Oxidative/nitrosative stress is a leading factor in the pathogenesis of peripheral diabetic neuropathy (PDN), and recent data suggest that peroxynitrite (a product of superoxide reaction with nitric oxide) is a major oxidant in diabetic nerve. Conventional antioxidants have a poor capacity to decompose peroxynitrite which at least partially explains their low efficacy in clinical neuropathy trials (e.g., lipoic acid). Our preliminary data obtained in two animal models of Type 1 diabetes indicate that the potent and specific peroxynitrite decomposition catalyst FP15 counteracts some manifestations of PDN at the very low doses of 3-5 mg/kg per day, p.o. THE OVERALL OBJECTIVE of this proposal is to provide detailed evaluation of FP15 in early PDN using the well-studied STZ-diabetic rat model and cultured endothelial and Schwann cells.
The SPECIFIC AIMS are 1) to evaluate FP15 on oxidative/nitrosative stress and poly(ADP-ribose) polymerase-1 (PARP-1) activation in peripheral nerve in STZ-diabetic rats and high glucose-exposed human endothelial and Schwann cells; 2) to assess FP15 on nerve conduction velocity, neurovascular function, thermal and mechanical algesia and key metabolic variables in STZ-diabetic rats. In animal experiments, we will assess motor and sensory nerve conduction velocities, thermal and mechanical algesia, tactile allodynia, sciatic endoneurial nutritive blood flow and endoneurial vascular conductance, vascular reactivity of epineurial arterioles, metabolic indices such as nerve cytosolic and mitochondrial NAD+/NADH redox state, energy state, sorbitol pathway intermediates, and measures of oxidative stress such as nitrotyrosine, poly(ADPribose), lipid peroxidation products, non-enzymatic antioxidants and antioxidative defense enzymes in peripheral nerve, and superoxide and nitrotyrosine in vasa nervorum. In cell culture experiments we will assess intracellular oxidative stress using DCF fluorescent probe/flow cytometry, and nitrotyrosine, PARP-1 and poly(ADP-ribose) by western blot analysis. The findings will generate new information regarding the role for nitrosative stress in the pathogenesis of PDN and may provide rationale for long-term studies of FP15 and further development of this compound for treatment of PDN.
