Elsevier

Life Sciences

Volume 80, Issue 13, 6 March 2007, Pages 1236-1244
Life Sciences

Effects of resveratrol on nerve functions, oxidative stress and DNA fragmentation in experimental diabetic neuropathy

https://doi.org/10.1016/j.lfs.2006.12.036Get rights and content

Abstract

Oxidative stress has been implicated in pathophysiology of diabetic neuropathy. All the pathways responsible for development of diabetic neuropathy are linked to oxidative stress in one way or the other. In the present study, we have targeted oxidative stress in diabetic neuropathy using resveratrol, a potent antioxidant. Eight weeks streptozotocin-diabetic rats developed neuropathy which was evident from significant reduction in motor nerve conduction velocity (MNCV), nerve blood flow (NBF) and increased thermal hyperalgesia. The 2-week treatment with resveratrol (10 and 20 mg/kg, i.p.) started 6 weeks after diabetes induction significantly ameliorated the alterations in MNCV, NBF, and hyperalgesia. Resveratrol also attenuated enhanced levels of malondialdehyde (MDA), peroxynitrite and produced increase in catalase levels in diabetic rats. There was marked reduction in DNA fragmentation observed after resveratrol treatment in diabetic rats as evident from decrease in Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positive cells in sciatic nerve sections. Results of the present study suggest the potential of resveratrol in treatment of diabetic neuropathy and its protective effect may be mediated through reduction in oxidative stress and DNA fragmentation.

Introduction

Diabetes mellitus is one of the serious problems in developing as well as developed countries. Between 1995 and 2025 the number of adult population affected by diabetes is projected to grow by 170%. India is projected to have the largest number (57 million) of people with diabetes by 2025 (www.who.org). Chronic hyperglycemia with diminished insulin production or function is the primary initiating factor responsible for this multifactorial cascade. Several clinical and preclinical evidences suggest that oxidative stress plays a central role in the development of diabetic complications including retinopathy, nephropathy and neuropathy. Diabetic neuropathy is one of the most common complications affecting more than 50–60% of diabetic patients and it is a common cause of non-traumatic amputation and autonomic failure.

The pathophysiology of diabetic neuropathy involves a very complex cascade of several interrelated mechanisms. Elevated blood glucose induced auto oxidative glycosylation, formation of glycation product, protein kinase-C activation; increased polyol pathway activity and hexosamine flux are the key components of the cascade. These pathways are responsible for generation of reactive oxygen species (superoxide, hydroxyl radical, hydrogen peroxide) and peroxynitrite, which ultimately contributes to the oxidative stress (Ahmed, 2005, Brownlee, 2001, Kaul and Ramarao, 2001, Sima, 2003, Wada and Yagihashi, 2005). Oxidative stress diminishes the activity of endogenous antioxidant enzyme defense system (SOD, Catalase and Glutathione peroxidase), which play a significant protective role. Oxidative stress exerts its devastating effects by directly damaging cellular proteins, lipids, and DNA, or indirectly, by affecting normal cellular signaling and gene regulation (Vincent et al., 2004). Vascular impairments due to long term oxidative stress results in endoneurial hypoxia and loss of neurotrophic support which ultimately leads to reduced nerve conduction velocity and impaired neural function (Stevens et al., 2000).

Several animal studies have demonstrated that antioxidants can prevent or reverse hyperglycemia induced nerve dysfunction. Several antioxidants (α-lipoic acid, taurine, M40403, β-carotene, vitamin C, vitamin E, and U83836E) have demonstrated amelioration in the nerve function deficit in experimental diabetic neuropathy (Sayyed et al., 2006, Sima, 2006, Vincent et al., 2004, Vinik, 2004). Resveratrol (3, 5, 4′-trihydroxystilbene), a naturally occurring polyphenol found in grapes and red wine, has been reported to have excellent antioxidant activity. It not only prevents free radical formation but also attenuates their toxicity by inhibiting the lipid peroxidation (Leiro et al., 2005). Protective effects of resveratrol has been reported in various disease conditions (Anekonda, in press, Sharma and Gupta, 2002, Zamin et al., 2006). However effects of resveratrol have not been studied on nerve functions, oxidative stress and DNA fragmentation in experimental diabetic neuropathy. Therefore in the present study we have investigated the effect of resveratrol on above parameters in diabetic neuropathic rats.

Section snippets

Animals

Healthy male Sprague–Dawley rats (250–270 g) were obtained from the Central Animal Facility, National Institute of Pharmaceutical Education and Research (NIPER). Animals were provided with standard diet and water ad libitum. They were housed in plastic cages (three in each) at a temperature of 24 ± 1 °C and humidity of 55 ± 5%, with 12-h light dark cycle. All animals were acclimatized for a minimum period of one-week prior to the beginning of the study. The experimental protocol was approved by the

Estimation of plasma glucose level

For glucose estimation 100 μl of blood was collected from the tail vein of each rat in a micro-centrifuge tubes containing 10 μl of heparin (1000 units/ml), mixed gently and this was centrifuged at 5000 rpm for 5 min and plasma was separated out. Then the glucose estimation was done using GOD-POD kit.

Motor nerve conduction velocity

Motor nerve conduction velocity (MNCV) was measured using Power Lab 8sp system as reported previously (Sayyed et al., 2006). Briefly the animals were anesthetized by 4% halothane in a mixture of nitrous oxide and O2 and anesthesia was maintained with 1% halothane, using gaseous anesthesia system (Harvard apparatus, UK). Core temperature was monitored using a rectal probe and maintained (37 ± 1 °C) with the help of homoeothermic blanket. Sciatic nerve was stimulated with 3 V proximally at sciatic

Measurement of sciatic nerve blood flow

Nerve blood flow measurement was done by laser-doppler flowmetry methods as previously reported (Sayyed et al., 2006) using LASER Doppler system (PeriFlux 4001, Perimed, Swedan). Briefly, animals were anaesthetized and sciatic nerve was exposed on the left flank and the laser probe (tip diameter 0.85 mm) was applied just in contact with an area of sciatic trunk as free as possible from epineurial or perineurial blood vessels. The exposed nerve was covered with normal saline to avoid tissue

Cold immersion test and hot immersion test

The animals were subjected to cold immersion test (10 °C) and hot immersion test (45 °C), which is immersion of the rat tail in water maintained at mentioned temperature and then tail flick latency or signs of struggle was observed. The cut off time was 15 s (Sayyed et al., 2006).

Measurement of allodynia

Measurement of allodynia was done using Electro-Vonfrey anaesthesiometer (IITC, USA). The animals were acclimatized for two days in the chamber with perforated base. Pressure was applied using rigid fiber supplied by

Measurement of lipid peroxidation

The MDA estimation was done according to the method of (Ohkawa et al., 1979) with some modifications. Briefly, 0.2 ml of plasma was added to 0.2 ml of 8.1% SDS, 1.5 ml of 20% acetic acid solution adjusted to pH 3.4 with NaOH and 1.5 ml of 0.8% of thiobarbituric acid. The mixture was made up to 4 ml with distilled water. The mixture was heated on water bath at 95 °C for 60 min. After that the mixture was cooled under tap water and was centrifuged at 10000 rpm. Absorbance was measured at 532 nm.

Plasma glucose levels and animal body weight

STZ treated rats showed significant rise in blood glucose levels with reduction in body weight as compared to the control rats. Treatment with resveratrol (10 and 20 mg/kg) did not produce any effect on blood glucose level and body weight of treated diabetic rats.

Motor nerve conduction velocity

In eighth week post diabetes, diabetic rats showed significant decrease (p < 0.001) in MNCV (35.56 ± 0.92 m/s) as compared to the age matched control rats (55.06 ± 0.92 m/s). Treatment with resveratrol 10 mg/kg as well as 20 mg/kg for two

Discussion

Diabetic neuropathy is usually marked by impaired MNCV, NBF and neuropathic pain. In the present study, we observed significant reduction in MNCV (35%), NBF (63%) with hyperalgesia and allodynia after eight weeks of diabetes induction. These results indicate development of diabetic neuropathy and are consistent with previous reports (Cotter et al., 1995, Patel and Gores, 1997, Stevens et al., 2000). Reduced NBF as a consequence to free radical induced vascular dysfunctions in diabetic

Acknowledgements

This study was supported by a grant from Council for Scientific and Industrial Research (CSIR), Government of India to Dr. S.S. Sharma. We would like to acknowledge the help of Mr. Manish Arora, M.S. (Pharm) student at NIPER for preparing the revised manuscript.

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