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Figure 4 Cumulative dose-response curves for changes in sciatic vasa nervorum vascular conductance after suffusion of saline containing increasing concentrations of norepinephrine. Blood flow in epi- and perineurial vessels was monitored by laser-Doppler flowmetry, and the results are expressed as a percentage of the maximum vascular conductance. Groups (n = 8-12): nondiabetic control (O); 8-week duration diabetic control (•); 8-week diabetic rats treated with deferoxamine (8 mg/kg/day) for the last 2 weeks (■). Data are mean ± SEM. Diabetes caused a leftward shift of the dose-response curve, indicating greatly enhanced sensitivity to vasoconstriction by norepinephrine. In nondiabetic rats, cosuffusion with the nitric oxide synthase inhibitor A^-nitro-l-arginine (100 |iM) caused a similar leftward shift (□). Treatment of diabetic rats with deferoxamine completely restored norepinephrine sensitivity, the interpretation being that it corrected a vasa nervorum NO deficit. (See Refs. 52 and 67 for further details.)

orum, the NO deficit markedly increases reactivity to norepinephrine (Fig. 4), and this was corrected by deferoxamine treatment (24,67). The relatively high potency of a-lipoic acid compared with other natural scavengers such as a-tocopherol could be due to the additional property of transition metal chelation (55). Thus, transition metal-catalyzed ROS production makes an important contribution to nerve and vascular dysfunction in experimental diabetes.

D. Nerve Growth, Regeneration, and Small Fiber Function

In addition to effects on NCV and blood flow, antioxidant treatment improves other aspects of nerve function, including growth and regenerative responses and the performance of small fibers (which do not normally contribute to NCV measurements). Thus, the hydrophilic scavenger /V-acetyl-L-cysteine allowed normal nerve maturation in young diabetic rats, preventing a reduction in mean nerve fiber size caused by impaired growth. JV-acetyl-L-cysteine improved the regenerative response to nerve trauma, which is blunted by diabetes (Fig. 5), inhibited an increase in plasma tumor necrosis factor activity, and prevented red cell lipid peroxidation (51,52). The lipophilic scavengers a-tocopherol and butylated hydroxytoluene and the metal chelator trientine also prevent blunted nerve growth and regeneration in young diabetic rats (53,54). Recently, improved regeneration, remyelination, and muscle reinnervation have been noted for a-lipoic acid treatment (Flint H, Cotter MA, and Cameron NE, unpublished observations, 1998). Interestingly, butylated hydroxytoluene and trientine also prevented nerve regeneration and growth deficits in the galactosemic rat model of enhanced polyol pathway and PKC activity (54). Vasodilator treatment had similar effects on these nerve growth parameters in diabetic rats (60); therefore, it is likely that antioxidant-mediated improvements in perfusion and their consequences for the supply of energy and nutrients to nerve fibers and cell

Postlesion time (days)

Figure 5 Effects of 4 weeks of diabetes and JV-acetyl-l-cysteine treatment on sciatic nerve myelinated fiber regeneration distance 9 and 14 days after a freeze lesion. Groups (n = 7-10): nondiabetic control (O); diabetic control (•); diabetic rats treated with 250 mg/kg/day /V-acetyl-l-cysteine from diabetes induction (■). Data are mean ± SEM. At both time points there was a significant regeneration deficit (p < 0.01) with untreated diabetes, which was completely prevented by /V-acetyl-L-cysteine treatment. (See Ref. 52 for further details.)

Postlesion time (days)

Figure 5 Effects of 4 weeks of diabetes and JV-acetyl-l-cysteine treatment on sciatic nerve myelinated fiber regeneration distance 9 and 14 days after a freeze lesion. Groups (n = 7-10): nondiabetic control (O); diabetic control (•); diabetic rats treated with 250 mg/kg/day /V-acetyl-l-cysteine from diabetes induction (■). Data are mean ± SEM. At both time points there was a significant regeneration deficit (p < 0.01) with untreated diabetes, which was completely prevented by /V-acetyl-L-cysteine treatment. (See Ref. 52 for further details.)

bodies are primarily responsible for their growth and regeneration-promoting actions.

The effects of antioxidant treatment on small fiber function has not been examined in as much detail as large fibers. However, in the isolated corpus cavernosum preparation from diabetic rats, a deficit in vasorelaxation to ni-trergic nerve fiber stimulation was completely prevented by chronic a-lipoic acid treatment and partially prevented by trientine (68).

E. Polyol Pathway, Oxidative Stress, and Neurovascular Dysfunction

Polyol pathway activity also contributes to oxidative stress; therefore, ARIs have an indirect antioxidant action. The reductions in nerve GSH content found in experimental diabetes and galactosemia were rapidly corrected by ARI treatment (69), which also prevented the elevation of nerve malondialde-hyde, a marker of lipid peroxidation, in diabetic rats (70). ARIs correct nerve blood flow and NCV defects in diabetic rats (71,72). They improve defective NO-mediated endothelium-dependent relaxation in vessels from diabetic animals and similar dysfunction resulting from acute hyperglycemic exposure of vessels from nondiabetic animals (3,6). The first half of the polyol pathway, catalyzed by aldose reductase, is much more important than the second half, catalyzed by sorbitol dehydrogenase, because sorbitol dehydrogenase inhibitors did not correct blood flow or NCV in diabetic rats (73) and did not rectify the nerve GSH deficit (Hohman TC, personal communication, 1997).

Aldose reductase requires NADPH as a cofactor, and NADPH is also used by glutathione reductase for maintaining GSH concentrations. Therefore, competition for NADPH in diabetes probably contributes to diminished GSH levels. A further potential polyol pathway action is to increase the formation of advanced glycation end products (AGEs). ARI treatment reduces tissue AGE accumulation, perhaps by inhibiting the synthesis of fructose or by decreasing elevated flux through the pentose phosphate pathway, processes that produce sugars that are considerably more potent glycating agents than glucose (74). Alternatively, the ARI-induced increases in tissue GSH and antioxidant capacity may be sufficient to oppose AGE formation by glycoxidation (75). AGE reactions are an important source of ROS; therefore, their reduction would decrease oxidative stress. Aminoguanidine, although not [directly] an antioxidant, irreversibly binds to reactive carbonyl intermediates, thus blocking AGE formation. Aminoguanidine treatment of diabetic rats has similar functional effects to ARIs and antioxidants in preventing and correcting NCV, nerve blood flow, and NO-mediated endothelium-dependent vasorelax-

Increased polyol pathway flux -> Impaired antioxidant protection

Figure 6 Relation of oxidative stress and nerve dysfunction to elevated polyol pathway activity and transition metal-catalyzed reactions in diabetes or galactosemia. The increased autoxidation and advanced glycation reactions produce ROS, and flux through the first half of the polyol pathway consumes NADPH. This impairs the glutathione redox cycle so that endogenous antioxidant protection is reduced.

Increased polyol pathway flux -> Impaired antioxidant protection

Figure 6 Relation of oxidative stress and nerve dysfunction to elevated polyol pathway activity and transition metal-catalyzed reactions in diabetes or galactosemia. The increased autoxidation and advanced glycation reactions produce ROS, and flux through the first half of the polyol pathway consumes NADPH. This impairs the glutathione redox cycle so that endogenous antioxidant protection is reduced.

ation deficits (3,4,40,76). The putative interrelations between the polyol pathway, advanced glycation, and autoxidation processes in the production of oxidative stress under hyperglycemic conditions are summarized in Figure 6.

F. Antioxidants, NF-kB, and PKC

One of the cellular events stimulated by oxidative stress-related biochemical changes in hyperglycemia, cither directly or via AGE receptors, oxidized low-density lipoprotein, or cytokine receptors, is the activation of NF-kB, an effect that may be prevented by antioxidant treatment with a-lipoic acid (77). This transcription factor is responsible for changes in gene expression that have important effects on vascular function relevant to diabetes, including elevated endothelin-1 synthesis and upregulation of intercellular and vascular cell adhesion molecules. It has also been linked to increased NADH oxidase activity (30,78).

PKC is another cell-signaling mechanism activated by diabetes, particularly in vascular tissue (79) although not in the nerve itself (80). In the retina of diabetic rats, there is an early reduction in blood flow paralleling that for nerve. In both retina and nerve, flow was restored by PKC inhibitor treatment

(18.81), and nerve NCV deficits were corrected. Diabetes activates PKC via increased de novo synthesis of diacylglycerol from glucose; however, even in the absence of hyperglycemia, PKC is also stimulated by oxidative stress (82). Antioxidants such as a-tocopherol inhibit PKC both directly and via stimulation of diacylglycerol kinase, which breaks down diacylglycerol

(79.82). When activated, PKC can modulate several important vascular systems. For example, it is involved in cell signaling mechanisms for endothelin-1 action and can also stimulate NF-kB, which increases endothelin-1 gene expression in endothelial cells (83). Phosphorylation by PKC controls endothelial constitutive NO synthase, reducing its activity (84). Furthermore, phosphorylation of vascular smooth muscle contractile proteins promotes vasoconstriction (85). Thus, PKC is at the heart of altered vascular responses in experimental diabetes and forms a major component of the dysfunctional mechanisms targeted by antioxidant treatment.

G. Therapeutic Implications

From this brief literature review, it is clear that antioxidant strategies based on the use of ROS scavengers and transition metal chelators can be very effective against experimental models of diabetic neuropathy and vasculopathy. The drawback with the scavenger approach is that very large doses of drug are required, one-two orders of magnitude greater than necessary if using transition metal chelators. However, it is possible that with lower blood glucose concentrations than normally found in the experimental models, more physiological doses, for example, of a-tocopherol, could be effective as an adjunct to tight metabolic control in patients. The use of agents with both scavenger and chelator properties, such as a-lipoic acid, or combined therapy with drugs that improve endogenous antioxidant protection mechanisms, such as ARIs, or drugs that target key cell signaling events, such as PKC inhibitors, could provide exciting future strategic approaches to the therapy of diabetic complications including neuropathy.

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