Viktor R. Drel

The leptin-deficient (ob/ob) mouse: a new animal model of peripheral neuropathy of type 2 diabetes and obesity.

Whereas functional, metabolic, neurotrophic, and morphological abnormalities of peripheral diabetic neuropathy (PDN) have been extensively explored in streptozotocin-induced diabetic rats and mice (models of type 1 diabetes), insufficient information is available on manifestations and pathogenetic mechanisms of PDN in type 2 diabetic models. The latter could constitute a problem for clinical trial design because the vast majority of subjects with diabetes have type 2 (non–insulin dependent) diabetes. This study was aimed at characterization of PDN in leptin-deficient (ob/ob) mice, a model of type 2 diabetes with relatively mild hyperglycemia and obesity. ob/ob mice (∼11 weeks old) clearly developed manifest sciatic motor nerve conduction velocity (MNCV) and hind-limb digital sensory nerve conduction velocity (SNCV) deficits, thermal hypoalgesia, tactile allodynia, and a remarkable (∼78%) loss of intraepidermal nerve fibers. They also had increased sorbitol pathway activity in the sciatic nerve and increased nitrotyrosine and poly(ADP-ribose) immunofluorescence in the sciatic nerve, spinal cord, and dorsal root ganglion (DRG). Aldose reductase inhibition with fidarestat (16 mg · kg−1 · d−1), administered to ob/ob mice for 6 weeks starting from 5 weeks of age, was associated with preservation of normal MNCV and SNCV and alleviation of thermal hypoalgesia and intraepidermal nerve fiber loss but not tactile allodynia. Sciatic nerve nitrotyrosine immunofluorescence and the number of poly(ADP-ribose)–positive nuclei in sciatic nerve, spinal cord, and DRGs of fidarestat-treated ob/ob mice did not differ from those in nondiabetic controls. In conclusion, the leptin-deficient ob/ob mouse is a new animal model that develops both large motor and sensory fiber and small sensory fiber PDN and responds to pathogenetic treatment. The results support the role for increased aldose reductase activity in functional and structural changes of PDN in type 2 diabetes.

High-Fat Diet–Induced Neuropathy of Pre-Diabetes and Obesity: Effects of “Healthy” Diet and Aldose Reductase Inhibition

OBJECTIVE—Subjects with dietary obesity and pre-diabetes have an increased risk for developing both nerve conduction slowing and small sensory fiber neuropathy. Animal models of this type of neuropathy have not been described. This study evaluated neuropathic changes and their amenability to dietary and pharmacological interventions in mice fed a high-fat diet (HFD), a model of pre-diabetes and alimentary obesity. RESEARCH DESIGN AND METHODS—Female C57BL6/J mice were fed normal diets or HFDs for 16 weeks. RESULTS—HFD-fed mice developed obesity, increased plasma FFA and insulin concentrations, and impaired glucose tolerance. They also had motor and sensory nerve conduction deficits, tactile allodynia, and thermal hypoalgesia in the absence of intraepidermal nerve fiber loss or axonal atrophy. Despite the absence of overt hyperglycemia, the mice displayed augmented sorbitol pathway activity in the peripheral nerve, as well as 4-hydroxynonenal adduct nitrotyrosine and poly(ADP-ribose) accumulation and 12/15-lipoxygenase overexpression in peripheral nerve and dorsal root ganglion neurons. A 6-week feeding with normal chow after 16 weeks on HFD alleviated tactile allodynia and essentially corrected thermal hypoalgesia and sensory nerve conduction deficit without affecting motor nerve conduction slowing. Normal chow containing the aldose reductase inhibitor fidarestat (16 mg · kg−1· day −1) corrected all functional changes of HFD-induced neuropathy. CONCLUSIONS—Similar to human subjects with pre-diabetes and obesity, HFD-fed mice develop peripheral nerve functional, but not structural, abnormalities and, therefore, are a suitable model for evaluating dietary and pharmacological approaches to halt progression and reverse diabetic neuropathy at the earliest stage of the disease.

Poly(ADP-ribose) polymerase inhibition alleviates experimental diabetic sensory neuropathy

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 · kg−1 · 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.

New Therapeutic and Biomarker Discovery for Peripheral Diabetic Neuropathy: PARP Inhibitor, Nitrotyrosine, and Tumor Necrosis Factor-α

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.

Nicotinamide Reverses Neurological and Neurovascular Deficits in Streptozotocin Diabetic Rats

In diabetes, activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) is an important effector of oxidative-nitrosative injury, which contributes to the development of experimental diabetic peripheral neuropathy (DPN). However, the potential toxicity of complete PARP inhibition necessitates the utilization of weaker PARP inhibitors with additional therapeutic properties. Nicotinamide (vitamin B3) is a weak PARP inhibitor, antioxidant, and calcium modulator and can improve energy status and inhibit cell death in ischemic tissues. We report the dose-dependent effects of nicotinamide in an established model of early DPN. Control and streptozotocin-diabetic rats were treated with 200 to 400 mg/kg/day nicotinamide (i.p.) for 2 weeks after 2 weeks of untreated diabetes. Sciatic endoneurial nutritive blood flow was measured by microelectrode polarography and hydrogen clearance, and sciatic motor and hind-limb digital sensory nerve conduction velocities and thermal and mechanical algesia were measured by standard electrophysiological and behavioral tests. Malondialdehyde plus 4-hydroxyalkenal concentration in the sciatic nerve and amino acid-(4)-hydroxynonenal adduct and poly(ADP-ribosyl)ated protein expression in human Schwann cells were assessed by a colorimetric method with N-methyl-2-phenyl indole and Western blot analysis, respectively. Nicotinamide corrected increased sciatic nerve lipid peroxidation in concert with nerve perfusion deficits and dose-dependently attenuated nerve conduction slowing, as well as mechanical and thermal hyperalgesia. Nicotinamide (25 mM) prevented high (30 mM) glucose-induced overexpression of amino acid-(4)-hydroxynonenal adducts and poly(ADP-ribosyl)ated proteins in human Schwann cells. In conclusion, nicotinamide deserves consideration as an attractive, nontoxic therapy for the treatment of DPN.

Poly(ADP-ribose)polymerase inhibition counteracts cataract formation and early retinal changes in streptozotocin-diabetic rats.

PURPOSE This study evaluated the role for poly(ADP-ribose) polymerase (PARP) in diabetes-induced cataractogenesis and early retinal changes. METHODS Control and streptozotocin (STZ)-diabetic rats were treated with or without the PARP inhibitors 1,5-isoquinolinediol (ISO; 3 mg kg(-1) d(-1) intraperitoneally) and 10-(4-methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-1 (GPI-15427, 30 mg kg(-1) d(-1) orally) for 10 weeks after the first 2 weeks without treatment. Lens clarity was evaluated by indirect ophthalmoscopy and slit lamp examination, and retinal changes were evaluated by immunohistochemistry and Western blot analysis. In in vitro studies, cultured human lens epithelial cells and bovine retinal pericytes and endothelial cells were exposed to high glucose or palmitate. RESULTS PARP is expressed in lens, and poly(ADP-ribosyl)ated proteins are primarily localized in the 38- to 87-kDa range of the protein spectrum, with several minor bands at 17 to 38 kDa. The 38- to 87-kDa and the 17- to 38-kDa poly(ADP-ribosyl)ated protein expression increased by 74% and 275%, respectively, after 4 weeks of diabetes and by approximately 65% early after exposure of lens epithelial cells to 30 mM glucose. Both PARP inhibitors delayed, but did not prevent, the formation of diabetic cataract. The number of TUNEL-positive nuclei in flatmounted retinas increased approximately 4-fold in STZ diabetic rats, and this increase was prevented by ISO and GPI-15427. Both PARP inhibitors reduced diabetes-induced retinal oxidative-nitrosative and endoplasmic reticulum stress and glial activation. GPI-15427 (20 microM) prevented oxidative-nitrosative stress and cell death in palmitate-exposed pericytes and endothelial cells. CONCLUSIONS PARP activation is implicated in the formation of diabetic cataract and in early retinal changes. These findings provide a rationale for the development of PARP inhibitors for the prevention of diabetic ocular complications.

Baicalein alleviates diabetic peripheral neuropathy through inhibition of oxidative–nitrosative stress and p38 MAPK activation

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.

Nitrosative stress and peripheral diabetic neuropathy in leptin-deficient (ob/ob) mice

Nitrosative stress contributes to nerve conduction slowing, thermal hypoalgesia, and impaired nitrergic innervation in animal models of Type 1 diabetes. The role for reactive nitrogen species in Type 2 diabetes-associated neuropathy remains unexplored. This study evaluated the role for nitrosative stress in functional and structural neuropathic changes in ob/ob mice, a model of Type 2 diabetes with mild hyperglycemia and obesity. Two structurally diverse peroxynitrite decomposition catalysts, Fe(III) tetrakis-2-(N-triethylene glycol monomethyl ether)-pyridyl porphyrin (FP15) and Fe(III) tetra-mesitylporphyrin octasulfonate (FeTMPS), were administered to control and 8-week-old ob/ob mice for 3 weeks at the doses of 5 mg kg(-1) day(-1) (FP15) and 5 and 10 mg kg(-1) day(-1) (FeTMPS). The 11-week-old ob/ob mice developed motor nerve conduction velocity (MNCV) and hind-limb digital sensory nerve conduction velocity (SNCV) deficits, thermal hypoalgesia, tactile allodynia, and a remarkable ( approximately 78%) loss of intraepidermal nerve fibers. They also had increased nitrotyrosine and poly(ADP-ribose) immunofluorescence in the sciatic nerve, spinal cord, and dorsal root ganglion neurons. Treatment with two structurally diverse peroxynitrite decomposition catalysts was associated with restoration of normal MNCV and SNCV, and alleviation of thermal hypoalgesia. Tactile response thresholds increased in response to peroxynitrite decomposition catalyst treatment, but still remained approximately 2.7- to 3.2-fold lower compared with non-diabetic controls. Intraepidermal nerve fiber loss was not alleviated by either FP15 or FeTMPS. Nitrotyrosine and poly(ADP-ribose) immunofluorescence in sciatic nerve, spinal cord, and dorsal root ganglia of peroxynitrite decomposition catalyst-treated ob/ob mice were essentially normal. In conclusion, nitrosative stress plays an important role in functional abnormalities associated with large motor, large sensory, and small sensory fiber neuropathy, but not in small sensory nerve fiber degeneration, in this animal model. Peroxynitrite decomposition catalysts alleviate Type 2 diabetes-associated sensory nerve dysfunction, likely by mechanism(s) not involving arrest of degenerative changes or enhanced regeneration of small sensory nerve fibers.

Different Roles of 12/15-Lipoxygenase in Diabetic Large and Small Fiber Peripheral and Autonomic Neuropathies

Up-regulation of 12/15-lipoxygenase, which converts arachidonic acid to 12(S)- and 15(S)-hydroxyeicosatetraenoic acids, causes impaired cell signaling, oxidative-nitrosative stress, and inflammation. This study evaluated the role for 12/15-lipoxygenase in diabetic large and small fiber peripheral and autonomic neuropathies. Control and streptozotocin-diabetic wild-type and 12/15-lipoxygenase-deficient mice were maintained for 14 to 16 weeks. 12/15-lipoxygenase gene deficiency did not affect weight gain or blood glucose concentrations. Diabetic wild-type mice displayed increased sciatic nerve 12/15-lipoxygenase and 12(S)-hydroxyeicosatetraenoic acid levels. 12/15-lipoxygenase deficiency prevented or alleviated diabetes-induced thermal hypoalgesia, tactile allodynia, motor and sensory nerve conduction velocity deficits, and reduction in tibial nerve myelinated fiber diameter, but not intraepidermal nerve fiber loss. The frequencies of superior mesenteric-celiac ganglion neuritic dystrophy, the hallmark of diabetic autonomic neuropathy in mouse prevertebral sympathetic ganglia, were increased 14.8-fold and 17.2-fold in diabetic wild-type and 12/15-lipoxygenase-deficient mice, respectively. In addition, both diabetic groups displayed small (<1%) numbers of degenerating sympathetic neurons. In conclusion, whereas 12/15-lipoxygenase up-regulation provides an important contribution to functional changes characteristic for both large and small fiber peripheral diabetic neuropathies and axonal atrophy of large myelinated fibers, its role in small sensory nerve fiber degeneration and neuritic dystrophy and neuronal degeneration characteristic for diabetic autonomic neuropathy is minor. This should be considered in the selection of endpoints for future clinical trials of 12/15-lipoxygenase inhibitors.

ROLE OF 12/15-LIPOXYGENASE IN NITROSATIVE STRESS AND PERIPHERAL PREDIABETIC AND DIABETIC NEUROPATHIES

This study evaluated the role of 12/15-lipoxygenase, which converts arachidonic acid to 12(S)- and 15(S)-hydroxyeicosatetraenoic acids, in nitrosative stress in the peripheral nervous system and peripheral prediabetic and diabetic neuropathies. The experiments were performed in C57BL6/J mice made diabetic with streptozotocin or fed a high-fat diet and in human Schwann cells cultured in 5.5 or 30 mM glucose. 12/15-Lipoxygenase overexpression and activation were present in sciatic nerve and spinal cord of diabetic and high-fat diet-fed mice, as well as in human Schwann cells cultured in high concentrations of D-, but not L-glucose. 12/15-Lipoxygenase inhibition by cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (8 mg kg(-1) day(-1) sc, for 4 weeks after 12 weeks without treatment) alleviated the accumulation of nitrated proteins in the sciatic nerve and spinal cord, and large and small nerve fiber dysfunction, but not intraepidermal nerve fiber loss. 12/15-Lipoxygenase gene deficiency alleviated nitrosative stress and nerve conduction deficit, but not small sensory fiber neuropathy, in high-fat diet-fed mice. In conclusion, 12/15-lipoxygenase is implicated in nitrosative stress and peripheral neuropathy in mouse models of type 1 and early type 2 diabetes. Its presence in human Schwann cells and upregulation by high glucose suggest a potential involvement in human disease.