Phenotyping animal models of diabetic neuropathy: a consensus statement of the diabetic neuropathy study group of the EASD (Neurodiab).

NIDDK, JDRF, and the Diabetic Neuropathy Study Group of EASD sponsored a meeting to explore the current status of animal models of diabetic peripheral neuropathy. The goal of the workshop was to develop a set of consensus criteria for the phenotyping of rodent models of diabetic neuropathy. The discussion was divided into five areas: (1) status of commonly used rodent models of diabetes, (2) nerve structure, (3) electrophysiological assessments of nerve function, (4) behavioral assessments of nerve function, and (5) the role of biomarkers in disease phenotyping. Participants discussed the current understanding of each area, gold standards (if applicable) for assessments of function, improvements of existing techniques, and utility of known and exploratory biomarkers. The research opportunities in each area were outlined, providing a possible roadmap for future studies. The meeting concluded with a discussion on the merits and limitations of a unified approach to phenotyping rodent models of diabetic neuropathy and a consensus formed on the definition of the minimum criteria required for establishing the presence of the disease. A neuropathy phenotype in rodents was defined as the presence of statistically different values between diabetic and control animals in 2 of 3 assessments (nocifensive behavior, nerve conduction velocities, or nerve structure). The participants propose that this framework would allow different research groups to compare and share data, with an emphasis on data targeted toward the therapeutic efficacy of drug interventions.

Pathophysiology of diabetic erectile dysfunction: potential contribution of vasa nervorum and advanced glycation endproducts.

Erectile dysfunction (ED) due to diabetes mellitus remains difficult to treat medically despite advances in pharmacotherapeutic approaches in the field. This unmet need has resulted in a recent re-focus on the pathophysiology, in order to understand the cellular and molecular mechanisms leading to ED in diabetes. Diabetes-induced ED is often resistant to PDE5 inhibitor treatment, thus there is a need to discover targets that may lead to novel approaches for a successful treatment. The aim of this brief review is to update the reader in some of the latest development on that front, with a particular focus on the role of impaired neuronal blood flow and the formation of advanced glycation endproducts.

Effects of interleukin-6 treatment on neurovascular function, nerve perfusion and vascular endothelium in diabetic rats.

AIM: Interleukin-6 (IL-6), a member of the neuropoietic cytokine family, participates in neural development and has neurotrophic activity. Recent research has also indicated actions to improve vasa nervorum function in diabetes. Both these facets are potentially relevant for treatment of diabetic neuropathy. The aim of this study was to determine whether IL-6 treatment corrected changes in neurovascular function in streptozotocin-induced diabetic rats. METHODS: After 1 month of diabetes, rats were given IL-6 for 1 month. The rats were subjected to sensory testing and measurements of nerve conduction velocities and nerve blood flow by hydrogen clearance microelectrode polarography. Further groups were used to study responses of the isolated gastric fundus and renal artery. Results were statistically analysed using ANOVA and post hoc tests. RESULTS: Diabetic rats showed mechanical hyperalgesia, thermal hyperalgesia, and tactile allodynia. The former was unaffected by IL-6 treatment, whereas the latter two measures were corrected. Immunohistochemical staining of dorsal root ganglia for IL-6 did not reveal any changes with diabetes or treatment. The results showed that 22 and 17.4% slowing of sciatic motor and saphenous sensory nerve conduction velocities, respectively, with diabetes were improved by IL-6. Sciatic endoneurial perfusion was halved by diabetes and corrected by IL-6. A 40.6% diabetic deficit in maximal non-adrenergic, non-cholinergic relaxation of gastric fundus to nerve stimulation was unaffected by IL-6. Renal artery endothelium-dependent relaxation was halved by diabetes, the endothelium-derived hyperpolarizing factor (EDHF) component being severely attenuated. IL-6 did not affect nitric oxide-mediated vasorelaxation, but markedly improved EDHF responses. CONCLUSIONS: IL-6 improved aspects of small and large nerve fibre and vascular endothelium dysfunction in diabetic rats. The functional benefits related to increased nerve blood flow via an EDHF mechanism, and IL-6 could have therapeutic potential in diabetic neuropathy and vasculopathy, which should be further evaluated.

Correction of nitrergic neurovascular dysfunction in diabetic mouse corpus cavernosum by p38 mitogen-activated protein kinase inhibition.

Increased p38 mitogen-activated protein kinase (MAPK) in response to stress stimuli, including hyperglycemia, contributes to diabetic somatic neuropathy. However, effects on autonomic nerve and vascular function have not been determined. The aim of this study was to investigate the effects of the p38 MAPK inhibitor, LY2161793, on penile neurovascular function in streptozotocin-induced diabetic mice. Diabetes duration was 6 weeks and intervention LY2161793 treatment was given for the final 2 weeks. In vitro measurements on phenylephrine-precontracted corpus cavernosum revealed a 32% reduction in maximum nitrergic nerve-mediated relaxation with diabetes that was 74% corrected by LY2161793 treatment. Maximum nitric oxide-mediated endothelium-dependent relaxation to acetylcholine was 42% attenuated by diabetes and 88% restored by LY2161793. Moreover, treatment partially corrected a diabetic deficit in endothelium-independent relaxation to a nitric oxide donor. Thus, p38 MAPK inhibition corrects nitric oxide-dependent indices of diabetic erectile autonomic neuropathy and vasculopathy, a therapeutic approach potentially worthy of consideration for clinical trials.

An in vitro investigation of aorta and corpus cavernosum from eNOS and nNOS gene-deficient mice.

In order to ascertain the relative contribution of the endothelial and neuronal nitric oxide (NO) synthase isoforms on NO-dependent vascular and nerve function in vitro, aorta and corpus cavernosum from mice deficient in their expression (eNOS-/- and nNOS-/-) were isolated in organ baths for tension measurements. Agonist or electrical field stimulation (EFS) evoked nerve-mediated responses were compared against wild-type controls. In aortas from nNOS-/- mice, contraction responses to phenylephrine were increased. Conversely, endothelium-dependent relaxation (EDR) to acetylcholine (ACh) was decreased. In contrast, eNOS-/- aortas showed decreased sensitivity to phenylephrine and developed a flurbiprofen-sensitive contraction to ACh, and sensitivity to the NO-donor sodium nitroprusside was increased. In cavernosum from eNOS-/- and nNOS-/- mice, maximum contractions to phenylephrine and EFS, and relaxation responses to nitroprusside, were increased. As in aorta, ACh addition led to a contractile response in eNOS-/- cavernosum. Maximum EFS induced non-adrenergic, non-cholinergic (NANC) nerve-mediated relaxation was increased in eNOS-/-, whilst being decreased in nNOS-/- cavernosum. These data suggest that whilst NO-dependent vascular function is primarily eNOS mediated, and nerve function nNOS mediated, aorta function may be at least partially reliant on nNOS-related mechanisms. In addition, mechanisms of physiological compensation were observed, which require further study.

Effect of the NAD(P)H oxidase inhibitor, apocynin, on peripheral nerve perfusion and function in diabetic rats.

Upregulation of vascular NAD(P)H oxidase has been considered an important source for elevated levels of reactive oxygen species that contribute to several cardiovascular disease states, including the vascular complications of diabetes mellitus. Previous studies have shown that treatment with antioxidants corrects impaired nerve function and blood flow in diabetic rats. The aim was to assess the degree of involvement of NAD(P)H oxidase in experimental diabetic neuropathy. To this end, after 6 weeks of untreated streptozotocin-diabetes, rats were treated for 2 weeks with the NAD(P)H oxidase, apocynin. Two high doses (15 and 100 mg/kg) were used to ensure that maximal effects were registered. Diabetes caused a 20% reduction in sciatic nerve motor conduction velocity, and a 14% deficit for sensory saphenous nerve. Apocynin treatment corrected these defects by 32% and 48%, respectively: there were no significant differences between the effects of the 2 doses. Sciatic nerve nutritive endoneurial perfusion was measured by hydrogen clearance microelectrode polarography. Blood flow and vascular conductance were 47% and 40% reduced by diabetes, respectively. Both doses of apocynin had similar effects, correcting the blood flow deficit by 31% and conductance by 47%. Thus, the data show that NAD(P)H oxidase contributes to the neurovascular deficits in diabetic rats. While only accounting for part of the elevated reactive oxygen species production in diabetes, this mechanism could provide a novel therapeutic candidate for further investigation in diabetic neuropathy and vasculopathy.

Corpus cavernosum dysfunction in diabetic rats: effects of combined alpha-lipoic acid and gamma-linolenic acid treatment.

BACKGROUND: The effects of streptozotocin-induced diabetes on nitric oxide (NO)-mediated relaxation of rat corpus cavernosum smooth muscle to neurogenic and endothelial stimulation was examined. The aim was to assess the effects of treatment with low doses of the antioxidant, alpha-lipoic acid, and the omega-6 essential fatty acid, gamma-linolenic acid, either separately or in combination. METHODS: Treatment was preventive from diabetes induction or corrective over 4 weeks after 4 weeks of untreated diabetes. Corpus cavernosum responses were examined in vitro. RESULTS: Neither diabetes nor treatment affected contractile responses to transmural electrical field stimulation of noradrenergic nerves. Stimulation of phenylephrine precontracted cavernosa in the presence of guanethidine and atropine caused relaxation via the nitrergic innervation. Maximum relaxation responses were 40% and 46% decreased after 4 and 8 weeks of diabetes, respectively. alpha-Lipoic acid, gamma-linolenic acid combination treatment fully prevented this deficit, and partially (52%) corrected the effect of 4 weeks of untreated diabetes. Neither alpha-lipoic acid nor gamma-linolenic components alone had significant effects, which suggests that there were synergistic interactions between the drugs. Both 4 and 8 weeks of untreated diabetes reduced maximum endothelium-dependent relaxation of phenylephrine precontracted cavernosa to acetylcholine by approximately 40%. While alpha-lipoic acid or gamma-linolenic acid were ineffective, joint treatment fully prevented and corrected this diabetic endothelial deficit. Neither diabetes nor treatment affected endothelium-independent relaxation to the NO donor, sodium nitroprusside. CONCLUSION: The data show that alpha-lipoic acid and gamma-linolenic acid interact synergistically to improve NO-mediated neurogenic and endothelium-dependent relaxation of corpus cavernosum in experimental diabetes.

Vascular factors and metabolic interactions in the pathogenesis of diabetic neuropathy.

Diabetes mellitus is a major cause of peripheral neuropathy, commonly manifested as distal symmetrical polyneuropathy. This review examines evidence for the importance of vascular factors and their metabolic substrate from human and animal studies. Diabetic neuropathy is associated with risk factors for macrovascular disease and with other microvascular complications such as poor metabolic control, dyslipidaemia, body mass index, smoking, microalbuminuria and retinopathy. Studies in human and animal models have shown reduced nerve perfusion and endoneurial hypoxia. Investigations on biopsy material from patients with mild to severe neuropathy show graded structural changes in nerve microvasculature including basement membrane thickening, pericyte degeneration and endothelial cell hyperplasia. Arterio-venous shunting also contributes to reduced endoneurial perfusion. These vascular changes strongly correlate with clinical defects and nerve pathology. Vasodilator treatment in patients and animals improves nerve function. Early vasa nervorum functional changes are caused by the metabolic insults of diabetes, the balance between vasodilation and vasoconstriction is altered. Vascular endothelium is particularly vulnerable, with deficits in the major endothelial vasodilators, nitric oxide, endothelium-derived hyperpolarising factor and prostacyclin. Hyperglycaemia and dyslipidaemia driven oxidative stress is a major contributor, enhanced by advanced glycation end product formation and polyol pathway activation. These are coupled to protein kinase C activation and omega-6 essential fatty acid dysmetabolism. Together, this complex of interacting metabolic factors accounts for endothelial dysfunction, reduced nerve perfusion and function. Thus, the evidence emphasises the importance of vascular dysfunction, driven by metabolic change, as a cause of diabetic neuropathy, and highlights potential therapeutic approaches.

Effect of the hydroxyl radical scavenger, dimethylthiourea, on peripheral nerve tissue perfusion, conduction velocity and nociception in experimental diabetes.

AIMS/HYPOTHESIS: Increased oxidative stress has been linked to diabetic neurovascular complications, which are reduced by antioxidants. Our aim was to assess the contribution of hydroxyl radicals to early neuropathic changes by examining the effects of treatment with the specific scavenger, dimethylthiourea, on nerve function and neural tissue blood flow in diabetic rats. METHODS: Diabetes was induced by streptozotocin. Measurements comprised sciatic nerve motor and saphenous nerve sensory conduction velocity. Responses to noxious mechanical and thermal stimuli were estimated by Randall-Sellito and Hargreaves tests respectively. Sciatic nerve and superior cervical ganglion blood flow were measured by hydrogen clearance microelectrode polarography. RESULTS: Eight weeks of diabetes reduced motor and sensory conduction velocity by 19.9% and 15.7% respectively, and these were completely corrected by 2 weeks of dimethylthiourea treatment. The ED50 for motor conduction was 9 mg kg(-1) x day(-1). Mechanical and thermal nociceptive sensitivities were 18.9% and 25.0% increased by diabetes, respectively, indicating hyperalgesia which was 70% reduced by dimethylthiourea. Sciatic endoneurial and superior cervical ganglion blood flows were 51.2% and 52.4% reduced by diabetes and there was an approximately 80% improvement with treatment. CONCLUSION/INTERPRETATION: Hydroxyl radicals seem to make a major contribution to neuropathy and vasculopathy in diabetic rats. Treatment with the hydroxyl scavenger, dimethylthiourea, was highly effective. The data suggest that the development of potent hydroxyl radical scavengers suitable for use in man could markedly enhance the potential therapeutic value of an antioxidant approach to the treatment of diabetic neuropathy and vascular disease.

The effects of treatment with alpha-lipoic acid or evening primrose oil on vascular hemostatic and lipid risk factors, blood flow, and peripheral nerve conduction in the streptozotocin-diabetic rat.

Oxidative stress and defective fatty acid metabolism in diabetes may lead to impaired nerve perfusion and contribute to the development of peripheral neuropathy. We studied the effects of 2-week treatments with evening primrose oil (EPO; n = 16) or the antioxidant alpha-lipoic acid (ALA; n = 16) on endoneurial blood flow, nerve conduction parameters, lipids, coagulation, and endothelial factors, in rats with streptozotocin-induced diabetes. Compared with their nondiabetic littermates, untreated diabetic rats had impaired sciatic motor and saphenous sensory nerve-conduction velocity (NCV; P <.001), reduced endoneurial blood flow (P <.001), and increased serum triglycerides (P <.01), cholesterol (P < 0.01), plasma factor VII (P <.0001), and von Willebrand factor (vWF; P <.0001). Plasma fibrinogen and serum high-density lipoprotein concentrations were not significantly different. Treatment with either ALA or EPO effectively corrected the deficits in NCV and endoneurial blood flow. ALA was associated with marked and statistically significant decreases in fibrinogen, factor VII, vWF, and triglycerides (P <.01, paired t tests before v after treatment). In contrast, EPO was associated with significant (P <.05) increases in fibrinogen, factor VII, vWF, triglycerides, and cholesterol and a significant decrease in high-density lipoprotein. Changes in levels of coagulation factors and lipids, qualitatively similar to those found with EPO, were obtained with a diet containing sunflower oil (to control for calorific and lipid content) or with a normal diet alone. Blood glucose and hematocrit levels were not significantly altered by treatments. These data suggest that although both ALA and EPO improve blood flow and nerve function, their actions on vascular factors differ. The marked effects of ALA in lowering lipid and hemostatic risk factors for cardiovascular disease indicate potential antithrombotic and antiatherosclerotic actions that could be of benefit in human diabetes and merit further study.

Diabetes causes an early reduction in autonomic ganglion blood flow in rats.

Impaired blood flow to peripheral nerve trunks makes a major contribution to the neuropathic complications of diabetes mellitus. Comparatively little attention has been paid to perfusion abnormalities for the cell bodies of origin of the autonomic and sensory nerves, although they are severely affected in diabetic neuropathy. The aim was to examine the time course of changes in superior cervical ganglion (SCG) perfusion in streptozotocin-induced diabetic rats. Ganglion blood flow, measured by hydrogen clearance microelectrode polarography, was approximately 70 ml min(-1) 100 g(-1). One week of diabetes caused a 46% perfusion deficit, which was maintained (54%) over 24 weeks. Thus, an early, profound, and long-lived reduction in ganglion perfusion may deleteriously affect neural cell body function and could contribute to autonomic neuropathy.

Effect of alpha-lipoic acid on vascular responses and nociception in diabetic rats.

Oxidative stress contributes to the vascular and neurological complications of diabetes mellitus. The aim was to evaluate the effects of treatment with the radical scavenger and transition metal chelator, alpha-lipoic acid, on endothelium-dependent relaxation of the mesenteric vasculature and on superior cervical ganglion blood flow in 8 week streptozotocin-induced diabetic rats. alpha-Lipoic acid effects on small nerve fiber-mediated nociception were also monitored. For the in vitro phenylephrine-precontracted mesenteric vascular bed, diabetes caused a 31% deficit in maximum endothelium-dependent relaxation to acetylcholine, and a 4-fold reduction in sensitivity. alpha-Lipoic acid gave 85% protection against these defects. Acetylcholine responses are mediated by nitric oxide and endothelium-derived hyperpolarizing factor: isolation of the latter by nitric oxide synthase blockade revealed a 74% diabetic deficit that was halved by alpha-lipoic acid. Superior cervical ganglion blood flow, 52% reduced by diabetes, was dose-dependently restored by alpha-lipoic acid (ED(50), 44 mg/kg/d). Diabetic rats exhibited mechanical and thermal hyperalgesia, which were abolished by alpha-lipoic acid treatment. Thus, diabetes impairs nitric oxide and endothelium-derived hyperpolarizing factor-mediated vasodilation. This contributes to reduced neural perfusion, and may be responsible for altered nociceptive function. The effect of alpha-lipoic acid strongly implicates oxidative stress in these events and suggests a potential therapeutic approach.

Neurovascular interactions between aldose reductase and angiotensin-converting enzyme inhibition in diabetic rats.

Increased polyol pathway flux has been linked to nerve complications in diabetic rats, which are attenuated by aldose reductase inhibitors, defective nitric oxide-mediated vasodilation being a particular target. Diabetes also elevates the endothelial angiotensin system, increasing vasa nervorum vasoconstriction. The aim was to assess whether promotion of vasodilation by treatment with the aldose reductase inhibitor, ZD5522 (3',5'-dimethyl-4'-nitromethylsulphonyl-2-(2-tolyl)acetanilide), coupled with reduced vasoconstriction using the angiotensin-converting enzyme inhibitor, lisinopril, interacted positively to improve neurovascular function. After 8 weeks of streptozotocin-induced diabetes, sciatic nerve blood flow and motor conduction velocity were 51% and 21% reduced, respectively. Two weeks of lisinopril treatment dose-dependently corrected the conduction deficit (ED(50) approximately 0.9 mg kg(-1)). Low-dose lisinopril (0.3 mg kg(-1)) or ZD5522 (0.25 mg kg(-1)) had modest corrective (10-20%) effects on nerve conduction and perfusion. However, when combined, blood flow and conduction velocity reached the nondiabetic range. The ZD5522 dose used gave a approximately 45% nerve sorbitol reduction but had no significant effect on fructose content; lisinopril co-treatment did not alter ZD5522 action on polyols. Thus, there was a marked neurovascular synergistic interaction between angiotensin-converting enzyme and aldose reductase inhibition in diabetic rats. This points to a potential therapeutic benefit, which requires evaluation in clinical trials.

Effects of an extracellular metal chelator on neurovascular function in diabetic rats.

AIMS/HYPOTHESIS: Increased oxidative stress has been causally linked to diabetic neurovascular complications, which are attenuated by antioxidants. There are several possible sources of reactive oxygen species in diabetes. Our aim was to assess the contribution of free radicals, produced by transition metal catalysed reactions, to early neuropathic changes. To this end, we examined, firstly, the effects of an extracellular high molecular weight chelator, hydroxyethyl starch-deferoxamine, which is expected to be confined to vascular space, on nerve perfusion and conduction deficits in diabetic rats and, secondly, the action of a single chelator dose. METHODS: Diabetes was induced by streptozotocin. In vivo measurements comprised sciatic nerve motor conduction velocity and endoneurial perfusion, monitored by hydrogen clearance microelectrode polarography. RESULTS: We found that 8 weeks of diabetes reduced sciatic blood flow and conduction velocity by 48.3 % and 19.9% respectively. Two weeks of intravenous treatment corrected these deficits. Starch vehicle was ineffective. The time-course of action of a single hydroxyethyl starch-deferoxamine injection was examined in diabetic rats. There was a rapid increase in nerve blood flow on day 1, which remained within the non-diabetic range for 9 days before declining to the diabetic level at day 27. In contrast, conduction velocity changes were slower, reaching the non-diabetic range at day 6 and declining to the diabetic level at day 27. CONCLUSION/INTERPRETATION: Extracellular transition metal catalysed reactions play a major role in the neurovascular deficits of experimental diabetes. Given the long-lasting effect of a single treatment, extracellular metal chelator therapy could be suitable for further assessment in clinical trials.

ATP-sensitive K(+) channel effects on nerve function, Na(+), K(+) ATPase, and glutathione in diabetic rats.

Some vasodilators correct nerve conduction velocity and endoneurial blood flow deficits in diabetic rats. It is not known whether vasa nervorum has ATP-sensitive K(+) (K(ATP)) channels that mediate vasodilation, or whether K(ATP) channels could modulate peripheral nerve function. Therefore, we examined the effects of 2 weeks treatment with the K(ATP) channel openers, celikalim and WAY135201 (R-4-[3, 4-dioxo-2-(1, 2, 2-trimethyl-propylamino)-cyclobut-1-1-enylamino]-3-methoxy-+ ++benzonitri le), on sciatic nerve blood flow, conduction velocity, Na(+)-K(+) ATPase activity and glutathione content after 6 weeks of untreated streptozotocin-diabetes in rats. Blood flow and motor conduction velocity, 47.6% and 20.3% reduced by diabetes, respectively, were completely restored by both celikalim and WAY135201 treatments. Diabetes diminished sciatic Na(+)-K(+) ATPase activity by 47.6% and this was 80-90% corrected by the K(ATP) channel openers. Sciatic nerve glutathione content, 30.3% reduced by diabetes, was unaffected by celikalim or WAY135201. Thus, K(ATP) channel openers had marked beneficial effects on nerve perfusion and function in experimental diabetic neuropathy, and may be suitable for further study in clinical trials.

Effects of aldose reductase inhibition on responses of the corpus cavernosum and mesenteric vascular bed of diabetic rats.

We examined the effects of 2 months of streptozotocin-induced diabetes mellitus in rats on relaxation and contraction of corpus cavernosum and the mesenteric vascular bed in vitro. A further diabetic group was treated from diabetes induction with 10 mg/kg/day of the aldose reductase inhibitor, WAY121509. For corpus cavernosum, maximal acetylcholine-induced relaxation was 35.5% reduced (p < 0.001) by diabetes, and this deficit was completely prevented by WAY121509 treatment. Neither diabetes nor treatment affected contractile responses to field stimulation of noradrenergic nerves; however, nonadrenergic noncholinergic nerve relaxation responses were 32.9% decreased by diabetes and WAY 121509 attenuated this by 84% (p < 0.001). For the mesenteric vascular bed, diabetes depressed maximal endothelium-dependent vasodilation to acetylcholine by 25.2% (p < 0.001), and this was partially (50.6%; p < 0.01) prevented by WAY121509. Nitric oxide synthase blockade revealed endothelium-derived hyperpolarising factor-mediated vasodilation to acetylcholine that was 73.5% (p < 0.001) depressed by diabetes; WAY121509 provided partial (43.4%; p < 0.001) protection. Neither diabetes nor treatment affected endothelium-independent vasorelaxation to the nitric oxide donor, sodium nitroprusside, in corpus cavernosum or mesenteric vessels. Thus the data show protective effects of WAY121509 on nitric oxide-mediated cavernosal vasorelaxation responses and on mesenteric endothelium-derived hyperpolarising factor responses. Together these findings could account for the beneficial effects of aldose reductase inhibition on diabetic complications in experimental models.

Pentoxifylline effects on nerve conduction velocity and blood flow in diabetic rats.

Pentoxifylline has several actions that improve blood rheology and tissue perfusion and may therefore potentially be applicable to diabetic neuropathy. The aims of this study were to ascertain whether 2 weeks of treatment with pentoxifylline could correct nerve conduction velocity and blood flow deficits in 6-week streptozotocin-diabetic rats and to examine whether the effects were blocked by co-treatment with the cyclooxygenase inhibitor, flurbiprofen, or the nitric oxide synthase inhibitor, NG-nitro-L-arginine. Diabetic deficits in sciatic motor and saphenous sensory nerve conduction velocity were 56.5% and 69.8% corrected, respectively, with pentoxifylline treatment. Sciatic endoneurial blood flow was approximately halved by diabetes and this deficit was 50.4% corrected by pentoxifylline. Flurbiprofen co-treatment markedly attenuated these actions of pentoxifylline on nerve conduction and blood flow whereas NG-nitro-L-arginine was without effect. Thus, pentoxifylline treatment confers neurovascular benefits in experimental diabetic neuropathy, which are linked at least in part to cyclooxygenase-mediated metabolism.

Effects of antioxidants on nerve and vascular dysfunction in experimental diabetes.

Reactive oxygen species (ROS) are elevated by metabolic changes in diabetes, including autoxidation and increased advanced glycation. Endogenous protection by the glutathione redox cycle is also compromised by the competing NADPH requirement of elevated polyol pathway flux. Antioxidant treatment strategies prevent or reverse nerve conduction velocity (NCV) deficits in diabetic rats. These include lipophilic scavengers such as butylated hydroxytoluene, probucol and vitamin E, more hydrophilic agents like alpha-lipoic acid and acetyl cysteine, and transition metal chelators that inhibit autoxidation. In the long-term, elevated ROS cause cumulative damage to neurons and Schwann cells, however, they also have a deleterious effect on nerve blood flow in the short term. This causes endoneurial hypoxia, which is responsible for early NCV deficits. Antioxidant treatment corrects the blood flow deficit and promotes normal endoneurial oxygenation. ROS cause antioxidant-preventable vascular endothelium abnormalities, neutralizing nitric oxide mediated vasodilation and increasing reactivity to vasoconstrictors. Unsaturated fatty acids are a major target for ROS and essential fatty acid metabolism is impaired by diabetes. Gamma-linolenic acid stimulates vasodilator prostanoid production, and there are marked synergistic interactions between gamma-linolenic acid and antioxidants. This has encouraged the development of novel drugs such as ascorbyl-gamma-linolenic acid and gamma-linolenic acid-lipoic acid with enhanced therapeutic potential.

Effects of chelator treatment on aorta and corpus cavernosum from diabetic rats.

Transition-metal catalyzed reactions contribute to oxidative stress, which has been implicated in the pathogenesis of diabetic complications. The aim was to evaluate the effects of treatment with the transition metal chelator trientine on endothelium-dependent relaxation of aorta and corpus cavernosum from streptozotocin-induced diabetes of 8 weeks duration in rats. Effects on cavernosum autonomic innervation were also examined. Diabetes caused a 30.1 +/- 3.8% reduction in maximum aorta endothelium-dependent relaxation to acetylcholine (ACh), which was markedly attenuated (72.7 +/- 10.6%) by trientine treatment. Reversal treatment (4 weeks untreated diabetes, 4 weeks trientine) did not effect endothelium-dependent relaxation compared with aortas from rats with 4 weeks of diabetes, however, there was a 22.5 +/- 6.2% improvement compared with 8 weeks of diabetes. Eight weeks of diabetes caused a 41.5 +/- 6.6% reduction in corpus cavernosum endothelium-dependent maximum relaxation to ACh that was 70.1 +/- 16.9% prevented by trientine. Cavernosum nonadrenergic, noncholinergic (NANC) nerve stimulation caused frequency-dependent relaxation to a maximum of 40.9 +/- 2.4%, which was reduced by diabetes to 24.2 +/- 2.1%. Trientine partially prevented this deficit, maximum relaxation being 31.9 +/- 2.3%. Thus, metal chelator treatment has beneficial effects on aorta and cavernosum endothelium-dependent relaxation and on cavernosum NANC innervation.

Protein kinase C effects on nerve function, perfusion, Na(+), K(+)-ATPase activity and glutathione content in diabetic rats.

AIMS/HYPOTHESIS: Increased protein kinase C activity has been linked to diabetic vascular complications in the retina and kidney, which were attenuated by protein kinase C antagonist treatment. Neuropathy has a vascular component, therefore, the aim was to assess whether treatment with WAY151 003 or chelerythrine, inhibitors of protein kinase C regulatory and catalytic domains respectively, could correct nerve blood flow, conduction velocity, Na(+),K(+)-ATPase, and glutathione deficits in diabetic rats. METHODS: Diabetes was induced by streptozotocin. Sciatic nerve conduction velocity was measured in vivo and sciatic endoneurial perfusion was monitored by microelectrode polarography and hydrogen clearance. Glutathione content and Na(+),K(+)-ATPase activity were measured in extracts from homogenised sciatic nerves. RESULTS: After 8 weeks of diabetes, sciatic blood flow was 50 % reduced. Two weeks of WAY151 003 (3 or 100 mg/kg) treatment completely corrected this deficit and chelerythrine dose-dependently improved nerve perfusion. The inhibitors dose-dependently corrected a 20 % diabetic motor conduction deficit, however, at high doses ( > 3.0 mg/kg WAY151003; > 0.1 mg/kg chelerythrine) conduction velocity was reduced towards the diabetic level. Sciatic Na(+),K(+)-ATPase activity, 42 % reduced by diabetes, was partially corrected by low but not high dose WAY151 003. In contrast, only a very high dose of chelerythrine partially restored Na(+),K(+)-ATPase activity. A 30 % diabetic deficit in sciatic glutathione content was unchanged by protein kinase C inhibition. The benefits of WAY151 003 on blood flow and conduction velocity were blocked by nitric oxide synthase inhibitor co-treatment. CONCLUSION/INTERPRETATION: Protein kinase C contributes to experimental diabetic neuropathy by a neurovascular mechanism rather than through Na(+),K(+)-ATPase defects.