Conduction Velocity Deficits Research Articles

ENDOPLASMIC RETICULUM STRESS AND PERIPHERAL PREDIABETIC AND DIABETIC NEUROPATHY

Endoplasmic reticulum (ER) stress caused by accumulation of unfolded proteins in the ER lumen, contributes to beta‐cell loss, insulin resistance, and plays an important role in the pathogenesis of both Type 1 and Type 2 diabetes. We evaluated the role of this phenomenon in the pathogenesis of prediabetic and diabetic peripheral neuropathy. The experiments have been performed in 1) Zucker lean and Zucker fa/fa rats, a model of obesity and Type 2 prediabetes, and 2) control and STZ‐diabetic rats, a model of Type 1 diabetes. Both 16‐wk‐old Zucker fa/fa rats and STZ‐diabetic rats with 12‐wk duration of STZ‐diabetes displayed ER stress response manifest by overexpression of BiP/GRP78 and GRP94 in the peripheral nerve. They also had nerve conduction velocity (NCV) deficit and small sensory nerve fiber dysfunction. Treatment with the chemical chaperone trimethylamine N‐oxide (TMAO) reversed sensory NCV deficit and alleviated thermal hypoalgesia and tactile allodynia in Zucker fa/fa rats. It also prevented sensory NCV deficit and partially prevented motor NCV deficit and small sensory nerve fiber dysfunction in STZ‐diabetic rats. In conclusion, ER stress is implicated in prediabetic and diabetic peripheral neuropathy. Studies of biochemical mechanisms of ER stress‐induced peripheral nerve damage are in progress.

Glutamate Carboxypeptidase Inhibition Reduces the Severity of Chemotherapy-Induced Peripheral Neurotoxicity in Rat

Chemotherapy is the most common method to treat cancer. The use of certain antineoplastic drugs, however, is associated with the development of peripheral neuropathy that can be dose-limiting. Excitotoxic glutamate release, leading to excessive glutamatergic neurotransmission and activation of N-methyl-D-aspartate (NMDA) receptors, is associated with neuronal damage and death in several nervous system disorders. N-Acetyl-aspartyl-glutamate (NAAG) is an abundant neuropeptide widely distributed in the central and peripheral nervous system which is physiologically hydrolyzed by the enzyme glutamate carboxypeptidase into N-Acetyl-aspartyl (NAA) and glutamate. Pharmacological inhibition of glutamate carboxypeptidase results in decreased glutamate and increased endogenous NAAG and has been shown to provide neuroprotection in several preclinical models. Here, we report the neuroprotective effect of an orally available glutamate carboxypeptidase inhibitor on three well-established animal models of chemotherapy (cisplatin, paclitaxel, bortezomib)-induced peripheral neuropathy. In all cases, glutamate carboxypeptidase inhibition significantly improved the chemotherapy-induced nerve conduction velocity deficits. In addition, morphological and morphometrical alterations induced by cisplatin and bortezomib in dorsal root ganglia (DRG) were improved by glutamate carboxypeptidase inhibition. Our data support a novel approach for the treatment of chemotherapy-induced peripheral neuropathy.

Dyslipidemia-Induced Neuropathy in Mice

OBJECTIVENeuropathy is a frequent and severe complication of diabetes. Multiple metabolic defects in type 2 diabetic patients result in oxidative injury of dorsal root ganglia (DRG) neurons. Our previous work focused on hyperglycemia clearly demonstrates induction of mitochondrial oxidative stress and acute injury in DRG neurons; however, this mechanism is not the only factor that produces neuropathy in vivo. Dyslipidemia also correlates with the development of neuropathy, even in pre-diabetic patients. This study was designed to explore the contribution of dyslipidemia in neuropathy.RESEARCH DESIGN AND METHODSMice (n = 10) were fed a control (10% kcal %fat) or high-fat (45% kcal %fat) diet to explore the impact of plasma lipids on the development of neuropathy. We also examined oxidized lipid–mediated injury in cultured DRG neurons from adult rat using oxidized LDLs (oxLDLs).RESULTSMice on a high-fat diet have increased oxLDLs and systemic and nerve oxidative stress. They develop nerve conduction velocity (NCV) and sensory deficits prior to impaired glucose tolerance. In vitro, oxLDLs lead to severe DRG neuron oxidative stress via interaction with the receptor lectin-like oxLDL receptor (LOX)-1 and subsequent NAD(P)H oxidase activity. Oxidative stress resulting from oxLDLs and high glucose is additive.CONCLUSIONSMultiple metabolic defects in type 2 diabetes directly injure DRG neurons through different mechanisms that all result in oxidative stress. Dyslipidemia leads to high levels of oxLDLs that may injure DRG neurons via LOX-1 and contribute to the development of diabetic neuropathy.

Peripheral neuropathy in mice with neuronal nitric oxide synthase gene deficiency

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.

Inducible nitric oxide synthase gene deficiency counteracts multiple manifestations of peripheral neuropathy in a streptozotocin-induced mouse model of diabetes

Evidence for the importance of peroxynitrite, a product of superoxide anion radical reaction with nitric oxide, in peripheral diabetic neuropathy is emerging. The role of specific nitric oxide synthase isoforms in diabetes-associated nitrosative stress and nerve fibre dysfunction and degeneration remains unknown. This study evaluated the contribution of inducible nitric oxide synthase (iNOS) to peroxynitrite injury to peripheral nerve and dorsal root ganglia and development of peripheral diabetic neuropathy. Control mice and mice with iNos (also known as Nos2) gene deficiency (iNos ( -/- )) were made diabetic with streptozotocin, and maintained for 6 weeks. Peroxynitrite injury was assessed by nitrotyrosine and poly(ADP-ribose) accumulation (immunohistochemistry). Thermal algesia was evaluated by paw withdrawal, tail-flick and hot plate tests, mechanical algesia by the Randall-Selitto test, and tactile allodynia by a von Frey filament test. Diabetic wild-type mice displayed peroxynitrite injury in peripheral nerve and dorsal root ganglion neurons. They also developed motor and sensory nerve conduction velocity deficits, thermal and mechanical hypoalgesia, tactile allodynia and approximately 36% loss of intraepidermal nerve fibres. Diabetic iNos ( -/- ) mice did not display nitrotyrosine and poly(ADP-ribose) accumulation in peripheral nerve, but were not protected from nitrosative stress in dorsal root ganglia. Despite this latter circumstance, diabetic iNos ( -/- ) mice preserved normal nerve conduction velocities. Small-fibre sensory neuropathy was also less severe in diabetic iNos ( -/- ) than in wild-type mice. iNOS plays a key role in peroxynitrite injury to peripheral nerve, and functional and structural changes of diabetic neuropathy. Nitrosative stress in axons and Schwann cells, rather than dorsal root ganglion neurons, underlies peripheral nerve dysfunction and degeneration.

Evaluation of the peroxynitrite decomposition catalyst Fe(III) tetra-mesitylporphyrin octasulfonate on peripheral neuropathy in a mouse model of type 1 diabetes

Whereas the important role of free radicals in diabetes-associated complications is well established, the contributions of the highly reactive oxidant peroxynitrite have not been properly explored. The present study used a pharmacological approach to evaluate the role of peroxynitrite in peripheral diabetic neuropathy. Control and STZ-diabetic mice were maintained with or without treatment with the potent peroxynitrite decomposition catalyst Fe(III) tetramesitylporphyrin octasulfonate (FeTMPS), at doses of 5 or 10 mg/kg/day in the drinking water for 3 weeks after an initial 3 weeks without treatment. Mice with a 6-week duration of diabetes developed clearly manifest motor (MNCV) and sensory nerve conduction velocity (SNCV) deficits, 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 44% loss of intraepidermal nerve fibers. They also had increased nitrotyrosine and poly(ADP-ribose) immunofluorescence in sciatic nerve, grey matter of the spinal cord, and dorsal root ganglion neurons. FeTMPS treatment alleviated or essentially corrected (at a dose of 10 mg/kg/day) MNCV and SNCV deficits, and was associated with less severe small sensory nerve fiber dysfunction and degeneration. Nitrotyrosine and poly(ADP-ribose) immunofluorescence in sciatic nerve, spinal cord, and dorsal root ganglion neurons in peroxynitrite decomposition catalyst-treated diabetic mice was markedly reduced. In conclusion, peroxynitrite contributes to large motor, large sensory, and small sensory fiber neuropathy in streptozotocin-diabetic mice. The findings provide rationale for development of potent peroxynitrite decomposition catalysts for the treatment of diabetic neuropathy.

Role of nitrosative stress in early neuropathy and vascular dysfunction in streptozotocin-diabetic rats

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.

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 (∼ 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 ∼ 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.

Leptin‐deficient (ob/ob) mouse‐ a new animal model of peripheral neuropathy of Type 2 diabetes and obesity

Whereas peripheral diabetic neuropathy (PDN) has extensively been explored in streptozotocin-diabetic rodents (Type 1 diabetes), insufficient information is available on PDN in Type 2 diabetic models. The latter represents a problem for clinical trial design, because the vast majority of diabetic patients have Type 2 (non-insulin-dependent) diabetes. We characterized PDN in leptin-deficient (ob/ob) mouse, a model of Type 2 diabetes with mild hyperglycemia and obesity. ~11-wk old ob/ob mice developed motor (MNCV) and sensory nerve conduction velocity (SNCV) deficits, thermal hypolagesia, tactile allodynia, and ~78% loss of intraepidermal nerve fibers. They also had increased sorbitol pathway activity in peripheral nerve, and increased nitrotyrosine and poly(ADP-ribose) immunofluorescence in nerve, spinal cord and dorsal root ganglia. Aldose reductase inhibition with fidarestat (16 mgkg−1d−1, for 6 wks, from 5 wks of age) preserved normal MNCV and SNCV, and alleviated thermal hypoalgesia and intraepidermal nerve fiber loss, but not tactile allodynia. Nitrotyrosine immunofluorescence and the number of poly(ADP-ribose) positive nuclei in nerve, spinal cord and DRGs of fidarestat-treated ob/ob mice did not differ from controls. In conclusion, ob/ob mouse is a new model that develops both large motor and sensory fiber and small sensory fiber PDN and responds to pathogenetic treatment.

The Leptin-Deficient (ob/ob) Mouse

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 ( approximately 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 ( approximately 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.

Effects of U83836E on nerve functions, hyperalgesia and oxidative stress in experimental diabetic neuropathy

Oxidative stress has been implicated to play an important role in the pathogenesis of diabetic neuropathy, which is the most common complication of diabetes mellitus affecting more than 50% of diabetic patients. In the present study, we have investigated the effect of U83836E [(−)-2-((4-(2,6-Di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl)methyl)-3,4-dihydro-2,3,7,8-tetramethyl-2 H-1-benzopyran-6-ol, 2HCl], a potent free radical scavenger in streptozotocin (STZ)-induced diabetic neuropathy in rats. STZ-induced diabetic rats showed significant deficit in motor nerve conduction velocity (MNCV), nerve blood flow (NBF) and thermal hyperalgesia after 8 weeks of diabetes induction, indicating development of diabetic neuropathy. Antioxidant enzyme (superoxide dismutase and catalase) levels were reduced and malondialdehyde (MDA) levels were significantly increased in diabetic rats as compared to the age-matched control rats, this indicates the involvement of oxidative stress in diabetic neuropathy. The 2-week treatment with U83836E (3 and 9 mg/kg, i.p.) started 6 weeks after diabetes induction significantly ameliorated the alterations in MNCV, NBF, hyperalgesia, MDA levels and antioxidant enzymes in diabetic rats. Results of the present study suggest the potential of U83836E in treatment of diabetic neuropathy.

Peripheral Nerve Dysfunction in Experimental Diabetes Is Mediated by Cyclooxygenase-2 and Oxidative Stress

Glucose-mediated oxidative stress and alterations in cyclooxygenase (COX) pathway activity with secondary deficits of endoneurial perfusion have been implicated in the pathogenesis of experimental diabetic neuropathy (EDN). We have previously reported that activation of the COX-2 pathway is an important mediator of neurochemical and neurovascular defects in EDN in a rat model. Considering that chemical COX inhibition may exert other pharmacological effects in addition to inhibition of COX activity, the aim of this study was to explore the role of COX-2 in experimental diabetic neuropathy, using a COX-2 knockout mouse model. Here we provide evidence that COX-2 inactivation had a protective effect against diabetes-induced motor and sensory nerve conduction slowing and impaired nerve antioxidative defense that were clearly manifest in the wild-type (COX-2(+/+)) diabetic mice. These preliminary data support the role of the activation of the COX-2 pathway in mediating sensory and motor nerve conduction velocity deficits in EDN. These findings also suggest that the COX-2 pathway seems to be an important modulator of oxidative stress in EDN.

Low-Dose Poly(ADP-Ribose) Polymerase Inhibitor-Containing Combination Therapies Reverse Early Peripheral Diabetic Neuropathy

Poly(ADP-ribose) polymerase (PARP) inhibition has recently been identified as a novel approach to treatment of experimental peripheral diabetic neuropathy (PDN). However, long-term inhibition of PARP, an enzyme involved in DNA repair, can potentially result in premature aging, loss of genome stability, and other side effects. This study evaluated potential synergistic interactions between low doses of the potent and specific PARP inhibitor 1,5-isoquinolinediol (ISO) and one of two vasodilators, the ACE inhibitor lisinopril (LIS) and the beta2-adrenoceptor agonist salbutamol (SAL) in the model of early PDN. Control and streptozotocin (STZ)-induced diabetic rats were treated with either ISO plus LIS or ISO plus SAL for 2 weeks after an initial 2 weeks without treatment. ISO (intraperitoneally) and LIS and SAL (both in the drinking water) were used in subtherapeutic doses, resulting in a minor correction of diabetes-associated sciatic motor and hind-limb digital sensory nerve conduction deficits when administered as monotherapies. Both combination treatments corrected endoneurial blood flow and vascular conductance deficits in STZ-induced diabetic rats. ISO plus SAL corrected all other changes of PDN, i.e., motor nerve conduction velocity (MNCV) and sensory nerve conduction velocity (SNCV) deficits as well as thermal and mechanical hyperalgesia. With ISO plus LIS, no significant correction of MNCV was observed, and the effect on thermal hyperalgesia was quite modest. SNCV and mechanical hyperalgesia were corrected. In vitro studies in human endothelial and Schwann cells showed early accumulation of poly(ADP-ribosyl)ated proteins (Western blot analysis) in response to high glucose, thus suggesting the importance of PARP activation in human PDN. In conclusion, low-dose PARP inhibitor-containing combination therapies may constitute a new approach for treatment of PDN.

Discovery of 3-[(4,5,7-Trifluorobenzothiazol-2-yl)methyl]indole-N-acetic Acid (Lidorestat) and Congeners as Highly Potent and Selective Inhibitors of Aldose Reductase for Treatment of Chronic Diabetic Complications

Recent efforts to identify treatments for chronic diabetic complications have resulted in the discovery of a novel series of highly potent and selective 3-[(benzothiazol-2-yl)methyl]indole-N-alkanoic acid aldose reductase inhibitors. The lead candidate, 3-[(4,5,7-trifluorobenzothiazol-2-yl)methyl]indole-N-acetic acid (lidorestat, 9) inhibits aldose reductase with an IC(50) of 5 nM, while being 5400 times less active against aldehyde reductase, a related enzyme involved in the detoxification of reactive aldehydes. It lowers nerve and lens sorbitol levels with ED(50)'s of 1.9 and 4.5 mg/kg/d po, respectively, in the 5-day STZ-induced diabetic rat model. In a 3-month diabetic intervention model (1 month of diabetes followed by 2 months of drug treatment at 5 mg/kg/d po), it normalizes polyols and reduces the motor nerve conduction velocity deficit by 59% relative to diabetic controls. It has a favorable pharmacokinetic profile (F, 82%; t(1/2), 5.6 h; Vd, 0.694 L/kg) with good drug penetration in target tissues (C(max) in sciatic nerve and eye are 2.36 and 1.45 mug equiv/g, respectively, when dosed with [(14)C]lidorestat at 10 mg/kg po).

Looking to the future: diabetic neuropathy and effects of rosuvastatin on neurovascular function in diabetes models

Vascular defects contribute substantially to neuropathic changes associated with metabolic abnormalities in diabetes. Statins appear to exert beneficial effects on vascular function independent of cholesterol lowering. In studies of the streptozotocin-induced diabetes rat model, rosuvastatin treatment was shown to correct sciatic motor and saphenous sensory nerve conduction velocity deficits (large fiber defects), thermal hyperalgesia (small fiber defects) and deficits in sciatic nerve and superior cervical ganglion blood flow. Rosuvastatin reversed deficits in nitrergic nerve–mediated vasodilation in the corpus cavernosum and corrected deficits in endothelium-derived hyperpolarising factor–mediated vasodilation in mesenteric vessels. Rosuvastatin did not alter plasma cholesterol in diabetic animals (a characteristic effect in this model), but significantly lowered triglycerides at high doses. Treatment with mevalonate reversed the beneficial effects of rosuvastatin on nerve function and blood flow, suggesting that the effects are mediated by inhibition of the cholesterol biosynthesis pathway in the absence of cholesterol reduction. Thus, rosuvastatin has wide-ranging neurovascular actions in diabetic rats that may be independent of lipid-lowering activity.

Transgenic mice overexpressing aldose reductase in Schwann cells show more severe nerve conduction velocity deficit and oxidative stress under hyperglycemic stress

To further understand the role of aldose reductase (AR) in the etiology of diabetic neuropathy, we generated transgenic mice that overexpress AR specifically in the Schwann cells under the control of the rat myelin protein zero (P 0) promoter. One of the transgenic mouse lines, which has overexpression of AR mRNA in the Schwann cell only and higher AR activity in the sciatic nerve, was used to examine the relationship between increased AR activity and motor nerve conduction velocity (MNCV) deficit under diabetic and galactosemic conditions. Under these conditions, nontransgenic mice showed a slight reduction in MNCV compared to those of controls. However, transgenic mice exhibited a significantly greater reduction in MNCV under these conditions, particularly under galactosemic condition, indicating that a Schwann cell-specific increase in aldose reductase activity is sufficient to produce the phenotype. Interestingly, under galactosemic condition where the difference in MNCV deficit between transgenic and nontransgenic mice was most pronounced, there was no significant difference in accumulated galactitol levels in the sciatic nerve between these mice. These results indicate that increase in AR activity leads to greater reduction of MNCV under galactosemic and diabetic conditions, but galactitol and sorbitol levels may not be good indicators of the severity of neuropathy. On the other hand, the level of reduced glutathione (GSH) in the sciatic nerve was found to be correlated with the severity of MNCV deficit under the diabetic condition. Diabetic AR transgenic mice showed significant reduction of GSH in their sciatic nerve, whereas the diabetic nontransgenic mice showed no reduction in GSH level compared to the nondiabetic control, suggesting that AR is a key contributor to oxidative stress under diabetic condition.

Effects of protein kinase Cbeta inhibition on neurovascular dysfunction in diabetic rats: interaction with oxidative stress and essential fatty acid dysmetabolism.

Elevated protein kinase C (PKC) activity is thought to play a substantial role in the aetiology of diabetic microvascular complications, the PKCbeta isoform being identified as particularly important. Neuropathy has a vascular component; therefore, one aim was to assess whether the PKCbeta inhibitor, LY333531, could correct nerve conduction velocity (NCV) and perfusion deficits in diabetic rats. Neurovascular dysfunction also depends on oxidant stress and impaired omega-6 essential fatty acid metabolism; correctable by antioxidant and gamma-linolenic acid (GLA) treatments, respectively. A second aim was to assess whether there were interactions between these mechanisms and PKCbeta-mediated effects. Diabetes was induced by streptozotocin; duration was 8 weeks. NCV was monitored and blood flow was assessed by hydrogen clearance microelectrode polarography. Diabetes caused 19.7% and 13.9% reductions in sciatic motor and saphenous sensory NCV, respectively. Two weeks of LY333531 treatment dose-dependently corrected these deficits. A dose of 10 mg kg(-1) day(-1) gave non-diabetic NCV values and also completely corrected a 50% diabetic reduction in sciatic endoneurial blood flow. Low-dose (0.25 mg kg(-1) day(-1)) LY333531 had modest effects ( approximately 20% correction) on NCV and sciatic perfusion. However, when combined with equi-effective doses of the antioxidants vitamin E or alpha-lipoic acid, or GLA, motor and sensory NCV and sciatic nerve perfusion were in the non-diabetic range. The joint effect was equivalent to that of the 10 mg kg(-1) day(-1) LY333531 dose, demonstrating synergism between PKCbeta, oxidative stress and essential fatty acid mechanisms. LY333531, alone or combined with antioxidants or GLA, could form the basis for therapeutic intervention in neuropathy, which requires assessment in clinical trials.

Workshop 5: NAAG and NAALADase: Functional Properties in the Central and Peripheral Nervous System. Naaladase (GCPII) knockout mice are less susceptible to neuropathy and stroke

Glutamate carboxypeptidase II (GCPII, also known as N‐acetylated‐alpha‐linked acidic dipeptidase or NAALADase) knockout (KO) mice were generated by inserting a GCPII targeting cassette containing a PGK‐Neo resistance marker and stop codons in exons 1 and 2, and removal of exons 1 and 2 intron/exon boundary sequence. Embryonic stem cells were injected into C57BL6 blastocysts, and chimeric offspring born. Germline transmission was confirmed by mating the chimeras to generate heterozygous KO mice. Crossing heterozygous mice generated F2 generation mice homozygous for the null mutant, as confirmed by loss of GCPII protein. NAAG hydrolyzing activity was minimal (0.07 pmol/mg/min) in KO tissue, with normal levels (4.82 pmol/mg/min) in wild types and intermediate levels (1.73 pmol/mg/min) in heterozygotes. Preliminary neuropathy experiments showed KO mice are less affected by nerve‐crush and recover faster from the damage‐induced neuropathy, as indicated by EMG recording and nerve morphology. Similarly, GCPII KO mice subjected to high dose vitamin B6 displayed less severe neuropathy than wild types, as indicated by reduced sensory nerve conduction velocity and morphological deficits. Also, in a transient middle cerebral artery occlusion model, GCPII KO mice were significantly more resistant to the effects of cerebral ischemia than their wildtype littermates. Findings support GCPII involvement in stroke and in mediating chronic neuropathic conditions and suggest GCPII inhibitors may be useful in treatment of brain ischemia as well as peripheral neuropathies.

NAALADase (GCP II) inhibition as a novel therapeutic target for neuropathic pain, diabetic neuropathy, and ALS

NAALADase (N‐acetylated‐α‐linked‐acidic dipeptidase; also termed glutamate carboxypeptidase II) is an enzyme that catalyzes hydrolysis of the abundant brain peptide N‐acetyl‐aspartyl‐glutamate (NAAG) to N acetyl‐aspartate (NAA) and glutamate. We have synthesized many potent and selective NAALADase inhibitors from distinct chemical classes. In rodents, selective inhibitors have been shown to increase NAAG and decrease glutamate extracellularly and provide therapeutic benefits in animal models of neurological diseases where excess glutamate neurotransmission has been implicated. Specifically, NAALADase inhibitors exhibit neuroprotection in rats following transient middle cerebral artery occlusion, attenuate neuropathic pain following chronic constrictive injury, prevent nerve conduction velocity deficits and sural nerve degeneration following chronic diabetes in STZ and BB/W rats, and delay clinical symptoms and mortality in the mouse SOD transgenic model of ALS. These biological effects are likely mediated by increases in extracellular NAAG and decreases in extracellular glutamate as a consequence of NAALADase inhibition. NAALADase inhibition may be useful in neurological disorders in which excessive excitatory amino acid transmission is pathogenic. Also, NAALADase inhibition may represent a novel glutamate regulating strategy devoid of the side‐effects that have hampered development of postsynaptic glutamate receptor antagonists. A lead NAALADase inhibitor termed GPI 5693 is currently completing Phase I clinical testing.

Reduced Ia‐afferent‐mediated Hoffman reflex in streptozotocin‐induced diabetic rats

In addition to reduced nerve conduction velocity, diabetic neuropathic patients often exhibit a reduction in the amplitude of the compound muscle action potential elicited by stimulation of the la‐afferent‐mediated reflex pathway (Hoffman or H wave) that can contribute to diminished or absent tendon reflexes. In contrast to nerve conduction velocity deficits, changes in H‐wave amplitudes have not been reproduced in diabetic animal models. Using electrophysiological techniques developed for repeated recordings in individual animals, we report H‐wave deficits in streptozotocin (STZ)‐treated insulin‐dependent diabetic rats. After 4 weeks of diabetes induced by STZ treatment, a 47% reduction in the H‐wave amplitude was demonstrated by recording compound muscle action potentials in foot muscles after stimulation of la afferents. Interestingly, we also demonstrate that the H‐wave amplitude gradually recovers to a 26% deficit after 12 weeks of experimental diabetes. The recovery of the H wave in STZ‐treated rats distinguishes this deficit mechanistically from other STZ‐induced electrophysiological changes and may model a similar recovery of the H wave reported in diabetic patients.