OBJECTIVE-Axonal regeneration is defective in both experimental and clinical diabetic neuropathy, contributing to loss of axonal extremities and neuronal dysfunction. The mechanisms behind this failure are not fully understood; however, a deficit in neurotrophic support and signaling has been implicated. RESEARCH DESIGN AND METHODS-We investigated the expression of neuritin (also known as candidate plasticity gene 15, cpg15) in the sensory nervous system of control rats and rats with streptozotocin (STZ)-induced diabetes using microarray PCR, Western blotting, and immunocytochemical analysis. The functional role of neuritin in sensory neurons in vitro was assessed using silencing RNA.RESULTS-Neuritin was expressed by a population of smalldiameter neurons in the dorsal root ganglia (DRG) and was anterogradely and retrogradely transported along the sciatic nerve in vivo. Nerve growth factor (NGF) treatment induced an increase in the transcription and translation of neuritin in sensory neurons in vitro. This increase was both time and dose dependent and occurred via mitogen-activated protein kinase or phosphatidylinositol-3 kinase activation. Inhibition of neuritin using silencing RNA abolished NGF-mediated neurite outgrowth, demonstrating the crucial role played by neuritin in mediating regeneration. Neuritin levels were reduced in both the DRG and sciatic nerve of rats with 12 weeks of STZ-induced diabetes, and these deficits were reversed in vivo by treatment with NGF.

Diabetes mellitus is associated with altered iron homeostasis in both human and animal diabetic models. Iron is a metal oxidant capable of generating reactive oxygen species (ROS) and has been postulated to contribute to diabetic nephropathy. Two proteins involved in iron metabolism that are expressed in the kidney are the divalent metal transporter, DMT1 (Slc11a2), and the Transferrin Receptor (TfR). Thus, we investigated whether renal DMT1 or TfR expression is altered in diabetes, as this could potentially affect ROS generation and contribute to diabetic nephropathy. Rats were rendered diabetic with streptozotocin (STZ-diabetes) and renal DMT1 and TfR expression studied using semi-quantitative immunoblotting and immunofluorescence. In STZ-diabetic Sprague-Dawley rats, renal DMT1 expression was significantly reduced and TfR expression increased after 2 weeks. DMT1 downregulation was observed in both proximal tubules and collecting ducts. Renal DMT1 expression was also decreased in Wistar rats following 12 weeks of STZ-diabetes, an effect that was fully corrected by insulin-replacement but not by cotreatment with the aldose reductase inhibitor, sorbinil. Increased renal TfR expression was also observed in STZ-diabetic Wistar rats together with elevated cellular iron accumulation. Together these data demonstrate renal DMT1 downregulation and TfR upregulation in STZ-diabetes. Whilst the consequence of altered DMT1 expression on renal iron handling and oxidant damage remains to be determined, the attenuation of the putative lysosomal iron exit pathway in proximal tubules could potentially explain lysosomal iron accumulation reported in human diabetes and STZ-diabetic animals.

Dysfunctional switches in neuronal phenotype are at the root of diabetic neuropathy and many aspects of neuronal phenotype are maintained by neurotrophic influences. Thus the failure of these and their mediating signals are fundamental to understanding and controlling the condition. In experimental diabetes, failure of the C fiber phenotype is secondary to reduced neurotrophic regulation by nerve growth factor (NGF). We have performed gene array profiling to identify candidate genes for other neurotrophic factors. This reveals sonic hedgehog as a neurotrophic regulator, with the capacity to maintain nerve conduction velocity in the normal range and to maintain normal gene expression for a range of influential candidate genes. As all known targets for sonic hedgehog are transcription dependent, this implicates impaired gene expression, rather than disturbed physiology, in the nerve conduction defects of diabetes and suggest the most successful method of prevention of the condition. W4B-02Oxidative stress in the pathogenesis of experimental diabetic neuropathy P. A. Low, A. Schmeichel, J. D. Schmelzer and M. Kishi Department of Neurology, Mayo Foundation, Rochester, MN 55905, USA, low@mayo.eduWe evaluated the effects of chronic hyperglycemia on L5 DRG neurons. Experimental diabetic neuropathy (EDN) was induced by streptozotocin. We studied peripheral nerve after 1, 3, 12 months of diabetes. A conduction deficit was present from the first month and persisted over 12 months, affecting mainly sensory fibers. 8-Hydroxy-deoxyguanosine labeling was significantly increased at all time points in DRG neurons, indicating oxidative injury. Caspase-3 labeling was increased at all three time-points, indicating commitment to the efferent limb of the apoptotic pathway. Apoptosis was confirmed by a significant increase in the percent of neurons undergoing apoptosis (TUNEL staining) at 1 month (8%), 3 months (7%) and 12 months (11%). Morphometry of DRG showed a selective loss (42%) of the largest neurons. These findings support the concept that oxidative stress leads to oxidative injury of DRG neurons, with mitochondrium as a specific target, leading to apoptosis and a predominantly sensory neuropathy. W4B-03Vascular changes in animal models of diabetic neuropathy N. E. Cameron and M. A. Cotter Biomedical Sciences, Institute of Medical Sciences, Aberdeen University, Aberdeen, UK, n.e.cameron@abdn.ac.uk Blood flow to neural tissues is reduced by diabetes. For peripheral nerve and ganglia, this occurs within a week of diabetes induction in experimental rat models. Some of the metabolic changes in diabetes target blood vessels, including vasa nervorum, resulting in attenuation of endothelial vasodilatory mechanisms based on nitric oxide, endothelium-derived hyperpolarizing factor, and prostanoids. Moreover, there is increased production of and reactivity to some vasoconstrictors, particularly angiotensin II and endothelin-1. Impaired vasa nervorum blood flow leads to endoneurial hypoxia, which causes or contributes of function defect...

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