David R. Tomlinson

A pharmacologic analysis of mechanical hyperalgesia in streptozotocin/diabetic rats

&NA; This study used streptozotocin (STZ; 50 mg/kg i.p.) diabetic rats and monitored weekly thermal and mechanical nociceptive thresholds for 8 weeks diabetes. Rats developed mechanical hyperalgesia as soon as 2 weeks after STZ injection. Thermal nociceptive threshold was not altered up to 8 weeks after STZ injection. Four week‐diabetic rat mechanical hyperalgesia showed reduced sensitivity to the antinociceptive effect of morphine (5–20 mg/kg i.p.). Furthermore, a reduced sensitivity to the antinociceptive effect of the GABAB agonist, (±)baclofen, was observed. A dose as high as 16 mg/kg i.p. of (±)baclofen was necessary to reverse 4 week‐diabetic rat hyperalgesia, whereas in control rats the highest antinociceptive dose devoid of muscle‐relaxant effect was 4 mg/kg i.p. The non‐peptide antagonist for the substance P, neurokinin1 (NK1) receptor, RP 67580 (3–9 mg/kg i.p.) was not effective in reversing the mechanical hyperalgesia associated with 4 week‐diabetes. A six day‐treatment with an antagonist for the N‐methyl‐d‐aspartate (NMDA) receptor for glutamate, (+)MK‐801 (0.1 mg/kg i.p. twice a day), gradually but completely reversed 4 week‐diabetes‐induced mechanical hyperalgesia. These data suggest that diabetes‐induced hyperalgesia may be the consequence of increased activity of primary afferent fibres leading to an increased excitatory tone within the spinal cord. An increased release of glutamate and activation of the NMDA receptor, would maintain the hyperalgesic state. Reduced activity of both opioidergic and GABABergic inhibitory systems, might exacerbate the increased excitation thus contributing to the ongoing pain. It is suggested that NMDA receptor antagonists may constitute an alternative therapy for diabetic neuropathic pain.

Prevention of defects of axonal transport and nerve conduction velocity by oral administration of myo-inositol or an aldose reductase inhibitor in streptozotocin-diabetic rats

SummaryThe effects of orally-administered myo-inositol have been compared with those of an aldose reductase inhibitor on acute neurological defects in experimentally diabetic rats. Three groups of streptozotocin-treated diabetic rats (50mg/kg, IP) together with three groups of age-matched controls (saline, IP) were compared. One pair of groups (control and diabetic) were untreated for 3 weeks, another pair of groups received daily oral myo-inositol (667 mg/kg) and the third pair received an aldose reductase inhibitor (ICI 105552; 50 mg·kg-1·day-1, orally). The untreated diabetic group showed statistically significant deficits in accumulation, proximal to 24 h sciatic nerve constrictions, of choline acetyltransferase activity by comparison with untreated controls (2.8±0.4 versus 5.1±0.4nmol acetylcholine ·h-1·nerve-1; p<0.001). The untreated diabetic rats also showed a fall in motor nerve conduction velocity of 6.2±0.7 m/s which was statistically significant (p<0.001). Treatment of the diabetic group with myo-inositol prevented the development of both defects of axonal transport and conduction velocity; both measurements were similar to those of the myo-inositol treated control rats. Likewise the diabetic rats which received aldose reductase inhibitor showed prevention of both defects. Nerves from untreated diabetic rats showed marked sorbitol accumulation and a statistically significant reduction in myo inositol content by comparison with the untreated controls (sorbitol, 1.56 ±0.22 versus 0.8±0.01 and myo-inositol, 1.47±0.10 versus 2.3±0.10 nmol/mg; p<0.001). Treatment of the diabetic rats with myo-inositol elevated the myo-inositol levels in nerve (3.05±0.22 nmol/mg) without affecting nerve sorbitol levels. Treatment of diabetic rats with aldose reductase inhibitor also normalised nerve myo-inositol (2.75±0.20 nmol/mg) and reduced nerve sorbitol (0.08±0.01 nmol/mg). These findings are consistent with the suggestion that both treatments prevent the development of the two functional defects by normalising the nerve myo-inositol content.

Role of Neurotrophins in Diabetic Neuropathy and Treatment with Nerve Growth Factors

In rodent models of diabetes, there are expression deficits in nerve growth factor (NGF) and in mRNA for its high-affinity receptor, trkA, leading to decreased retrograde axonal transport of NGF and decreased support of NGF-dependent sensory neurons, with reduced expression of their neuropeptides, substance P and calcitonin gene-related peptide (CGRP). Treatment of diabetic rats with intensive insulin normalized these deficits, and treatment with exogenous NGF caused dose-related increases, giving levels of NGF and neuropeptides that were greater than those of controls. Neurotrophin-3 (NT-3) mRNA was also deficient in leg muscle from diabetic rats, and administration of recombinant NT-3 to diabetic rats increased the conduction velocity of sensory nerves without affecting motor conduction velocity. In regenerating nerves after experimental crush injury, expression of NGF in the nerve trunk is increased in diabetes to a greater extent than in controls, but this is offset by a greater reduction in the neuronal expression of trkA in dorsal root ganglia of diabetic rats. Nonetheless, targeted administration of exogenous NGF via impregnated conduits stimulated regeneration in both control and diabetic rats. These findings implicate deficient neurotrophic support in diabetic neuropathy and suggest that its correction should be a paramount therapeutic target.

Aldose reductase inhibitors and their potential for the treatment of diabetic complications.

Aldose reductase converts glucose to sorbitol, which is further processed to fructose. The enzyme is present in most tissues and its possible physiological role is to produce an electrically neutral, non-diffusible osmolyte in cells exposed to hypertonicity, as typified by the renal medullary cells of the loop of Henlé. The enzyme has a low affinity for glucose, and under normal conditions it processes little substrate. However, in diabetes mellitus, the marked rise in intracellular glucose that occurs in some cells causes marked production of sorbitol. The increased flux and accumulation of sorbitol is damaging to cells and may result in some of the long-term complications of diabetes. In this review, David Tomlinson, Elizabeth Stevens and Lara Diemel discuss the role of aldose reductase and the potential of its inhibitors as therapeutic agents targeted at chronic diabetic complications.

Diabetic neuropathy, nerve growth factor and other neurotrophic factors

Diabetic neuropathy typically presents as an insidious symmetrical distal degenerative disease of peripheral nerves. A failure of neurotrophic factors to regulate neuronal phenotype might be expected to result in such a clinical picture. Experimentally, diabetic rats show reduced expression of target-derived nerve growth factor as well as reduced expression of neuronal genes that are responsive to nerve growth factor. The latter is corrected by administration of exogenous nerve growth factor. Thus, deficient neurotrophic support might contribute to the pathogenesis of diabetic neuropathy, and any successful treatment might include exogenous neurotrophins or other strategies to correct their deficiency of action.

Insulin and insulin-like growth factor I enhance regeneration in cultured adult rat sensory neurones

Insulin and the insulin-like growth factors (IGFs) may directly affect the growth, development, and maintenance of the vertebrate nervous system. Previous in vitro studies have focused on embryonic nervous tissue. In this study the effects of insulin, IGF-I, IGF-II and nerve growth factor (NGF) on regeneration and neuronal survival were studied in cultured adult rat sensory neurones in a cell culture environment that limited non-neuronal cell mediated effects. Regeneration, as assessed by neurite outgrowth, was significantly enhanced by insulin and IGF-I in a dose-dependent manner. The half-maximally effective concentrations, ED50s, were approximately 1 nM and 0.1 nM for insulin and IGF-I, respectively. Concentrations of IGF-I as low as 10pM were active. There was some evidence that IGF-II stimulated regeneration, although this failed to reach statistical significance. NGF also promoted regeneration, confirming previous studies, exhibiting an ED50 of approximately 0.3 ng/ml and inducing a maximal response 2-fold greater than that observed with insulin or IGF-I. Combined treatment with NGF and insulin had an additive effect. Specific anti-NGF antiserum inhibited the regenerative response to NGF but failed to block the response to IGF-I, supporting the view that IGF-I was acting directly on sensory neurones rather than stimulating NGF production by non-neuronal cells. Insulin, IGF-I and NGF had no effect on neuronal survival in this culture system. These results show that adult sensory neurones can respond with enhanced regenerative growth to insulin and IGF-I, in addition to NGF although the response to IGF-II was less clear.

Expression of neuropeptides in experimental diabetes; effects of treatment with nerve growth factor or brain-derived neurotrophic factor ☆

Rats with streptozotocin-induced diabetes of 4 to 6 weeks duration showed a depletion of both substance P (P < 0.01) and calcitonin gene-related peptide (P < 0.01) in the sciatic nerve. Since expression of both peptides is sensitive to nerve growth factor (NGF) in vitro we examined the effect of treatment of diabetic rats with NGF, which significantly increased the levels of both peptides in treated diabetic animals (P < 0.01 for both). Treatment of non-diabetic rats with a similar NGF regime raised the mean peptide levels to a value similar to that seen in treated diabetic rats but the change was not statistically significant. In vehicle-treated diabetic rats the depletions of sciatic nerve neuropeptides were accompanied by a significant (P < 0.05) reduction in the level of CGRP mRNA in the 4th and 5th lumbar dorsal root ganglia, this was accompanied by an analogous reduction in the mRNA for gamma-preprotachykinin A (gamma-PPT), which did not attain statistical significance. Treatment of diabetic rats with NGF also prevented the deficits in the levels of CGRP and gamma-PPT mRNA in the lumbar dorsal root ganglia (P < 0.05). Treatment of other diabetic rats with the related neurotrophin, brain-derived neurotrophic factor (BDNF), had no effect on the levels of substance P and calcitonin gene-related peptide in the sciatic nerve.

Myo-inositol and sorbitol metabolism in relation to peripheral nerve function in experimental diabetes in the rat: The effect of aldose reductase inhibition

SummaryA possible relationship between increased sorbitol concentration and decreased myo-inositol concentration in peripheral nerves of diabetic rats has been examined. To this end, sorbinil, an aldose reductase inhibitor, was used either to prevent or reverse elevation of nerve sorbitol concentration in diabetic rats. Sorbinil treatment at 20 mg · kg-1 · day-1 prevented elevation of nerve sorbitol levels in early diabetes and reduced sorbitol concentration from 2.38 to 0.51 μmol/g in rats diabetic for 10 weeks. This treatment reduced the increase in nerve fructose concentration and prevented the reduced myo-inositol concentration found in diabetic rat nerve (control 3.63, diabetic 2.40, diabetic/sorbinil, 3.56 μmol/g). Sorbniil treatment did not prevent a significant slowing of motornerve conduction velocity at 10 weeks although treatment reduced the extent of slowing. Sorbinil treatment at 25 mg · kg-1 · day-1 reduced elevated sorbitol and fructose concentrations in diabetic rat nerve and normalised myo-inositol concentration. Myo-inositol treatment at 650 mg · kg-1 · day-1 did not affect the elevated concentrations of sorbitol, fructose or glucose in peripheral nerves of diabetic rats, but it did restore reduced myo-inositol concentration. Both sorbinil and myo-inositol treatment partially reversed the slowing of motor-nerve conduction velocity in diabetic rats. These results are discussed in relation to the involvement of sorbitol and myo-inositol metabolism in the aetiology of diabetic neuropathy.

Effects of anti-oxidant treatment on sciatic nerve dysfunction in streptozotocin-diabetic rats; comparison with essential fatty acids

SummaryIn Study 1, the effects of treatment of streptozotocin-diabetic rats with the antioxidants, probucol or vitamin E were compared. Untreated diabetic rats showed a reduction of 45% (p<0.01) in nerve laser Doppler flux, which was used as an index of nerve blood flow. In diabetic rats treated with either probucol or vitamin E nerve Doppler flux was reduced by only 13 or 16%, respectively (p<0.01 for either compared to untreated diabetic rats). A second study examined the effects of treatment with evening primrose oil either alone or in combination with probucol. Reduced nerve Doppler flux was reproduced in untreated diabetic rats (47%; p<0.01). In parallel diabetic groups, nerve Doppler flux was reduced by only 14% with evening primrose oil alone and by 8% with evening primrose oil plus probucol (both p<0.01 vs untreated diabetic rats). Both treatments were also associated with marked attenuation of motor and sensory nerve conduction velocity deficits. Measurements on plasma from rats showed normalisation of triglyceride levels by probucol treatment without an effect on those of cholesterol in Study 1. In Study 2, the converse was true for evening primrose oil treatment, whilst the combined treatment lowered both plasma triglycerides and cholesterol. This work indicates similar effects of antioxidants and evening primrose oil against reduced nerve Doppler flux and conduction velocity in diabetic rats, with dissimilar actions on plasma triglycerides and cholesterol.

Reversal, by treatment with an aldose reductase inhibitor, of impaired axonal transport and motor nerve conduction velocity in experimental diabetes mellitus

Abstract Streptozotocin-diabetic rats showed reduced motor nerve conduction velocity, impaired orthograde axonal transport of choline acetyltransferase, and accumulated sorbitol in the sciatic nerve compared with age-matched controls. Another age-matched group, with diabetes of similar intensity, but treated daily with an aldose reductase inhibitor, showed none of these neurological defects. These findings implicate aldose reductase activity in the development of acute conduction and axonal transport defects in experimental diabetes.