电穿孔介导质粒DNA肿瘤内转移抑制恶性肿瘤生长与转移

利用携带绿色荧光蛋白（green fluorescent protein, GFP）编码基因的表达质粒,测试电穿孔方法介导目的基因活体组织内转移的效率并优化电击参数.在此基础上采用电穿孔技术直接将编码白介素12（IL-12）、白介素2（IL-2）、粒单细胞克隆刺激因子（GM-CSF）等免疫调节因子或反义血管内皮细胞生长因子121（VEGF₁₂₁）、可溶性血管内皮细胞膜受体（sFlk-1及ExTek）等血管生成抑制因子表达质粒转移至肿瘤局部.实验结果表明电穿孔介导GFP表达质粒肌肉内转移的效率较高,GFP可在肌细胞内持续高水平表达3周以上,而在肿瘤细胞内只能表达4～6 d,但高电压短脉冲电击组肿瘤内GFP阳性细胞数比低电压长脉冲组高2.68倍.多次电击介导IL-12表达质粒转移至肿瘤组织内,可有效地抑制小鼠膀胱癌BTT-gfp、人乳腺癌MCF-7及肝癌SMMC 7721-gfp的生长.MCF-7对血管生成抑制因子基因转移治疗较敏感,单独应用反义VEGF₁₂₁、sFlk-1或ExTek即显示明确的治疗效果.SMMC 7721-gfp单独应用sFlk-1有效.小鼠膀胱癌对单独应用反义VEGF₁₂₁、sFlk-1或ExTek治疗效果不理想,但联合应用sFlk-1和ExTek仍然可以有效地抑制肿瘤生长与转移,甚至使肿瘤缩小或消失.提示电穿孔技术是一项高效、安全、经济的体内基因转移方法.     

Targeting Apoptosis Signalling Kinase-1 (ASK-1) Does Not Prevent the Development of Neuropathy in Streptozotocin-Induced Diabetic Mice

Apoptosis signal-regulating kinase-1 (ASK1) is a mitogen-activated protein 3 kinase (MAPKKK/MAP3K) which lies upstream of the stress-activated MAPKs, JNK and p38. ASK1 may be activated by a variety of extracellular and intracellular stimuli. MAP kinase activation in the sensory nervous system as a result of diabetes has been shown in numerous preclinical and clinical studies. As a common upstream activator of both p38 and JNK, we hypothesised that activation of ASK1 contributes to nerve dysfunction in diabetic neuropathy. We therefore wanted to characterize the expression of ASK1 in sensory neurons, and determine whether the absence of functional ASK1 would protect against the development of neuropathy in a mouse model of experimental diabetes. ASK1 mRNA and protein is constitutively expressed by multiple populations of sensory neurons of the adult mouse lumbar DRG. Diabetes was induced in male C57BL/6 and transgenic ASK1 kinase-inactive (ASK1n) mice using streptozotocin. Levels of ASK1 do not change in the DRG, spinal cord, or sciatic nerve following induction of diabetes. However, levels of ASK2 mRNA increase in the spinal cord at 4 weeks of diabetes, which could represent a future target for this field. Neither motor nerve conduction velocity deficits, nor thermal or mechanical hypoalgesia were prevented or ameliorated in diabetic ASK1n mice. These results suggest that activation of ASK1 is not responsible for the nerve deficits observed in this mouse model of diabetic neuropathy.     

Transplantation of bone marrow-derived mononuclear cells improves mechanical hyperalgesia, cold allodynia and nerve function in diabetic neuropathy

Relief from painful diabetic neuropathy is an important clinical issue. We have previously shown that the transplantation of cultured endothelial progenitor cells or mesenchymal stem cells ameliorated diabetic neuropathy in rats. In this study, we investigated whether transplantation of freshly isolated bone marrow-derived mononuclear cells (BM-MNCs) alleviates neuropathic pain in the early stage of streptozotocin-induced diabetic rats. Two weeks after STZ injection, BM-MNCs or vehicle saline were injected into the unilateral hind limb muscles. Mechanical hyperalgesia and cold allodynia in SD rats were measured as the number of foot withdrawals to von Frey hair stimulation and acetone application, respectively. Two weeks after the BM-MNC transplantation, sciatic motor nerve conduction velocity (MNCV), sensory nerve conduction velocity (SNCV), sciatic nerve blood flow (SNBF), mRNA expressions and histology were assessed. The BM-MNC transplantation significantly ameliorated mechanical hyperalgesia and cold allodynia in the BM-MNC-injected side. Furthermore, the slowed MNCV/SNCV and decreased SNBF in diabetic rats were improved in the BM-MNC-injected side. BM-MNC transplantation improved the decreased mRNA expression of NT-3 and number of microvessels in the hind limb muscles. There was no distinct effect of BM-MNC transplantation on the intraepidermal nerve fiber density. These results suggest that autologous transplantation of BM-MNCs could be a novel strategy for the treatment of painful diabetic neuropathy.     

PARP inhibition or gene deficiency counteracts intraepidermal nerve fiber loss and neuropathic pain in advanced diabetic neuropathy

Evidence that poly(ADP-ribose) polymerase (PARP) activation plays an important role in diabetic complications is emerging. This study evaluated the role of PARP in rat and mouse models of advanced diabetic neuropathy. The orally active PARP inhibitor 10-(4-methylpiperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one (GPI-15427; formulated as a mesilate salt, 30 mg kg(-1) day(-1) in the drinking water for 10 weeks after the first 2 weeks without treatment) at least partially prevented PARP activation in peripheral nerve and DRG neurons, as well as thermal hypoalgesia, mechanical hyperalgesia, tactile allodynia, exaggerated response to formalin, and, most importantly, intraepidermal nerve fiber degeneration in streptozotocin-diabetic rats. These findings are consistent with the lack of small sensory nerve fiber dysfunction in diabetic PARP -/- mice. Furthermore, whereas diabetic PARP +/+ mice displayed approximately 46% intraepidermal nerve fiber loss, diabetic PARP -/- mice retained completely normal intraepidermal nerve fiber density. In conclusion, PARP activation is an important contributor to intraepidermal nerve fiber degeneration and functional changes associated with advanced Type 1 diabetic neuropathy. The results support a rationale for the development of potent and low-toxicity PARP inhibitors and PARP inhibitor-containing combination therapies.     

Tanshinone IIA improves impaired nerve functions in experimental diabetic rats

Diabetic neuropathy is one of the most common complications in diabetes mellitus. Thus far, effective therapeutic agents for restoring the impaired motor and sensory nerve functions in diabetic neuropathy are still lacking. The antioxidant and neuroprotective properties of tanshinone IIA make it a promising candidate for the treatment of diabetic neuropathy. Therefore, the present study investigated the possible beneficial effect of tanshinone IIA on the impaired nerve functions displayed by a rat diabetic model. Insulin-dependent diabetes in rats was developed by a single dose of streptozotocin (STZ) at 50 mg/kg. The diabetic rats were randomly divided into four groups (n = 10 in each group), and were intraperitoneally administrated daily for 4 weeks with tanshinone IIA (20 mg/kg, 50 mg/kg and 100 mg/kg), or normal saline from the fourth day after STZ injection, respectively. At the end of tanshinone IIA administration, thermal and mechanical nociceptive threshold were determined by a hot plate test and Von Frey hairs; motor nerve conducting velocity (MNCV) was determined by an electrophysiological method; nerve blood flow (NBF) was detected using a laser Doppler flow meter; Na⁺,K⁺ATPase activity, the level of superoxide dismutase (SOD), catalase and malondialdehyde (MDA) in sciatic nerves, and the serum total antioxidant capability were also determined. We found that tanshinone IIA was capable of restoring diabetes-induced deficit in nerve functions (MNCV and NBF), and impairment in thermal and mechanical nociceptive capability. In addition, tanshinone IIA significantly increased the serum total antioxidant capability, improved the activities of Na⁺,K⁺ATPase, increased the levels of SOD and catalase, and reduced the MDA level in sciatic nerves in diabetic rats. All the findings indicate the beneficial effect of tanshinone IIA on impaired nerve functions and raise the possibility of developing tanshinone IIA as a therapeutic agent for diabetic neuropathy.     

Favorable effect of VEGF gene transfer on ischemic peripheral neuropathy

Ischemic peripheral neuropathy is a frequent, irreversible complication of lower extremity vascular insufficiency. We investigated whether ischemic peripheral neuropathy could be prevented and/or reversed by gene transfer of an endothelial cell mitogen designed to promote therapeutic angiogenesis. Intramuscular gene transfer of naked DNA encoding vascular endothelial growth factor (VEGF) simultaneously with induction of hindlimb ischemia in rabbits abrogated the substantial decrease in motor and sensory nerve parameters, and nerve function recovered promptly. When gene transfer was administered 10 days after induction of ischemia, nerve function was restored earlier and/or recovered faster than in untreated rabbits. These findings are due in part to enhanced hindlimb perfusion. In addition, however, the demonstration of functional VEGF receptor expression by Schwann cells indicates a direct effect of VEGF on neural integrity as well. These findings thus constitute a new paradigm for the treatment of ischemic peripheral neuropathy.     

Inhibiting heat-shock protein 90 reverses sensory hypoalgesia in diabetic mice

Increasing the expression of Hsp70 (heat-shock protein 70) can inhibit sensory neuron degeneration after axotomy. Since the onset of DPN (diabetic peripheral neuropathy) is associated with the gradual decline of sensory neuron function, we evaluated whether increasing Hsp70 was sufficient to improve several indices of neuronal function. Hsp90 is the master regulator of the heat-shock response and its inhibition can up-regulate Hsp70. KU-32 (N-{7-[(2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyl-tetrahydro-2H-pyran-2-yloxy]-8-methyl-2-oxo-2H-chromen-3-yl}acetamide) was developed as a novel, novobiocin-based, C-terminal inhibitor of Hsp90 whose ability to increase Hsp70 expression is linked to the presence of an acetamide substitution of the prenylated benzamide moiety of novobiocin. KU-32 protected against glucose-induced death of embryonic DRG (dorsal root ganglia) neurons cultured for 3 days in vitro. Similarly, KU-32 significantly decreased neuregulin 1-induced degeneration of myelinated Schwann cell DRG neuron co-cultures prepared from WT (wild-type) mice. This protection was lost if the co-cultures were prepared from Hsp70.1 and Hsp70.3 KO (knockout) mice. KU-32 is readily bioavailable and was administered once a week for 6 weeks at a dose of 20 mg/kg to WT and Hsp70 KO mice that had been rendered diabetic with streptozotocin for 12 weeks. After 12 weeks of diabetes, both WT and Hsp70 KO mice developed deficits in NCV (nerve conduction velocity) and a sensory hypoalgesia. Although KU-32 did not improve glucose levels, HbA1c (glycated haemoglobin) or insulin levels, it reversed the NCV and sensory deficits in WT but not Hsp70 KO mice. These studies provide the first evidence that targeting molecular chaperones reverses the sensory hypoalgesia associated with DPN.     

Normal blood flow but lower oxygen tension in diabetes of young rats: microenvironment and the influence of sympathectomy.

Studies of rats with experimental streptozotocin (STZ)-induced diabetes at 4 months have identified sciatic nerve trunk oligemia and hypoxia, but it is uncertain how early these abnormalities develop or which develops first. We studied young (4-week-old) rats after 6 or 16 weeks of STZ-induced diabetes (or after citrate buffer injection in controls) by recording multi-fiber conduction in three different nerve territories and by measuring sciatic endoneurial blood flow (NBF) and oxygen tension (PnO2) at end point. To evaluate the impact of sympathectomy on this diabetic model, separate animal groups were treated for 5 weeks with guanethidine monosulfate given at the onset of diabetes (group 1, end point 6 weeks) or after 6 weeks of diabetes (group 2, end point 16 weeks). Diabetes was associated with deficits in sensory and motor caudal conduction and increased resistance to ischemic conduction failure (RICF). NBF was comparable to control animals at both time points and was within the published normal range of NBF. In contrast, oxygen tensions were shifted to lower values in diabetic animals. Sympathectomy was associated with blunting of the RICF increase in group 2 but worsened caudal sensory conduction despite evidence of modest improvement in sciatic nerve oxygenation. Our findings support the concept that neuropathy occurs early in diabetes and that hypoxia develops before oligemia. Sympathectomy did not benefit this diabetic model.     

Suppression of NF-κB and NF-κB regulated oxidative stress and neuroinflammation by BAY 11-7082 (IκB phosphorylation inhibitor) in experimental diabetic neuropathy

Inflammation is an emerging patho-mechanism of diabetes and its complications. NF-κB pathway is one of the central machinery initiating and propagating inflammatory responses. The present study envisaged the involvement of NF-κB inflammatory cascade in the pathophysiology of diabetic neuropathy using BAY 11-7082, an IκB phosphorylation inhibitor. Streptozotocin was used to induce diabetes in Sprauge Dawley rats. BAY 11-7082 (1 &; 3 mg/kg) was administered to diabetic rats for 14 days starting from the end of six weeks post diabetic induction. Diabetic rats developed deficits in nerve functions and altered nociceptive parameters and also showed elevated expression of NF-κB (p65), IκB and p-IκB along with increased levels of IL-6 &; TNF-α and inducible enzymes (COX-2 and iNOS). Furthermore, there was an increase in oxidative stress and decrease in Nrf2/HO-1 expression. We observed that BAY 11-7082 alleviated abnormal sensory responses and deficits in nerve functions. BAY 11-7082 also ameliorated the increase in expression of NF-κB, IκB and p-IκB. BAY 11-7082 curbed down the levels of IL-6, TNF-α, COX-2 and iNOS in the sciatic nerve. Lowering of lipid peroxidation and improvement in GSH levels was also seen along with increased expression of Nrf2/HO-1. Thus it can be concluded that NF-κB expression and downstream expression of proinflammatory mediators are prominent features of nerve damage leading to inflammation and oxidative stress and BAY 11-7082 was able to ameliorate experimental diabetic neuropathy by modulating neuroinflammation and improving antioxidant defence.     

Sildenafil Ameliorates Long Term Peripheral Neuropathy in Type II Diabetic Mice

Diabetic peripheral neuropathy is a common complication of long-standing diabetes mellitus. To mimic clinical trials in which patients with diabetes enrolled have advanced peripheral neuropathy, we investigated the effect of sildenafil, a specific inhibitor of phosphodiesterase type 5 enzyme, on long term peripheral neuropathy in middle aged male mice with type II diabetes. Treatment of diabetic mice (BKS.Cg-m+/+Lepr^db/J, db/db) at age 36 weeks with sildenafil significantly increased functional blood vessels and regional blood flow in the sciatic nerve, concurrently with augmentation of intra-epidermal nerve fiber density in the skin and myelinated axons in the sciatic nerve. Functional analysis showed that the sildenafil treatment considerably improved motor and sensory conduction velocities in the sciatic nerve and peripheral thermal stimulus sensitivity compared with the saline treatment. In vitro studies showed that mouse dermal endothelial cells (MDE) cultured under high glucose levels exhibited significant down regulation of angiopoietin 1 (Ang1) expression and reduction of capillary-like tube formation, which were completely reversed by sildenafil. In addition, incubation of dorsal root ganglia (DRG) neurons with conditioned medium harvested from MDE under high glucose levels suppressed neurite outgrowth, where as conditional medium harvested from MDE treated with sildenafil under high glucose levels did not inhibit neurite outgrowth of DRG neurons. Moreover, blockage of the Ang1 receptor, Tie2, with a neutralized antibody against Tie2 abolished the beneficial effect of sildenafil on tube formation and neurite outgrowth. Collectively, our data indicate that sildenafil has a therapeutic effect on long term peripheral neuropathy of middle aged diabetic mice and that improvement of neurovascular dysfunction by sildenafil likely contributes to the amelioration of nerve function. The Ang1/Tie2 signaling pathway may play an important role in these restorative processes.     

Mechanism of in vivo DNA transport into cells by electroporation: electrophoresis across the plasma membrane may not be involved

BACKGROUND: Recently, in vivo gene transfer with electroporation (electro-gene transfer) has emerged as a leading technology for developing nonviral gene therapies and nucleic acid vaccines. The widely hypothesized mechanism is that electroporation induces structural defects in the membrane and provides an electrophoretic force to facilitate DNA crossing the permeabilized membrane. In this study, we have designed a device and experiments to test the hypothesis. METHODS: In this study, we have designed a device that alternates the polarity of the applied electric field to elucidate the mechanism of in vivo electro-gene transfer. We also designed experiments to challenge the theory that the low-voltage (LV) pulses cannot permeabilize the membrane and are only involved in DNA electrophoresis, and answer the arguments that (1) the reversed polarity pulses can cause opposing sides of the cell membrane to become permeabilized and provide the electrophoresis for DNA entry; or (2) once DNA enters cytoplasmic/endosomal compartments after electroporation, it may bind to cellular entities and might not be reversibly extracted. Thus a gradual buildup of the DNA in the cell still seems quite possible even under the condition of the rapid reversal of polarity. RESULTS: Our results indicate that electrophoresis does not play an important role in in vivo electro-gene transfer. CONCLUSIONS: This study provides new insights into the mechanism of electro-gene transfer, and may allow the definition of newer and more efficient conditions for in vivo electroporation.     

Concurrent targeting of nitrosative stress-PARP pathway corrects functional, behavioral and biochemical deficits in experimental diabetic neuropathy

Peroxynitrite mediated nitrosative stress, an indisputable initiator of DNA damage and overactivation of poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated after sensing DNA damage, are two crucial pathogenetic mechanisms in diabetic neuropathy. The intent of the present study was to investigate the effect of combination of a peroxynitrite decomposition catalyst (PDC), FeTMPyP and a PARP inhibitor, 4-ANI against diabetic peripheral neuropathy. The end points of evaluation of the study included motor nerve conduction velocity (MNCV) and nerve blood flow (NBF) for evaluating nerve functions; thermal hyperalgesia and mechanical allodynia for assessing nociceptive alterations, malondialdehyde and peroxynitrite levels to detect oxidative stress-nitrosative stress; NAD concentration in sciatic nerve to assess overactivation of PARP. Additionally immunohistochemical studies for nitrotyrosine and Poly(ADP-ribose) (PAR) was also performed. Treatment with the combination of FeTMPyP and 4-ANI led to significant improvement in nerve functions and pain parameters and also attenuated the oxidative-nitrosative stress markers. Further, the combination also reduced the overactivation of PARP as evident from increased NAD levels and decreased PAR immunopositivity in sciatic nerve microsections. Thus, it can be concluded that treatment with the combination of a PDC and PARP inhibitor attenuates alteration in peripheral nerves in diabetic neuropathy (DN).     

Synergistic vasculogenic effects of AMD3100 and stromal-cell-derived factor-1α in vasa nervorum of the sciatic nerve of mice with diabetic peripheral neuropathy

Autologous endothelial progenitor cell (EPC) transplantation has been suggested as a potential therapeutic approach in diabetic neuropathy (DN). However, such treatment might be limited by safety concerns regarding possible unwanted proliferation or differentiation of the transplanted stem cells. An alternative approach is the stimulation of endogenous bone-marrow-derived EPC (BM-EPC) recruitment into ischemic lesions by the administration of stem cell mobilization agents or chemokines. We first tested the EPC mobilization effect of vascular endothelial growth factor (VEGF) and AMD3100 in a mouse model of diabetes and found that AMD3100 was effective as an EPC mobilization agent, whereas VEGF did not increase circulating EPCs in these animals. Because recent studies have suggested that deceased local expression of stromal-cell-derived factor (SDF)-1α in diabetes is the main cause of defective EPC migration, AMD3100 was administrated systemically to stimulate EPC mobilization and SDF-1α was injected locally to enhance its migration into the streptozotocin-induced DN mice model. This combined therapy increased local expression levels of vasculogenesis-associated factors and newly formed endothelial cells in the sciatic nerve, resulting in the restoration of the sciatic vasa nervorum. The treatment also improved the impaired conduction velocity of the sciatic nerve in DN mice. Thus, AMD3100 might be an effective EPC mobilization agent in diabetes, with local SDF-1α injection synergistically increasing vascularity in diabetic nerves. This represents a novel potential therapeutic option for DN patients.     

PARP inhibition alleviates diabetes-induced systemic oxidative stress and neural tissue 4-hydroxynonenal adduct accumulation: correlation with peripheral nerve function

This study evaluated the role of poly(ADP-ribose) polymerase (PARP) in systemic oxidative stress and 4-hydoxynonenal adduct accumulation in diabetic peripheral neuropathy. Control and streptozotocin-diabetic rats were maintained with or without treatment with the PARP inhibitor, 1,5-isoquinolinediol, 3 mg kg(-1) day(-1), for 10 weeks after an initial 2 weeks. Treatment efficacy was evaluated by poly(ADP-ribosyl)ated protein content in peripheral nerve and spinal cord (Western blot analysis) and dorsal root ganglion neurons and nonneuronal cells (fluorescence immunohistochemistry), as well as by indices of peripheral nerve function. Diabetic rats displayed increased urinary isoprostane and 8-hydroxy-2'-deoxyguanosine excretion (ELISA) and 4-hydroxynonenal adduct accumulation 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 (double-label fluorescence immunohistochemistry), as well as motor and sensory nerve conduction velocity deficits, thermal hypoalgesia, and tactile allodynia. PARP inhibition counteracted diabetes-induced systemic oxidative stress and 4-hydroxynonenal adduct accumulation in peripheral nerve and spinal cord (Western blot analysis) and dorsal root ganglion neurons (perikarya, fluorescence immunohistochemistry), which correlated with improvement of large and small nerve fiber function. The findings reveal the important role of PARP activation in systemic oxidative stress and 4-hydroxynonenal adduct accumulation in diabetic peripheral neuropathy.     

Sensory Nerve Innervation of Epineurial Arterioles of the Sciatic Nerve Containing Calcitonin Gene–Related Peptide: Effect of Streptozotocin-Induced Diabetes

The authors have determined that epineurial arterioles of the sciatic nerve are innervated by nonadrenergic, noncholinergic nerves that contribute to the regulation of vasodilation. Using immunohistochemistry, the authors determined that nerves innervating epineurial arterioles contain the neuropeptide calcitonin gene–related peptide (CGRP). Using streptozotocin-induced diabetic rats, the authors demonstrated that CGRP content in sensory nerves innervating epineurial arterioles and vasodilation in response to exogenous CGRP was decreased. In summary, epineurial arterioles of the sciatic nerve are innervated by sensory nerves containing the neuropeptide CGRP. The diabetes-like condition induced by streptozotocin reduces the content of CGRP in these nerves and exogenous CGRPmediated vasodilation. CGRP is likely an important regulator of vascular tone and compromising its function could contribute to nerve ischemia and diabetic neuropathy.     

A comprehensive study of optimal conditions for naked plasmid DNA transfer into skeletal muscle by electroporation

Efficient gene transfer is a key factor in gene therapy. Reducing the damage caused by gene transfer to muscle by electroporation is very important for its clinical application. Extensive investigation of optimal conditions for gene transfer by electroporation is required. The parameters used for electroporation, including plasmid concentration; injection volume; the plasmid dose of the injection; the concentration of saline media; the size of plasmid DNA; the age of the mice; the lag time between plasmid injection and electroporation; and the effect of repeated gene transfer by electroporation, were systematically investigated in the present study. The efficiencies of gene transfer by electroporation in normal and rodent models of diabetes were also evaluated. We found that electroporation used for non-viral gene transfer could be repeated in the same place in the muscle, but the expression efficiency was closely related to the muscle damage. Increasing pulse times could enhance the efficiency of gene transfer with a lower strength of electric field. It was better to use a higher plasmid concentration than to use a larger dose of plasmid and repeated injection to achieve a high level of transgene expression. Optimal conditions varied in different animal models, being milder for diabetic mice than for normal mice, and it was also shown that the conditions that worked well on these small rodents were not necessarily suitable for larger animals. Our results provide a comprehensive view of the factors that affect the efficiency of gene transfer into skeletal muscle by electroporation.     

Pentoxifylline Effects on Nerve Conduction Velocity and Blood Flow in Diabetic Rats

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

Formononetin Ameliorates Diabetic Neuropathy by Increasing Expression of SIRT1 and NGF

Diabetic neuropathy is commonly observed complication in more than 50 % of type 2 diabetic patients. Histone deacetylases including SIRT1 have significant role to protect neuron from hyperglycemia induced damage. Formononetin (FMNT) is known for its effect to control hyperglycemia and also activate SIRT1. In present study, we evaluated effect of FMNT as SIRT1 activator in type 2 diabetic neuropathy. Type 2 diabetic neuropathy was induced in rats by modification of diet for 15 days using high fat diet and administration of streptozotocin (35 mg/kg/day, i. p.). FMNT treatment was initiated after confirmation of type 2 diabetes. Treatment was given for 16 weeks at 10, 20 and 40 mg/kg/day dose orally. FMNT treatment‐controlled hypoglycemia and reduced insulin resistance significantly in diabetic animals. FMNT treatment reduced oxidative stress in sciatic nerve tissue. FMNT treatment also reduced thermal hyperalgesia and mechanical allodynia significantly. It improved conduction velocity in nerve and unregulated SIRT1 and NGF expression in sciatic nerve tissue. Results of present study indicate that continuous administration of FMNT protected diabetic animals from hyperglycemia induced neuronal damage by controlling hyperglycemia and increasing SIRT1 and NGF expression in nerve tissue. Thus, FMNT can be an effective candidate for treatment of type 2 diabetic neuropathy.     

Effects of resveratrol on nerve functions, oxidative stress and DNA fragmentation in experimental diabetic neuropathy

Oxidative stress has been implicated in pathophysiology of diabetic neuropathy. All the pathways responsible for development of diabetic neuropathy are linked to oxidative stress in one way or the other. In the present study, we have targeted oxidative stress in diabetic neuropathy using resveratrol, a potent antioxidant. Eight weeks streptozotocin-diabetic rats developed neuropathy which was evident from significant reduction in motor nerve conduction velocity (MNCV), nerve blood flow (NBF) and increased thermal hyperalgesia. The 2-week treatment with resveratrol (10 and 20 mg/kg, i.p.) started 6 weeks after diabetes induction significantly ameliorated the alterations in MNCV, NBF, and hyperalgesia. Resveratrol also attenuated enhanced levels of malondialdehyde (MDA), peroxynitrite and produced increase in catalase levels in diabetic rats. There was marked reduction in DNA fragmentation observed after resveratrol treatment in diabetic rats as evident from decrease in Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positive cells in sciatic nerve sections. Results of the present study suggest the potential of resveratrol in treatment of diabetic neuropathy and its protective effect may be mediated through reduction in oxidative stress and DNA fragmentation.