NGF protects paravertebral but not prevertebral sympathetic neurons against exposure to high glucose in vitro.

In diabetes, sympathetic neuroaxonal dystrophy occurs in prevertebral celiac/superior mesenteric ganglia (CG/SMG) but not in paravertebral superior cervical ganglia (SCG). Changes in neurotrophic support by NGF occur during postnatal development and are implicated in diabetic neuropathy. Therefore, our aim was to compare the effects of age and NGF on the responses of CG/SMG and SCG neurons to high glucose levels in vitro. Neurons were dissociated from neonatal (5 days) and adult (12 weeks) rat ganglia and maintained in serum-free media containing glucose (10-100 mM) in the presence or absence of NGF (50 ng/ml) for 48 h. Cultures were immunostained for the pan neuronal marker, PGP9.5, and TUNEL. Neurons were assessed for viability, the presence of neurite outgrowth and for TUNEL-positive nuclei as a marker of apoptosis. Glucose caused significant concentration-dependent decreases in both viability and the proportion of neurons developing neurites together with significant increases in TUNEL-positive staining. Neonatal SCG neurons with neurites were significantly more susceptible to high glucose than adult SCG neurons whereas postnatal age had no influence on the response of CG/SMG neurons to high glucose. NGF protected adult SCG but not adult CG/SMG neurite-bearing neurons against the induction of TUNEL staining by high glucose. In the presence of NGF, CG/SMG neurons were markedly more susceptible to high glucose than SCG neurons. The greater susceptibility of CG/SMG neurons to diabetic neuropathy may be due to a selective inability of NGF to protect this particular population of sympathetic neurons against hyperglycaemia.

Abnormal connexin expression underlies delayed wound healing in diabetic skin.

OBJECTIVE: Dynamically regulated expression of the gap junction protein connexin (Cx)43 plays pivotal roles in wound healing. Cx43 is normally downregulated and Cx26 upregulated in keratinocytes at the edge of the wound as they adopt a migratory phenotype. We have examined the dynamics of Cx expression during wound healing in diabetic rats, which is known to be slow. RESEARCH DESIGN AND METHODS: We induced diabetes with streptozotocin and examined Cx expression and communication in intact and healing skin. RESULTS: We found that diabetes decreased Cx43 and Cx26 protein and communication in the intact epidermis and increased Cx43 protein and communication in the intact dermis. Diabetes also altered the dynamic changes of Cxs associated with wound healing. Within 24 h, Cx43 was upregulated in a thickened bulb of keratinocytes at the wound edge (rather than downregulated as in controls, which formed a thin process of migratory cells). Cx43 decline was delayed until 48 h, when reepithelialization began. Although Cx26 was upregulated as normal after wounding in diabetic skin, its distribution at the wound edge was abnormal, being more widespread. Application of Cx43-specific antisense gel to diabetic wounds prevented the abnormal upregulation of Cx43 and doubled the rate of reepithelialization, which exceeded control levels. CONCLUSIONS: Cx expression in diabetic skin is abnormal, as is the dynamic response of Cx43 to injury, which may underlie the delayed healing of diabetic wounds. Preventing the upregulation of Cx43 in diabetic wounds significantly improves the rate of healing and clearly has potential therapeutic value.

Effect of aminoguanidine treatment on diabetes-induced changes in the myenteric plexus of rat ileum.

The aim of this study was to investigate the ability of aminoguanidine (AG) to prevent diabetes-induced changes in nitric oxide synthase- (nNOS), vasoactive intestinal polypeptide- (VIP) and noradrenaline- (NA) containing nerves of the rat ileum using immunohistochemical and biochemical techniques. Diabetes was induced in adult male Wistar rats by a single intraperitoneal injection of streptozotocin (65 mg/kg). AG was administered in the drinking water to control (1.8 g/l) and diabetic (0.9 g/l) rats over a period of 8 weeks. Diabetes caused a significant increase in the thickness of nNOS-containing nerve fibres (p<0.001) in the circular muscle, in nNOS activity (p<0.05) and in the size distribution of nNOS-containing myenteric neurons (p<0.001). The thickness of VIP-containing nerve fibres was significantly greater (p<0.01) and there was a significant increase in varicosity size (p<0.01) and proportion of VIP-positive myenteric neurons (p<0.01) in diabetes. NA levels were significantly reduced (p<0.01) and the size of varicosities containing tyrosine hydroxylase (TH) was significantly increased (p<0.001) in diabetes. AG treatment completely or partially prevented the diabetes-induced increase in nNOS activity, in VIP-containing varicosity size, and in fibre width of both VIP- and nNOS-containing fibres in the circular muscle but had no effect on the diabetes-induced increase in nNOS-containing neuronal size or proportion of VIP-containing myenteric neurons. In contrast to VIP, AG treatment had no effect on the increase in TH-containing varicosity size in diabetes and also failed to prevent the decrease in NA levels induced by diabetes. These results indicate that AG treatment for neuropathy is not equally effective for all autonomic nerves supplying the ileum and that diabetes-induced changes in NA-containing nerves are particularly difficult to treat.

Selective susceptibility of different populations of sympathetic neurons to diabetic neuropathy in vivo is reflected by increased vulnerability to oxidative stress in vitro.

Diabetes is the major cause of autonomic neuropathy in humans. Sympathetic neurons from the celiac/superior mesenteric ganglia (CG/SMG) develop neuropathic changes in diabetes whereas sympathetic superior cervical ganglion (SCG) neurons do not. Glucose-induced oxidative stress is proposed as a major factor in the development of diabetic neuropathy. The aim of this study was to investigate whether sympathetic neurons that develop neuropathy in diabetes are more susceptible to oxidative stress. Explants of CG/SMG and SCG from control adult rats were cultured in media free of serum and NGF, exposed to menadione for 48 h to induce oxidative stress and assessed for neuronal viability, TUNEL-positive nuclei and tyrosine hydroxylase- (TH)-immunoreactivity. TH-immunoreactivity was also assessed in ganglia from control and 8 week streptozotocin-diabetic rats. Menadione caused a concentration-dependent loss of neuronal viability and increase in TUNEL staining in both ganglia. However, at low concentrations, menadione had a significantly greater effect (p<0.01) on CG/SMG neurons than SCG neurons. At 1 nM, menadione caused a significant increase (p<0.05) in the number of CG/SMG neurons containing intense TH-immunoreactivity without affecting SCG neurons. Similarly, 8 weeks streptozotocin-induced diabetes resulted in a significant increase (p<0.05) in intensely fluorescent TH-containing CG/SMG neurons but not SCG neurons. This is the first demonstration that oxidative stress in vitro causes the same accumulation of TH in CG/SMG neurons as is observed following streptozotocin-induced diabetes in vivo. Furthermore, the selective vulnerability of CG/SMG neurons to diabetes is reflected by increased sensitivity to oxidative stress.

Diabetes only affects nitric oxide synthase-containing myenteric neurons that do not contain heme oxygenase 2.

It has been demonstrated that subpopulations of myenteric neurons are differentially susceptible to the development of neuropathy in diabetes. Within the myenteric plexus are neurons that contain neuronal nitric oxide synthase (nNOS). However, these are not a homogeneous population. Some of the nNOS-containing neurons also contain heme oxygenase 2 (HO2). Therefore, the aim of this study was to compare the effects of diabetes on HO2- and nNOS-containing neurons within the myenteric plexus of the rat ileum. Diabetes was induced in male Wistar rats (350-400 g) by a single i.p. injection of buffered streptozotocin (65 mg/kg). After 12 weeks, immunostaining of wholemount preparations of ileum revealed that diabetes induced a significant shift (P < 0.001, chi-squared test for trend) towards increased neuronal cell body size in nNOS-immunoreactive neurons while HO2-immunoreactive neurons remained unaffected. Double-labeling studies revealed that approximately 50% of nNOS-containing neurons also contained HO2 and that the diabetes-induced change in size was confined to nNOS-immunoreactive neurons that did not contain HO2 (P < 0.01). No change in the size distribution occurred in neurons in which nNOS and HO2 were colocalized. Differences in the response of these two subpopulations of nNOS-containing neurons to diabetes could occur because they supply different targets within the gastrointestinal tract or indicate that the antioxidant, HO2, protects those nNOS-containing neurons in which it is colocalized, against oxidative stress that occurs in diabetes.

Prevention and partial reversal of diabetes-induced changes in enteric nerves of the rat ileum by combined treatment with alpha-lipoic acid and evening primrose oil.

Treatment with alpha-lipoic acid (LA) or evening primrose oil (EPO), individually, fails to prevent diabetes-induced changes in enteric nerves. Since synergy between these treatments has been reported, the aim was to investigate the effectiveness of combined LA/EPO treatment. LA and EPO were administered in the diet (approximately 80 and 200 mg/kg/day, respectively) to control and diabetic (induced by streptozotocin, 65 mg/kg, i.p.) rats. For prevention, treatment started after 1 week and lasted 7 weeks. For reversal, treatment lasted 4 weeks and was initiated after 8 weeks. Nerves supplying the ileum containing vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP) and noradrenaline (NA) were examined immunohistochemically or biochemically. Diabetes caused a significant increase in VIP-containing cell bodies (p<0.001), decrease in NA content (p<0.01) and loss of CGRP-immunoreactivity. LA/EPO treatment totally prevented diabetes-induced changes in VIP (p<0.001) and CGRP and partially reversed (p<0.05) these changes once they had been allowed to develop. In contrast, treatment had no effect on diabetes-induced changes in NA-containing nerves. Therefore, LA and EPO are only effective at treating diabetes-induced changes in some enteric nerves when administered in combination. However, diabetes-induced changes in NA-containing nerves are resistant to treatment.

Comparative effects of high glucose on different adult sympathetic neurons in culture.

In vivo, diabetes causes neuropathy in the sympathetic celiac/superior mesenteric ganglion (CG/SMG) but not the superior cervical ganglion (SCG). Therefore, our aim was to compare the effects of high glucose in vitro on adult rat SCG and CG/SMG neurons. High glucose decreased viability and neurite outgrowth and increased TUNEL staining in freshly dissociated neurons from both ganglia. In cultures established for 24 h before exposure to high glucose, a significant (p < 0.01) decrease in neurite-bearing neurons from CG/SMG but not SCG occurred. CG/SMG neurons took longer (p < 0.05) to initiate neurite outgrowth and had lower (p < 0.002) anti-oxidant defence enzyme activity. These properties may contribute to the selective development of neuropathy in CG/SMG in diabetes in vivo.

P2X and P2Y purinoceptor expression in pancreas from streptozotocin-diabetic rats.

The expression of the nucleotide receptors P2X1, P2X2, P2X7, P2Y1, P2Y2 and P2Y4, in the pancreas of the streptozotocin-induced diabetic rat was investigated using immunohistochemistry. In diabetic animals, P2X7 receptor expression, normally located in the outer periphery of the islet, was increased and located inside the islet. Double-labelling experiments, using antibodies raised against insulin, somatostatin and glucagon, showed, for the first time, an increase in immunostaining for P2X7 receptors on islet glucagon-containing alpha cells (which had migrated to the interior), while no P2X7 receptors were found in beta and delta cells. P2Y1 receptors were present in intra-islet capillaries, while P2Y4 receptors were found on both alpha and beta cells. P2Y1 and P2Y2 receptor expression was also found in pancreatic duct cells and P2X1, P2X2, P2Y1 and P2Y2 receptors were identified in small blood vessels.

The effectiveness of treatments of diabetic autonomic neuropathy is not the same in autonomic nerves supplying different organs.

The aim of the study was to investigate antioxidant (alpha-lipoic acid [LA]) and gamma-linolenic acid treatments in the prevention of changes in autonomic nerves induced in streptozotocin-diabetic rats. Autonomic nerves supplying the heart, penis, and gut were examined using immunohistochemical and biochemical techniques. LA and gamma-linolenic acid (present in evening primrose oil [EPO]) were administered as dietary supplements ( approximately 80 and 200 mg. kg(-1). day(-1), respectively). LA treatment prevented the diabetes-induced decrease of norepinephrine (NA) in the heart and of type I nitric oxide synthase (NOS-I) expression in erectile tissue of the penis but failed to prevent diabetes-induced changes in NA-, vasoactive intestinal polypeptide-, or calcitonin gene-related peptide-containing nerves supplying the ileum. LA partially prevented and EPO totally prevented the increase in NOS-I activity induced by diabetes in the ileum. EPO treatment failed to prevent any other diabetes-induced changes in the heart, penis, or ileum. These results demonstrate that, whereas LA treatment is more effective than EPO in preventing diabetes-induced changes in autonomic nerves, the effectiveness of LA treatment varies with the target organ studied. Diabetes-induced changes in nerves supplying the ileum are more resistant to treatment than those of the heart and penis.