Neuroprotective potential of combination of resveratrol and 4-amino 1,8 naphthalimide in experimental diabetic neuropathy: Focus on functional,sensorimotor and biochemical changes

The present study investigated whether combination of resveratrol and 4-amino 1,8 naphthalimide (4-ANI) is effective in the development of diabetic neuropathy (DN). After 6 weeks of diabetes induction, rats were treated for 2 weeks with resveratrol and 4-amino 1,8 naphthalimide (4-ANI) either alone or in combination. Experimental end points included functional, behavioural and biochemical parameters along with PAR immunohistochemistry and were performed at the end of treatment. Combination of resveratrol (10 mg/kg) and 4-ANI (3 mg/kg) attenuated conduction and nerve blood flow deficits and resulted in amelioration of diabetic neuropathic pain. Significant reversal of biochemical alterations (peroxynitrite, MDA and NAD levels) were also observed, as well as PAR accumulation in the sciatic nerve. This study suggests the beneficial effect of combining resveratrol and 4-ANI in experimental diabetic neuropathy.     

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).     

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.     

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-2H-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.     

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.     

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.     

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.     

The effects of zenarestat, an aldose reductase inhibitor, on minimal F-wave latency and nerve blood flow in streptozotocin-induced diabetic rats

The effects of zenarestat, 3-(4-bromo-2-fluorobenzyl)-7-chloro-3,4-dihydro-2,4-dioxo-1(2H)-quinazolineacetic acid, an aldose reductase inhibitor (ARI), on F-wave conduction abnormalities, nerve blood flow (NBF) reduction and sorbitol accumulation were studied in streptozotocin-induced diabetic rats. Two weeks after the induction of diabetes, zenarestat was given once a day for two weeks. In diabetic control rats, marked accumulation of sorbitol, reduction of NBF and prolongation of minimal F-wave latency (FWL) were observed as compared to normal rats. Zenarestat, at a dose of 32 mg/kg, inhibited sorbitol concentration to nearly the normal rat level and significantly improved not only NBF but also minimal FWL. At a dose of 3.2 mg/kg, sorbitol accumulation was inhibited by approximately 40% and there was a tendency to increase in NBF; however, minimal FWL was not improved at all. These data suggest that a highly inhibition of the nerve sorbitol accumulation is requisite for the treatment of diabetic peripheral 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.     

Protective effect of stobadine on NCV in streptozotocin-diabetic rats: augmentation by vitamin E

Hyperglycaemia-induced oxidative stress makes an important contribution to the aetiology of diabetic neuropathy. Elevated reactive oxygen species (ROS) cause cumulative damage to neurons and Schwan cells, however, they also have a deleterious effect on nerve blood flow causing endoneurial hypoxia, which is responsible for early nerve conduction velocity (NCV) deficits and contributes to an increase in resistance to ischaemic conduction failure (RICF). We tested whether antioxidants - stobadine, vitamin E or the combination of these drugs, could prevent the early signs of neural dysfunction in animal model of diabetes in 8-9 weeks old male Wistar rats, made diabetic by streptozotocin (55 mg/kg i.v.) 4 months prior to testing. Neuropathy was evaluated electrophysiologically by measuring motor NCV and RICF of sciatic nerve in vitro. We observed that treatment with the combination of stobadine and vitamin E significantly (p < 0.001) reduced the NCV slowing in diabetic rats, although it did not fully prevent the NCV impairment. Significant effect (p < 0.05) was observed also in stobadine monotherapy. The RICF elevated in diabetic animals was not affected by any drug applied. This study confirmed that treatment with appropriate antioxidants, especially their combination could partially prevented the decrease in NCV in diabetic rats.     

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.     

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

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

Correction of protein kinase C activity and macrophage migration in peripheral nerve by pioglitazone, peroxisome proliferator activated-gamma-ligand, in insulin-deficient diabetic rats

Pioglitazone, one of thiazolidinediones, a peroxisome proliferator-activated receptor (PPAR)-γ ligand, is known to have beneficial effects on macrovascular complications in diabetes, but the effect on diabetic neuropathy is not well addressed. We demonstrated the expression of PPAR-γ in Schwann cells and vascular walls in peripheral nerve and then evaluated the effect of pioglitazone treatment for 12 weeks (10 mg/kg/day, orally) on neuropathy in streptozotocin-diabetic rats. At end, pioglitazone treatment improved nerve conduction delay in diabetic rats without affecting the expression of PPAR-γ. Diabetic rats showed suppressed protein kinase C (PKC) activity of endoneurial membrane fraction with decreased expression of PKC-α. These alterations were normalized in the treated group. Enhanced expression of phosphorylated extracellular signal-regulated kinase detected in diabetic rats was inhibited by the treatment. Increased numbers of macrophages positive for ED-1 and 8-hydroxydeoxyguanosine-positive Schwann cells in diabetic rats were also corrected by the treatment. Pioglitazone lowered blood lipid levels of diabetic rats, but blood glucose and nerve sorbitol levels were not affected by the treatment. In conclusion, our study showed that pioglitazone was beneficial for experimental diabetic neuropathy via correction of impaired PKC pathway and proinflammatory process, independent of polyol pathway.     

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.     

An Early Diagnostic Tool for Diabetic Peripheral Neuropathy in Rats

The skin’s rewarming rate of diabetic patients is used as a diagnostic tool for early diagnosis of diabetic neuropathy. At present, the relationship between microvascular changes in the skin and diabetic neuropathy is unclear in streptozotocin (STZ) diabetic rats. The aim of this study was to investigate whether the skin rewarming rate in diabetic rats is related to microvascular changes and whether this is accompanied by changes observed in classical diagnostic methods for diabetic peripheral neuropathy. Computer-assisted infrared thermography was used to assess the rewarming rate after cold exposure on the plantar skin of STZ diabetic rats’ hind paws. Peripheral neuropathy was determined by the density of intra-epidermal nerve fibers (IENFs), mechanical sensitivity, and electrophysiological recordings. Data were obtained in diabetic rats at four, six, and eight weeks after the induction of diabetes and in controls. Four weeks after the induction of diabetes, a delayed rewarming rate, decreased skin blood flow and decreased density of IENFs were observed. However, the mechanical hyposensitivity and decreased motor nerve conduction velocity (MNCV) developed 6 and 8 weeks after the induction of diabetes. Our study shows that the skin rewarming rate is related to microvascular changes in diabetic rats. Moreover, the skin rewarming rate is a non-invasive method that provides more information for an earlier diagnosis of peripheral neuropathy than the classical monofilament test and MNCV in STZ induced diabetic rats.     

MiR-214-3p plays a protective role in diabetic neuropathic rats by regulating Nav1.3 and TLR4

This study aimed to investigate the functions of miR-214-3p in diabetic neuropathic rodents. The diabetic neuropathy was induced by intraperitoneal injection of streptozotocin (STZ) in rats, and miR-214-3p was delivered via tail vein injection of lentivirus. Hot or cold stimulus tests demonstrated that STZ induced thermal hyperalgesia. Neurophysiological measurements revealed that motor and sensory nerve conduction velocity and nerve blood flow were decreased in diabetic neuropathic rats. However, the STZ-induced hyperalgesia, and reduced nerve conduction velocity and nerve blood flow were all significantly reversed by miR-214-3p administration. HE staining, TUNEL, ELISA, and immunoblotting demonstrated that STZ led to obvious pathological lesion, cell apoptosis, and inflammation in dorsal root ganglion (DRG), evidenced by altered levels of apoptosis-related protein molecules and inflammatory factors, and activation of Toll-like receptor 4 (TLR4)/myeloid differentiation primary response gene 88/nuclear factor kappa B signaling. The pathological alterations in diabetic neuropathic rats in DRG were significantly ameliorated by miR-214-3p application. In addition, sodium channel protein type 3 subunit alpha isoform 1 (Nav1.3) and TLR4 were identified as targets of miR-214-3p via dual-luciferase reporter assay. MiR-214-3p may play its roles by downregulating Nav1.3 and TLR4. In summary, miR-214-3p alleviated diabetes-induced nerve injury, and pathological lesion, cell apoptosis, and inflammation in DRG by regulating Nav1.3 and TLR4 in STZ-induced rats. These findings may provide novel therapeutic targets for clinical treatment of diabetic neuropathy.     

Effects of poly(ADP-ribose) polymerase inhibition on dysfunction of non-adrenergic non-cholinergic neurotransmission in gastric fundus in diabetic rats

Diabetes mellitus compromises nitric oxide (NO)-mediated endothelium-dependent relaxation of blood vessels, which has been linked to the excessive generation of reactive oxygen species. There are also deleterious effect on nitrergic innervation, contributing to autonomic neuropathy symptoms such as impotence and gastroporesis. Poly(ADP-ribose) polymerase (PARP) is a nuclear protein stimulated by DNA damage, caused, for example, by oxidative stress. Activation has been linked to impaired endothelial nitric oxide synthase (eNOS)-mediated vasodilation in experimental diabetes. There is no information on the potential role of PARP in nitrergic nerve dysfunction, therefore, the aim was to examine the effects of PARP inhibition, using 3-aminobenzamide (3-AB) on neurally mediated gastric fundus relaxation in streptozotocin-induced diabetic rats. Eight weeks of diabetes caused a 42.5% deficit in maximum relaxation of in vitro gastric fundus strips to electrical stimulation of the non-adrenergic non-cholinergic innervation. This was largely prevented or corrected (4 weeks of treatment following 4 weeks of untreated diabetes) by 3-AB. Diabetes also markedly attenuated the maintenance of relaxation responses to prolonged stimulation, and this was partially corrected by 3-AB treatment. Experiments in the presence of the NOS inhibitor, N(G)-nitro-L-arginine, and/or blockade of the co-transmitter, vasoactive intestinal polypeptide, by alpha-chymotrypsin, showed that the beneficial effects of 3-AB were primarily due to improved nitrergic neurotransmission. Thus, PARP plays an important role in defective nitrergic neurotransmission in experimental diabetes, which may have therapeutic implications for treatment of aspects of diabetic autonomic neuropathy.     

Contribution of hyperglycemia on diabetic complications in obese type 2 diabetic SDT fatty rats: effects of SGLT inhibitor phlorizin

The spontaneously diabetic torii (SDT) fatty rat is a new model of type 2 diabetes showing overt obesity, hyperglycemia and hyperlipidemia. With early onset of diabetes mellitus, diabetic microvascular complications, including nephropathy, peripheral neuropathy and retinopathy, are observed at young ages. In the present study, blood glucose levels of female SDT fatty rats were controlled with phlorizin, a non-selective SGLT inhibitor, to examine whether and how these complications are caused by hyperglycemia. Phlorizin treatment adequately controlled plasma glucose levels during the experiment. At 29 weeks of age, urinary albumin excretion considerably increased in SDT fatty rats. Glomerulosclerosis and tubular pathological findings also indicate diabetic nephropathy. These renal parameters tended to decrease with phlorizin; however, effects were partial. Sciatic nerve conduction velocities were significantly delayed in SDT fatty rats compared with Sprague-Dawley (SD) rats. Intraepidermal nerve fiber density, an indicator of subclinical small nerve fiber neuropathy, significantly decreased in SDT fatty rats. Retinal dysfunction (prolongation of peak latency for oscillatory potential in electroretinograms) and histopathological eye abnormalities, including retinal folding and mature cataracts were also observed. Both nerve and eye disorders were prevented with phlorizin. These findings indicate that severe hyperglycemia mainly causes diabetic complications in SDT fatty rats. However, other factors, such as hyperlipidemia and hypertension, may affect diabetic nephropathy. These characteristics of diabetic complications will become helpful in evaluating new drugs for diabetic complications using SDT fatty rats.     

Impaired capsaicin-induced decrease in heart rate and coronary flow in isolated heart of diabetic rats

The effect of capsaicin (0.1 microM) on heart rate and coronary flow was studied in Langendorff-perfused heart from streptozotocin-induced (50 mg/kg i.v.) diabetic rats where sensory neuropathy developed. In hearts from animals 4- and 8-week diabetes baseline heart rate and coronary flow decreased from 317.9 +/- 2.9 b.p.m. and 13.4 +/- 0.7 m/min to 255.1 +/- 12.7 and 219.8 +/- 2.8 b.p.m. and 8.9 +/- 0.6 and 10.0 +/- 0.1 ml/min (P<0.05), respectively. Capsaicin significantly decreased both variables in either normal or 4-week diabetic animals its effects, however, on coronary flow or heart rate were missing in preparations from 8-week diabetic rats. Endothelin-1 (0.1 nM), the putative mediator of the capsaicin effect, significantly decreased heart rate and coronary flow irrespective of the presence or absence of diabetes. In the femoral nerve of streptozotocin-treated animals conduction velocity involving both fast conducting A- and slow-conducting C-fibres was decreased proportional to the duration of the pre-existing diabetic state. It is concluded that in insulin deficient diabetes the diminished responses evoked by capsaicin on heart rate and coronary flow are signs of sensory neuropathy. This is related to a feeble endothelin release from sensory nerve endings without changes in post-receptor mechanisms mediating the endothelin effects.     

Effect of Hyperglycemia and Its Prevention by Insulin Treatment on the Incorporation of 32P into Polyphosphoinositides and Other Phospholipids in Peripheral Nerve of the Streptozotocin Diabetic Rat

The influence of varying doses of streptozotocin and preventive insulin treatment on phospholipid metabolism in sciatic nerve in vitro from diabetic rats was studied. Animals were given 30, 45, and 60 mg/kg injections of streptozotocin and 10 weeks later nerves were removed and incubated in the presence of [32P]-orthophosphate. The quantity of isotope incorporated into phosphatidylinositol-4,5-bisphosphate (PIP2) was progressively greater with increasing drug dosage, whereas uptake of label into other phospholipids was unchanged. Rats were made diabetic and within 72 h were implanted with long-acting, insulin-containing osmotic minipumps and the incorporation of [32P]orthophosphate into phospholipids of intact and epineurium-free nerves was examined 8 weeks later. For whole nerve, increased labeling in nerves from diabetic animals occurred only in PIP2 and phosphatidylinositol-4-phosphate (PIP) and was completely prevented by insulin treatment. Isotope incorporation into polyphosphoinositides was also markedly elevated (greater than or equal to 100%) in desheathed diabetic nerves, but not in nerves from insulin-treated animals. Other phospholipids in epineurium-free nerves displayed some rise in isotope uptake, but the increases were not prevented by insulin treatment and appeared unrelated to hyperglycemia. Morphological examination of nerves extended previous findings that prolonged insulin treatment produces axonal degeneration. These observations indicate that abnormal nerve polyphosphoinositide metabolism is at least in part a consequence of hyperglycemia. The metabolic alterations may be intimately involved in reduced nerve conduction velocity, which is characteristic of diabetic neuropathy.