3-nitrotyrosine and Diabetic-Neuropathies

Macro- and microvascular disease are the most common causes of morbidity and mortality in patients with diabetes mellitus. Diabetic cardiovascular dysfunction represents a problem of great clinical importance underlying the development of various severe complications including retinopathy, nephropathy, neuropathy and increase the risk of stroke, hypertension and myocardial infarction. Hyperglycemic episodes, which complicate even well-controlled cases of diabetes, are closely associated with increased oxidative and nitrosative stress, which can trigger the development of diabetic complications. Hyperglycemia stimulates the production of advanced glycosylated end products, activates protein kinase C, and enhances the polyol pathway leading to increased superoxide anion formation. Superoxide anion interacts with nitric oxide, forming the potent cytotoxin peroxynitrite, which attacks various biomolecules in the vascular endothelium, vascular smooth muscle and myocardium, leading to cardiovascular dysfunction. The pathogenetic role of nitrosative stress and peroxynitrite, and downstream mechanisms including poly(ADP-ribose) polymerase (PARP) activation, is not limited to the diabetes-induced cardiovascular dysfunction, but also contributes to the development and progression of diabetic nephropathy, retinopathy and neuropathy. Accordingly, neutralization of peroxynitrite or pharmacological inhibition of PARP is a promising new approach in the therapy and prevention of diabetic complications. This review focuses on the role of nitrosative stress and downstream mechanisms including activation of PARP in diabetic complications and on novel emerging therapeutical strategies offered by neutralization of peroxynitrite and inhibition of PARP.

Topics: Animals; Cardiomyopathies; Diabetes Complications; Diabetic Angiopathies; Diabetic Nephropathies; Diabetic Neuropathies; Diabetic Retinopathy; Endothelium, Vascular; Humans; Nitric Oxide; Oxidative Stress; Peroxynitrous Acid; Poly(ADP-ribose) Polymerases; Superoxides; Tyrosine

We determined the impact diet-induced obesity (DIO) and types 1 and 2 diabetes have on peripheral neuropathy with emphasis on corneal nerve structural changes in C57Bl/6J mice. Endpoints examined included nerve conduction velocity, response to thermal and mechanical stimuli and innervation of the skin and cornea. DIO mice and to a greater extent type 2 diabetic mice were insulin resistant. DIO and both types 1 and 2 diabetic mice developed motor and sensory nerve conduction deficits. In the cornea of DIO and type 2 diabetic mice there was a decrease in sub-epithelial corneal nerves, innervation of the corneal epithelium, and corneal sensitivity. Type 1 diabetic mice did not present with any significant changes in corneal nerve structure until after 20 weeks of hyperglycemia. DIO and type 2 diabetic mice developed corneal structural damage more rapidly than type 1 diabetic mice although hemoglobin A1 C values were significantly higher in type 1 diabetic mice. This suggests that DIO with or without hyperglycemia contributes to development and progression of peripheral neuropathy and nerve structural damage in the cornea.

Topics: Aldehydes; Animals; Cornea; Diabetes Mellitus, Experimental; Diabetic Neuropathies; Diet; Ganglia, Spinal; Glucose Tolerance Test; Mice; Mice, Inbred C57BL; Neural Conduction; Obesity; Tyrosine

Recently a new rat model for type 2 diabetes the Zucker diabetic Sprague-Dawley (ZDSD/Pco) was created. In this study we sought to characterize the development of diabetic neuropathy in ZDSD rats using age-matched Sprague-Dawley rats as a control. Rats were examined at 34 weeks of age 12 weeks after the onset of hyperglycemia in ZDSD rats. At this time ZDSD rats were severely insulin resistant with slowing of both motor and sensory nerve conduction velocities. ZDSD rats also had fatty livers, elevated serum free fatty acids, triglycerides, and cholesterol, and elevated sciatic nerve nitrotyrosine levels. The corneas of ZDSD rats exhibited a decrease in subbasal epithelial corneal nerves and sensitivity. ZDSD rats were hypoalgesic but intraepidermal nerve fibers in the skin of the hindpaw were normal compared to Sprague-Dawley rats. However, the number of Langerhans cells was decreased. Vascular reactivity of epineurial arterioles, blood vessels that provide circulation to the sciatic nerve, to acetylcholine and calcitonin gene-related peptide was impaired in ZDSD rats. These data indicate that ZDSD rats develop many of the neural complications associated with type 2 diabetes and are a good animal model for preclinical investigations of drug development for diabetic neuropathy.

Topics: Age Factors; Animals; Arterioles; Blood Glucose; Cholesterol; Diabetes Mellitus, Type 2; Diabetic Angiopathies; Diabetic Neuropathies; Disease Models, Animal; Fatty Acids, Nonesterified; Fatty Liver; Langerhans Cells; Male; Neural Conduction; Nociception; Rats, Sprague-Dawley; Rats, Zucker; Sciatic Nerve; Time Factors; Triglycerides; Tyrosine; Vasodilation

Evidence for an important role for Na(+)/H(+) exchangers in diabetic complications is emerging. The aim of this study was to evaluate whether Na(+)/H(+) exchanger 1 inhibition reverses experimental peripheral diabetic neuropathy. Control and streptozotocin-diabetic rats were treated with the specific Na(+)/H(+) exchanger 1 inhibitor cariporide for 4 wk after 12 wk without treatment. Neuropathy end points included sciatic motor and sensory nerve conduction velocities, endoneurial nutritive blood flow, vascular reactivity of epineurial arterioles, thermal nociception, tactile allodynia, and intraepidermal nerve fiber density. Advanced glycation end product and markers of oxidative stress, including nitrated protein levels in sciatic nerve, were evaluated by Western blot. Rats with 12-wk duration of diabetes developed motor and sensory nerve conduction deficits, thermal hypoalgesia, tactile allodynia, and intraepidermal nerve fiber loss. All these changes, including impairment of nerve blood flow and vascular reactivity of epineurial arterioles, were partially reversed by 4 wk of cariporide treatment. Na(+)/H(+) exchanger 1 inhibition was also associated with reduction of diabetes-induced accumulation of advanced glycation endproduct, oxidative stress, and nitrated proteins in sciatic nerve. In conclusion, these findings support an important role for Na(+)/H(+) exchanger 1 in functional, structural, and biochemical manifestations of peripheral diabetic neuropathy and provide the rationale for development of Na(+)/H(+) exchanger 1 inhibitors for treatment of diabetic vascular and neural complications.

Topics: Aldehydes; Animals; Arterioles; Behavior, Animal; Blood Glucose; Blotting, Western; Body Weight; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Neuropathies; Glycation End Products, Advanced; Guanidines; Male; Nerve Fibers; Nitrates; Pain Measurement; Pyruvaldehyde; Rats; Rats, Wistar; Reduced Folate Carrier Protein; Sciatic Nerve; Skin; Sulfones; Superoxides; Tyrosine

Diabetic neuropathy (DN) is the most common complication of diabetes and is characterized by distal-to-proximal loss of peripheral nerve axons. The idea of tissue-specific pathological alterations in energy metabolism in diabetic complications-prone tissues is emerging. Altered nerve metabolism in type 1 diabetes models is observed; however, therapeutic strategies based on these models offer limited efficacy to type 2 diabetic patients with DN. Therefore, understanding how peripheral nerves metabolically adapt to the unique type 2 diabetic environment is critical to develop disease-modifying treatments. In the current study, we utilized targeted liquid chromatography-tandem mass spectrometry (LC/MS/MS) to characterize the glycolytic and tricarboxylic acid (TCA) cycle metabolomes in sural nerve, sciatic nerve, and dorsal root ganglia (DRG) from male type 2 diabetic mice (BKS.Cg-m+/+Lepr(db); db/db) and controls (db/+). We report depletion of glycolytic intermediates in diabetic sural nerve and sciatic nerve (glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate (sural nerve only), 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, and lactate), with no significant changes in DRG. Citrate and isocitrate TCA cycle intermediates were decreased in sural nerve, sciatic nerve, and DRG from diabetic mice. Utilizing LC/electrospray ionization/MS/MS and HPLC methods, we also observed increased protein and lipid oxidation (nitrotyrosine; hydroxyoctadecadienoic acids) in db/db tissue, with a proximal-to-distal increase in oxidative stress, with associated decreased aconitase enzyme activity. We propose a preliminary model, whereby the greater change in metabolomic profile, increase in oxidative stress, and decrease in TCA cycle enzyme activity may cause distal peripheral nerves to rely on truncated TCA cycle metabolism in the type 2 diabetes environment.

Topics: Aconitate Hydratase; Animals; Citric Acid Cycle; Diabetes Mellitus, Type 2; Diabetic Neuropathies; Disease Models, Animal; Down-Regulation; Ganglia, Spinal; Glycolysis; Lipid Peroxidation; Male; Mice; Mice, Mutant Strains; Neurons; Oxidative Stress; Peripheral Nervous System; Receptors, Leptin; Sciatic Nerve; Sural Nerve; Tyrosine

Micro and macrovascular disease is the most frequent cause of morbidity and mortality of the patients with diabetes mellitus. The recent investigations have pointed out the relationship between endothelial dysfunction and the progression of these diabetic complications, suggesting the crucial role of nitric oxide, vasodilator molecule of endothelial origin, in these events, including diabetic symmetric polyneuropathy.. The present study encompassed 100 individuals with diabetes mellitus type II and diabetic distal symmetric polyneuropathy (DSP). Nitrate+nitrite concentration, 3-nitrotyrosine, S-nitrosothiols, ADMA and SDMA levels and arginase activity were determined compared to the control group consisted of 50 age and sex matched voluntary blood donors.. NO(2)+NO(3) concentrations, as well as 3-nitrotyrosine, S-nitrosothiol, ADMA and SDMA levels were significantly higher in patients with DSP compared to the control group. Plasma arginase activity in the patients with diabetic DSP was significantly lower compared to the values in plasma of control subjects.. The obtained results confirmed that nitrate+nitrite, 3-nitrotyrosine, S-nitrosothiols, ADMA, SDMA and arginase activity determination in plasma of patients with diabetic DSP could be useful in monitoring the disease development and in assesing the therapy effects.

Topics: Aged; Arginase; Arginine; Blood Glucose; Chromatography, High Pressure Liquid; Diabetes Mellitus, Type 2; Diabetic Neuropathies; Electrodiagnosis; Female; Fructosamine; Glycated Hemoglobin; Humans; Male; Methylation; Middle Aged; Nitrates; Nitrites; S-Nitrosothiols; Tyrosine

To observe the effect of Jinmaitong capsule (JMT), a compound traditional Chinese medicine, on expressions of inducible nitric oxide synthase (iNOS) and nitro tyrosine (NT) protein in streptozocin-induced diabetic (STZ-DM) rats.. Intraperitoneal injection of streptozocin in rats to establish a model. STZ-DM rats were randomly divided into the model control group (distilled water), the small-dose JMT group (JMT at dose of 0.45 g x kg(-1) x d(-1)), the medium-dose JMT group (JMT at dose of 0.88 g x kg(-1) x d(-1)), the large-dose JMT group (JMT at dose of 1.75 g x kg(-1) x d(-1)) and Vitamin C group (VC at dose of 0.05 g x kg(-1) x d(-1)). Ten normal rats with matching weight and age were selected as the normal control group (distilled water). After intragastric administration for 16 weeks, the expressions of iNOS and NT in sciatic nerve were detected by the immunohistochemistry method.. The expression levels of iNOS and NT protein in diabetic rats were higher than those in normal rats (P<0.05, P<0.01). Compared with the model group, the levels of iNOS and NT protein in JMT and VC groups were significantly decreased (P<0.05, P<0.01). Particularly, the medium-dose JMT group showed a better effect than the VC group (P<0.05, P<0.01).. JMT could down-regulate the expressions of iNOS and NT protein of sciatic nerve in diabetic rats.

Topics: Animals; Blood Glucose; Body Weight; Capsules; Diabetes Mellitus, Experimental; Diabetic Neuropathies; Drugs, Chinese Herbal; Enzyme Activation; Male; Nitric Oxide Synthase Type II; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Tyrosine

Schwann cells may be involved in the pathogenesis of several neuropathies, such as those linked to an excess of d-glucose. Indeed, hyperglicemic condition can often result in the production of high reactive/nitrosative oxygen species concentration and possible damage of several cell structures. In the present work attention has been focused on the possible nitrosative effect of hyperglycemia on RT4 Schwannoma cell lines.. Cells were cultured for 72hrs in the presence of 180 mM D-glucose. Morphology, growth rate, cell viability, catalase evaluation and Western blot were performed.. In D-glucose-exposed cells, 3-Nitrotyrosine increase and subsequent modifications in cell morphology, growth rate, viability and catalase activity were found.. Our findings suggested a possible primary role played by Schwann cells in the hyperglicemic neuropathy pathogenesis, through the excessive production of RNS and a decrease in antioxidant defense systems, bearing out the importance of the "nitrosative hypothesis" in the hyperglicemic-induced nervous system complications.

Topics: Animals; Catalase; Cell Line, Tumor; Cell Proliferation; Cell Survival; Diabetic Neuropathies; Glucose; Hyperglycemia; Neurilemmoma; Oxidative Stress; Rats; Reactive Nitrogen Species; Schwann Cells; Tyrosine

Diabetic peripheral neuropathy is common and causes significant morbidity. Obstructive sleep apnea (OSA) is also common in patients with type 2 diabetes. Because OSA is associated with inflammation and oxidative stress, we hypothesized that OSA is associated with peripheral neuropathy in type 2 diabetes.. To assess the relationship between OSA and peripheral neuropathy in patients with type 2 diabetes.. A cross-sectional study of adults with type 2 diabetes recruited randomly from the diabetes clinic of two UK hospitals.. Peripheral neuropathy was diagnosed using the Michigan Neuropathy Screening Instrument. OSA (apnea-hypopnea index ≥ 5 events/h) was assessed using home-based, multichannel respiratory monitoring. Serum nitrotyrosine was measured by ELISA, lipid peroxide by spectrophotometer, and microvascular function by laser speckle contrast imaging. Two hundred thirty-four patients (mean [SD] age, 57 [12] yr) were analyzed. OSA prevalence was 65% (median apnea-hypopnea index, 7.2; range, 0-93), 40% of which were moderate to severe. Neuropathy prevalence was higher in patients with OSA than those without (60% vs. 27%, P < 0.001). After adjustment for possible confounders, OSA remained independently associated with diabetic neuropathy (odds ratio, 2.82; 95% confidence interval, 1.44-5.52; P = 0.0034). Nitrotyrosine and lipid peroxide levels (n = 102, 74 with OSA) were higher in OSA and correlated with hypoxemia severity. Cutaneous microvascular function (n = 71, 47 with OSA) was impaired in OSA.. We describe a novel independent association between diabetic peripheral neuropathy and OSA. We identified increased nitrosative/oxidative stress and impaired microvascular regulation as potential mechanisms. Prospective and interventional studies are needed to assess the impact of OSA and its treatment on peripheral neuropathy development and progression in patients with type 2 diabetes.

Topics: Adult; Aged; Biomarkers; Cross-Sectional Studies; Diabetes Mellitus, Type 2; Diabetic Neuropathies; Female; Humans; Linear Models; Lipid Peroxides; Male; Microcirculation; Middle Aged; Multivariate Analysis; Oxidative Stress; Prevalence; Severity of Illness Index; Skin; Sleep Apnea, Obstructive; Tyrosine

Metanx is a product containing L-methylfolate, pyridoxal 5'-phosphate, and methylcobalamin for management of endothelial dysfunction. Metanx ingredients counteract endothelial nitric oxide synthase uncoupling and oxidative stress in vascular endothelium and peripheral nerve. This study evaluates Metanx on diabetic peripheral neuropathy in ZDF rats, a model of type 2 diabetes. Metanx was administered to 15-week-old ZDF and ZDF lean rats at either 4.87 mg ⋅ kg(-1) ⋅ day(-1) (a body weight-based equivalent of human dose) or 24.35 mg ⋅ kg(-1) ⋅ day(-1) by oral gavage two times a day for 4 weeks. Both doses alleviated hind limb digital sensory, but not sciatic motor, nerve conduction slowing and thermal and mechanical hypoalgesia in the absence of any reduction of hyperglycemia. Low-dose Metanx increased intraepidermal nerve fiber density but did not prevent morphometric changes in distal tibial nerve myelinated fibers. Metanx treatment counteracted endothelial nitric oxide synthase uncoupling, inducible nitric oxide synthase upregulation, and methylglyoxal-derived advanced glycation end product, nitrotyrosine, and nitrite/nitrate accumulation in the peripheral nerve. In conclusion, Metanx, at a body weight-based equivalent of human dose, increased intraepidermal nerve fiber density and improved multiple parameters of peripheral nerve function in ZDF rats. Clinical studies are needed to determine if Metanx finds use in management of diabetic peripheral neuropathy.

Topics: Animals; Diabetes Mellitus, Type 2; Diabetic Neuropathies; Disease Models, Animal; Folic Acid; Hyperalgesia; Male; Nerve Fibers; Neural Conduction; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Pyridoxal Phosphate; Rats; Rats, Zucker; Sciatic Nerve; Tibial Nerve; Tyrosine; Vitamin B 12

With the consideration of the multifactorial etiology of diabetic peripheral neuropathy, an ideal drug or drug combination should target at least several key pathogenetic mechanisms. The flavonoid baicalein (5,6,7-trihydroxyflavone) has been reported to counteract sorbitol accumulation, activation of 12/15-lipoxygenase, oxidative-nitrosative stress, inflammation, and impaired signaling in models of chronic disease. This study evaluated baicalein on diabetic peripheral neuropathy. Control and streptozotocin-diabetic C57Bl6/J mice were maintained with or without baicalein treatment (30 mg kg(-1) d(-1), i.p., for 4 weeks after 12 weeks without treatment). Neuropathy was evaluated by sciatic motor and hind-limb digital sensory nerve conduction velocities, thermal algesia (Hargreaves test), tactile response threshold (flexible von Frey filament test), and intraepidermal nerve fiber density (fluorescent immunohistochemistry with confocal microscopy). Sciatic nerve and spinal cord 12/15-lipoxygenase and total and phosphorylated p38 mitogen-activated protein kinase expression and nitrated protein levels were evaluated by Western blot analysis, 12(S)hydroxyeicosatetraenoic acid concentration (a measure of 12/15-lipoxygenase activity) by ELISA, and glucose and sorbitol pathway intermediate concentrations by enzymatic spectrofluorometric assays. Baicalein did not affect diabetic hyperglycemia, and alleviated nerve conduction deficit and small sensory nerve fiber dysfunction, but not intraepidermal nerve fiber loss. It counteracted diabetes-associated p38 mitogen-activated protein kinase phosphorylation, oxidative-nitrosative stress, and 12/15-lipoxygenase overexpression and activation, but not glucose or sorbitol pathway intermediate accumulation. In conclusion, baicalein targets several mechanisms implicated in diabetic peripheral neuropathy. The findings provide rationale for studying hydroxyflavones with an improved pharmacological profile as potential treatments for diabetic neuropathy and other diabetic complications.

Topics: Animals; Antioxidants; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Blood Glucose; Body Weight; Diabetes Mellitus, Experimental; Diabetic Neuropathies; Disease Models, Animal; Flavanones; Gene Expression Regulation; Hyperalgesia; Mice; Mice, Inbred C57BL; Neural Conduction; Oligonucleotides, Antisense; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Reaction Time; Receptors, Eicosanoid; Sciatic Nerve; Spinal Cord; Statistics, Nonparametric; Tyrosine

Oxidative stress resulting in excessive generation of ROS is a compelling initiator of DNA damage along with damage to various cellular proteins and other macromolecules. Poly(ADP-ribose) polymerase (PARP) activation in response to DNA damage, stirs an energy-consuming cellular metabolic cycle; culminating into cell death. The present study was designed to determine the effect of combining an antioxidant, Melatonin and a PARP inhibitor, Nicotinamide on the hallmark deficits developing in diabetic neuropathy (DN). Streptozotocin (STZ, 55 mg/kg, i.p.) was administered to induce diabetes. Six weeks post diabetes induction, two week treatment with Melatonin (3 and 10 mg/kg) and Nicotinamide (100 and 300 mg/kg) either alone or in combination was given. Effect of these interventions on the functional, behavioral and biochemical changes caused by hyperglycemia were studied in treated animals. Melatonin and Nicotinamide alone as well as in combination ameliorated the functional deficits along with improvement in pain parameters. The combination also demonstrated an essential reversal of biochemical alterations. Nitrotyrosine and Poly ADP Ribose (PAR) immunopositivity was significantly decreased in sciatic nerve micro-sections of treatment group. The results of this study advocate that simultaneous inhibition of oxidative stress-PARP activation cascade may prove useful for the pharmacotherapy of DN.

Topics: Animals; Antioxidants; Blood Glucose; Body Weight; Diabetic Neuropathies; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Hyperalgesia; Lipid Peroxidation; Male; Melatonin; Neural Conduction; Niacinamide; Pain Measurement; Pain Threshold; Poly(ADP-ribose) Polymerases; Rats; Rats, Sprague-Dawley; Regional Blood Flow; Sciatic Nerve; Streptozocin; Tyrosine; Vitamin B Complex

This study evaluated poly(ADP-ribose) polymerase (PARP) inhibition as a new therapeutic approach for peripheral diabetic neuropathy using clinically relevant animal model and endpoints, and nitrotyrosine (NT), TNF-alpha, and nitrite/nitrate as potential biomarkers of the disease. Control and streptozotocin-diabetic rats were maintained with or without treatment with orally active PARP inhibitor 10-(4-methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one (GPI-15,427), 30 mg kg(-1) d(-1), for 10 wk after first 2 wk without treatment. Therapeutic efficacy was evaluated by poly(ADP-ribosyl)ated protein expression (Western blot analysis), motor and sensory nerve conduction velocities, and tibial nerve morphometry. Sciatic nerve and spinal cord NT, TNF-alpha, and nitrite/nitrate concentrations were measured by ELISA. NT localization in peripheral nervous system was evaluated by double-label fluorescent immunohistochemistry. A PARP inhibitor treatment counteracted diabetes-induced motor and sensory nerve conduction slowing, axonal atrophy of large myelinated fibers, and increase in sciatic nerve and spinal cord NT and TNF-alpha concentrations. Sciatic nerve NT and TNF-alpha concentrations inversely correlated with motor and sensory nerve conduction velocities and myelin thickness, whereas nitrite/nitrate concentrations were indistinguishable between control and diabetic groups. NT accumulation was identified in endothelial and Schwann cells of the peripheral nerve, neurons, astrocytes, and oligodendrocytes of the spinal cord, and neurons and glial cells of the dorsal root ganglia. The findings identify PARP as a compelling drug target for prevention and treatment of both functional and structural manifestations of peripheral diabetic neuropathy and provide rationale for detailed evaluation of NT and TNF-alpha as potential biomarkers of its presence, severity, and progression.

Topics: Animals; Blotting, Western; Diabetic Neuropathies; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Immunohistochemistry; Male; Nitrates; Nitrites; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Rats; Rats, Wistar; Tumor Necrosis Factor-alpha; Tyrosine

Evidence for the important role of the potent oxidant peroxynitrite in peripheral diabetic neuropathy and neuropathic pain is emerging. This study evaluated the contribution of neuronal nitric oxide synthase (nNOS) to diabetes-induced nitrosative stress in peripheral nerve and dorsal root ganglia, and peripheral nerve dysfunction and degeneration. Control and nNOS-/- mice were made diabetic with streptozotocin, and maintained for 6 weeks. Peroxynitrite injury was assessed by nitrotyrosine and poly(ADP-ribose) immunoreactivities. Peripheral diabetic neuropathy was evaluated by measurements of sciatic motor and hind-limb digital sensory nerve conduction velocities, thermal algesia, tactile allodynia, and intraepidermal nerve fiber density. Control nNOS-/- mice displayed normal motor nerve conduction velocity and thermal response latency, whereas sensory nerve conduction velocity was slightly lower compared with non-diabetic wild-type mice, and tactile response threshold and intraepidermal nerve fiber density were reduced by 47 and 38%, respectively. Both diabetic wild-type and nNOS-/- mice displayed enhanced nitrosative stress in peripheral nerve. In contrast to diabetic wild-type mice, diabetic nNOS-/- mice had near normal nitrotyrosine and poly(ADP-ribose) immunofluorescence in dorsal root ganglia. Both diabetic wild-type and nNOS-/- mice developed motor and sensory nerve conduction velocity deficits and thermal hypoalgesia although nNOS gene deficiency slightly reduced severity of the three disorders. Tactile response thresholds were similarly decreased in control and diabetic nNOS-/- mice compared with non-diabetic wild-type mice. Intraepidermal nerve fiber density was lower by 27% in diabetic nNOS-/- mice compared with the corresponding non-diabetic group, and by 20% in diabetic nNOS-/- mice compared with diabetic wild-type mice. In conclusion, nNOS is required for maintaining the normal peripheral nerve function and small sensory nerve fibre innervation. nNOS gene deficiency does not protect from development of nerve conduction deficit, sensory neuropathy and intraepidermal nerve fiber loss.

Topics: Animals; Behavior, Animal; Diabetes Mellitus, Experimental; Diabetic Neuropathies; Hindlimb; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neural Conduction; Nitric Oxide Synthase Type I; Pain Measurement; Sciatic Nerve; Streptozocin; Tyrosine

Diabetic polyneuropathy (DPN) and cold-induced nerve injury share several pathogenic mechanisms. This study explores whether cold exposure contributes to the development of DPN. Streptozotocin-induced diabetic rats and controls were exposed to a room temperature (23 degrees C) or cold environment (10 degrees C). H-reflex, tail and sciatic motor, and sensory nerve conduction studies were performed. Analyses of sural nerve, intraepidermal nerve fibers, and skin and nerve nitrotyrosine ELISAs were performed. Diabetic animals exposed to a cold environment had an increased H-reflex four weeks earlier than diabetic room temperature animals (P = .03). Cold-exposed diabetic animals also had greater reduction in motor conduction velocities at 20 weeks (P = .017), decreased skin nerve fiber density (P = .037), and increased skin nitrotyrosine levels (P = .047). Cold exposure appears to hasten the development of DPN in the rat STZ model of diabetes. These findings support that further study into the relationship between ambient temperature and DPN is warranted.

Topics: Animals; Blood Glucose; Cold Temperature; Diabetes Mellitus, Experimental; Diabetic Neuropathies; Disease Models, Animal; H-Reflex; Male; Motor Neurons; Neural Conduction; Rats; Sensory Receptor Cells; Skin; Streptozocin; Tyrosine

Diabetic neuropathy, a major complication of diabetes, affects more than 60% of diabetic patients. Recently, involvement of peroxynitrite has been postulated in diabetic neuropathy. In the present study, we have studied the effects of peroxynitrite decomposition catalysts (PDC's)-5,10,15,20-tetrakis(4-sulfonatophenyl) porphyrinato iron(III) [FeTPPS] and 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrinato iron(III) [FeTMPyP]-in experimental diabetic neuropathy. Male Sprague-Dawley rats, with six weeks of untreated diabetes were treated for two weeks with peroxynitrite decomposition catalysts. Diabetic animals showed a significant decrease in motor nerve conduction velocity and nerve blood flow, nociception as evident from decreased tail flick latency (hyperalgesia) and increased paw withdrawal pressure (mechanical allodynia) along with elevation in peroxynitrite and reduction in nerve glutathione levels. Two weeks treatment with PDC's significantly improved all the above stated functional and biochemical deficits. Aftermath of this study advocates the beneficial effects of peroxynitrite decomposition catalysts in experimental diabetic neuropathy.

Topics: Animals; Blood Glucose; Catalysis; Diabetic Neuropathies; Glutathione; Male; Metalloporphyrins; Neural Conduction; Peroxynitrous Acid; Poly Adenosine Diphosphate Ribose; Rats; Rats, Sprague-Dawley; Regional Blood Flow; Sciatic Nerve; Tyrosine

Recently we have shown that diabetes-induced retinal neurodegeneration positively correlates with oxidative stress and peroxynitrite. Studies also show that peroxynitrite impairs nerve growth factor (NGF) survival signaling in sensory neurons. However, the causal role of peroxynitrite and the impact of tyrosine nitration on diabetes-induced retinal neurodegeneration and NGF survival signaling have not been elucidated.. Expression of NGF and its receptors was examined in retinas from human and streptozotocin-induced diabetic rats and retinal ganglion cells (RGCs). Diabetic animals were treated with FeTPPS (15 mg x kg(-1) x day(-1) ip), which catalytically decomposes peroxynitrite to nitrate. After 4 weeks of diabetes, retinal cell death was determined by TUNEL assay. Lipid peroxidation and nitrotyrosine were determined using MDA assay, immunofluorescence, and Slot-Blot analysis. Expression of NGF and its receptors was determined by enzyme-linked immunosorbent assay (ELISA), real-time PCR, immunoprecipitation, and Western blot analyses.. Analyses of retinal neuronal death and NGF showed ninefold and twofold increases, respectively, in diabetic retinas compared with controls. Diabetes also induced increases in lipid peroxidation, nitrotyrosine, and the pro-apoptotic p75(NTR) receptor in human and rat retinas. These effects were associated with tyrosine nitration of the pro-survival TrkA receptor, resulting in diminished phosphorylation of TrkA and its downstream target, Akt. Furthermore, peroxynitrite induced neuronal death, TrkA nitration, and activation of p38 mitogen-activated protein kinase (MAPK) in RGCs, even in the presence of exogenous NGF. FeTPPS prevented tyrosine nitration, restored NGF survival signal, and prevented neuronal death in vitro and in vivo.. Together, these data suggest that diabetes-induced peroxynitrite impairs NGF neuronal survival by nitrating TrkA receptor and enhancing p75(NTR) expression.

Topics: Animals; Cadaver; Cell Death; Diabetes Mellitus; Diabetes Mellitus, Experimental; Diabetic Neuropathies; Humans; Lipid Peroxidation; Middle Aged; Nerve Degeneration; Nerve Growth Factor; Optic Nerve; Peroxynitrous Acid; Rats; Reference Values; Retinal Ganglion Cells; RNA, Messenger; Streptozocin; Tyrosine

Whereas an important role of free radicals and oxidants in peripheral diabetic neuropathy is well established, the contribution of nitrosative stress and, in particular, of the highly reactive oxidant peroxynitrite, has not been properly explored. Our previous findings implicate peroxynitrite in diabetes-associated motor and sensory nerve conduction deficits and peripheral nerve energy deficiency and poly(ADP-ribose) polymerase activation associated with Type 1 diabetes. In this study the role of nitrosative stress in diabetic sensory neuropathy is evaluated. The peroxynitrite decomposition catalyst Fe(III) tetrakis-2-(N-triethylene glycol monomethyl ether)pyridyl porphyrin (FP15) was administered to control and streptozotocin (STZ)-diabetic mice at the dose of 5 mg kg(-1) day(-1) (FP15), for 3 weeks after initial 3 weeks without treatment. Mice with 6-week duration of diabetes developed clearly manifest thermal hypoalgesia (paw withdrawal, tail-flick, and hot plate tests), mechanical hypoalgesia (tail pressure Randall-Sellito test), tactile allodynia (flexible von Frey filament test), and approximately 38% loss of intraepidermal nerve fibers. They also had increased nitrotyrosine and poly(ADP-ribose) immunofluorescence in the sciatic nerve, grey matter of spinal cord, and dorsal root ganglion neurons. FP15 treatment was associated with alleviation of thermal and mechanical hypoalgesia. Tactile response threshold tended to increase in response to peroxynitrite decomposition catalyst treatment, but still remained approximately 59% lower compared with non-diabetic controls. Intraepidermal nerve fiber density was 25% higher in FP15-treated than in untreated diabetic rats, but the difference between two groups did not achieve statistical significance (p=0.054). Nitrotyrosine and poly(ADP-ribose) immunofluorescence in sciatic nerve, spinal cord, and dorsal root ganglion neurons of peroxynitrite decomposition catalyst-treated diabetic mice were markedly reduced. In conclusion, nitrosative stress plays an important role in sensory neuropathy associated with Type 1 diabetes. The findings provide rationale for further studies of peroxynitrite decomposition catalysts in a long-term diabetic model.

Topics: Animals; Behavior, Animal; Body Weight; Diabetes Mellitus, Experimental; Diabetic Neuropathies; Fluorescent Antibody Technique; Hot Temperature; Hyperalgesia; Immunohistochemistry; Male; Metalloporphyrins; Mice; Mice, Inbred C57BL; Nerve Fibers; Neurons; Pain Threshold; Peroxynitrous Acid; Poly Adenosine Diphosphate Ribose; Reaction Time; Sciatic Nerve; Spinal Nerve Roots; Streptozocin; Tyrosine

Diabetic neuropathy (DN) is a debilitating complication of type 1 and type 2 diabetes. Rodent models of DN do not fully replicate the pathology observed in human patients. We examined DN in streptozotocin (STZ)-induced [B6] and spontaneous type 1 diabetes [B6Ins2(Akita)] and spontaneous type 2 diabetes [B6-db/db, BKS-db/db]. Despite persistent hyperglycemia, the STZ-treated B6 and B6Ins2(Akita) mice were resistant to the development of DN. In contrast, DN developed in both type 2 diabetes models: the B6-db/db and BKS-db/db mice. The persistence of hyperglycemia and development of DN in the B6-db/db mice required an increased fat diet while the BKS-db/db mice developed severe DN and remained hyperglycemic on standard mouse chow. Our data support the hypothesis that genetic background and diet influence the development of DN and should be considered when developing new models of DN.

Topics: Animals; Diabetic Neuropathies; Disease Models, Animal; DNA Fragmentation; Hyperalgesia; In Situ Nick-End Labeling; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Inbred NOD; Nerve Fibers; Neural Conduction; Pain Measurement; Reaction Time; Streptozocin; Tyrosine; Ubiquitin Thiolesterase

Evidence for important roles of the highly reactive oxidant peroxynitrite in diabetic complications is emerging. We evaluated the role of peroxynitrite in early peripheral neuropathy and vascular dysfunction in STZ-diabetic rats. In the first dose-finding study, control and STZ-diabetic rats were maintained with or without the potent peroxynitrite decomposition catalyst Fe(III)tetrakis-2-(N-triethylene glycol monomethyl ether) pyridyl porphyrin (FP15) at 3, 5, or 10 mg.kg(-1).day(-1) in the drinking water for 4 wk after an initial 2 wk without treatment for assessment of early neuropathy. In the second study with similar experimental design, control and STZ-diabetic rats were maintained with or without FP15, 5 mg.kg(-1).day(-1), for vascular studies. Rats with 6-wk duration of diabetes developed motor and sensory nerve conduction velocity deficits, mechanical hyperalgesia, and tactile allodynia in the absence of small sensory nerve fiber degeneration. They also had increased nitrotyrosine and poly(ADP-ribose) immunofluorescence in the sciatic nerve and dorsal root ganglia. All these variables were dose-dependently corrected by FP15, with minimal differences between the 5 and 10 mg.kg(-1).day(-1) doses. FP15, 5 mg.kg(-1).day(-1), also corrected endoneurial nutritive blood flow and nitrotyrosine, but not superoxide, fluorescence in aorta and epineurial arterioles. Diabetes-induced decreases in acetylcholine-mediated relaxation by epineurial arterioles and coronary and mesenteric arteries, as well as bradykinin-induced relaxation by coronary and mesenteric arteries, were alleviated by FP15 treatment. The findings reveal the important role of nitrosative stress in early neuropathy and vasculopathy and provide the rationale for further studies of peroxynitrite decomposition catalysts in long-term diabetic models.

Topics: Animals; Aorta; Arterioles; Blood Glucose; Body Weight; Coronary Vessels; Diabetes Mellitus, Experimental; Diabetic Angiopathies; Diabetic Neuropathies; Ganglia, Spinal; Hyperalgesia; Hypoglycemic Agents; Male; Mesenteric Arteries; Metalloporphyrins; Neural Conduction; Peroxynitrous Acid; Poly Adenosine Diphosphate Ribose; Rats; Rats, Wistar; Regional Blood Flow; Sciatic Nerve; Superoxides; Tyrosine; Vasodilator Agents

Poly(ADP-ribose) polymerase (PARP) activation is emerging as a fundamental mechanism in the pathogenesis of diabetes complications including diabetic neuropathy. This study evaluated the role of PARP in diabetic sensory neuropathy. The experiments were performed in control and streptozotocin-induced diabetic rats treated with or without the PARP inhibitor 1,5-isoquinolinediol (ISO; 3 mg x kg(-1) x day(-1) i.p.) for 2 weeks after 2 weeks without treatment. Diabetic rats developed thermal hyperalgesia (assessed by paw-withdrawal and tail-flick tests), mechanical hyperalgesia (von Frey anesthesiometer/rigid filaments and Randall-Sellito tests), tactile allodynia (flexible von Frey filaments), and increased flinching behavior in phases 1 and 2 of the 2% formalin pain test. They also had clearly manifest increase in nitrotyrosine and poly(ADP-ribose) immunoreactivities in the sciatic nerve and increased superoxide formation (hydroxyethidine method) and nitrotyrosine immunoreactivity in vasa nervorum. ISO treatment alleviated abnormal sensory responses, including thermal and mechanical hyperalgesia and tactile allodynia as well as exaggerated formalin flinching behavior in diabetic rats, without affecting the aforementioned variables in the control group. Poly(ADP-ribose) and, to a lesser extent, nitrotyrosine abundance in sciatic nerve, as well as superoxide and nitrotyrosine formation in vasa nervorum, were markedly reduced by ISO therapy. Apoptosis in dorsal root ganglion neurons (transferase-mediated dUTP nick-end labeling assay) was not detected in any of the groups. In conclusion, PARP activation contributes to early diabetic sensory neuropathy by mechanisms that may include oxidative stress but not neuronal apoptosis.

Topics: Animals; Apoptosis; Arthropathy, Neurogenic; Blood Glucose; Body Weight; Diabetes Mellitus, Experimental; Diabetic Neuropathies; Ganglia, Spinal; Hyperalgesia; Immunohistochemistry; Isoquinolines; Male; Neural Conduction; Neurons; Oxidative Stress; Poly(ADP-ribose) Polymerase Inhibitors; Quinolines; Rats; Rats, Wistar; Streptozocin; Tyrosine

Nitrosative stress, that is, enhanced peroxynitrite formation, has been documented in both experimental and clinical diabetic neuropathy (DN), but its pathogenetic role remains unexplored. This study evaluated the role for nitrosative stress in two animal models of type 1 diabetes: streptozotocin-diabetic mice and diabetic NOD mice. Control (C) and streptozotocin-diabetic (D) mice were treated with and without the potent peroxynitrite decomposition catalyst FP15 (5 mg kg(-1) d(-1)) for 1 wk after 8 wk without treatment. Sciatic nerve nitrotyrosine (a marker of peroxynitrite-induced injury) and poly(ADP-ribose) immunoreactivities were present in D and absent in C and D+FP15. FP15 treatment corrected sciatic motor and hind-limb digital sensory nerve conduction deficits and sciatic nerve energy state in D, without affecting those variables in C. Nerve glucose and sorbitol pathway intermediate concentrations were similarly elevated in D and D+FP15 vs C. In diabetic NOD mice, a 7-day treatment with either 1 or 3 mg kg(-1) d(-1) FP15 reversed increased tail-flick latency (a sign of reduced pain sensitivity); the effect of the higher dose was significant as early as 3 days after beginning of the treatment. In conclusion, nitrosative stress plays a major role in DN in, at least, type 1 diabetes. This provides the rationale for development of agents counteracting peroxynitrite formation and promoting peroxynitrite decomposition, and their evaluation in DN.

Topics: Animals; Blood Glucose; Creatine; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Neuropathies; Metalloporphyrins; Mice; Mice, Inbred NOD; Neural Conduction; Neurons, Afferent; Oxidative Stress; Peroxynitrous Acid; Phosphocreatine; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Reactive Nitrogen Species; Sciatic Nerve; Tyrosine; Weight Gain