of clinical benefit for improvement of impaired endothelial functions in diabetes, but its direct effect on nitric oxide (NO) available in endothelial cells during diabetes as yet remains unclear. Objective: To examine the correlation between level of bioavailable NO and arteriolar vasodilation under the direct effect of vitamin C, using a novel NO-sensitive fluorescent indicator in diabetic rats. Methods: Male Spraque-Dawley rats (200-250g body weight) were used for the experiment. The rats were divided into control (CON) and diabetic rats (DM). The diabetes was induced in rats by injection of streptozotocin (STZ) (50 mg/kg body weight). The direct effects of vitamin C administration on NO bioavailability were examined using a buffer solution containing 4,5-diaminofluorescein-diacetate (DAF-2DA) and acetylcholine (Ach) with or without vitamin C in Krebs-Ringer solution. Twenty minutes after the buffer solution was superfused on the rat mesentery, the changes in DAF-2T fluorescence intensities were examined along the arteriolar walls. By visualizing the microcirculation using FITC-dextran, Ach-induced vasodilation of the arterioles (15 to 35 μm in diameter) was measured using Image Pro-Plus Software. The analysis was made after pre-constriction with norepinephrine, and after topical application of vitamin C (ascorbic acid) on the mesentery and following topical application of Ach. Results: DAF-2DF was a NO-sensitive fluorescent indicator that did not make the microvascular walls visible without endothelial activation by Ach. The administration of vitamin C increased Ach-evoked vasodilation in CON and DM groups. Twenty minutes after vitamin C administration, the DAF-2T fluorescence intensities increased significantly in both groups. Conclusion: Vitamin C improved diabetes-induced endothelial dysfunction by enhancing NO bioavailability. The level of bioavailable NO and endothelium-dependent vasodilation were highly correlated.