Ultrastructural observations on myelinated fibres in experimental diabetes: effect of the aldose reductase inhibitor ponalrestat given alone or in conjunction with insulin therapy

Six groups of rats were studied over a 12-week period: onset and end controls, untreated diabetics, ponalrestat-treated diabetics, insulin-treated diabetics, and diabetics treated with ponalrestat and insulin. The concentrations of glucose, sorbitol and fructose significantly increased and that of myo-inositol significantly decreased in the sciatic nerve of untreated diabetic animals. Ponalrestat administration completely normalized sorbitol levels and partially corrected fructose and myo-inositol concentrations without altering nerve glucose levels. The biochemical abnormalities were also corrected in both the insulin-treated and insulin and ponalrestat-treated diabetic animals. Myelinated fibre cross-sectional areas and axonal areas were significantly less in the tibial nerve of diabetic animals as compared with age-matched controls. Insulin treatment partially corrected the reduction in fibre and axonal area but teased fibre preparations showed an excess of axonal degeneration as compared with controls, untreated diabetics and ponalrestat-treated diabetics. Ponalrestat given alone or in conjunction with insulin therapy did not correct the reduction in fibre or axonal area and single isolated fibres from diabetic animals treated with ponalrestat and insulin showed a marked excess of axonal degeneration, probably related to hypoglycaemia. The study fails to reveal any significant beneficial effect of aldose reductase inhibition on the structural abnormalities in peripheral nerve in experimental diabetes.     

The effect of insulin treatment on myelinated nerve fibre maturation and integrity and on body growth in streptozotocin-diabetic rats

Diabetes mellitus was produced in rats by the administration of streptozotocin and observations made over a period of 2 months. Four groups of animals were studied: onset and end controls, untreated diabetic rats and rats treated daily with a long-acting insulin preparation. Body weight increased in the end controls and insulin-treated diabetic animals to a similar degree over the observation period but was reduced in the untreated diabetic rats. Skeletal growth, assessed by measurements of tibial length, was also reduced in the untreated diabetic rats and partially corrected by insulin treatment. Myelinated fibre diameter in the tibial and sural nerves increased over the observation period in the controls, but the increase was less in the untreated animals and the growth deficit was not corrected by insulin treatment. Myelinated fibre numbers did not alter in the tibial or sural nerves between the onset and end controls. Numbers were significantly less in the tibial nerves of both the untreated and insulin-treated diabetic rats as compared with the two control groups; in the sural nerves, fibre numbers did not differ significantly between the four groups. Finally, the number of degenerating axons, assessed in teased fibre preparations, was very small in the control and untreated diabetic animals but was significantly increased in the insulin-treated group. Measurements of plasma glucose concentrations did not suggest that the axonal degeneration could be related to hypoglycaemia. The explanation for this paradoxical effect of insulin therapy is uncertain. It may be dependent upon fluctuations in blood glucose levels or other metabolic actions of insulin apart from its hypoglycaemic effect.     

Effect of alpha-tocopherol supplementation on the ultrastructural abnormalities of peripheral nerves in experimental diabetes

Ultrastructural observations were made on myelinated fibers in the tibial nerves in order to investigate the beneficial effects of alpha-tocopherol administration in streptozotocin-diabetic rats. Male Wistar rats, aged 12 weeks and weighing between 250 g to 300 g were studied. Six onset control rats were used to obtain the baseline parameters for this strain and age. Further 3 groups--untreated diabetic animals, diabetic animals treated with alpha-tocopherol, and age-matched controls--were studied over a 3-month period. In the diabetic animal, administration of alpha-tocopherol resulted in a significant increase (p < 0.05) in total plasma vitamin E levels when compared with other groups. Myelinated fiber cross-sectional area (p < 0.05), axonal area (p < 0.01) and myelin sheath area (p < 0.05) were significantly less in the tibial nerve of diabetic animals than in age-matched controls, but not different from those of onset controls. In the alpha-tocopherol treated diabetic animals, the values for these parameters were intermediate without showing significant difference when compared with age-matched controls and untreated diabetics. The "g" ratio (axon to fiber area) did not differ between any experimental groups. The number of large myelinated fibers were less in the untreated diabetic animals, but in the alpha-tocopherol-treated diabetics, the values were significantly higher (p < 0.05) than with untreated diabetics and were similar to those of age-matched controls. In conclusion, this ultrastructural study reiterated the fact that structural abnormalities of myelinated fibers occur in experimental diabetes and that alpha-tocopherol administration may be useful in preventing the development of these abnormalities.     

Hypoglycemic neuropathy in experimental diabetes

Morphological and electrophysiological observations were made over 4 weeks on 5 groups of 8-week-old male Sprague-Dawley rats. These were comprised of controls, untreated diabetics, and diabetic animals in which sustained hypoglycemia, moderate hypoglycemia, or normoglycemia was induced by continuous subcutaneous insulin infusion (CSII) therapy. Teased fiber studies showed a marked increase in the number of myelinated fibers undergoing axonal degeneration and regeneration in the tibial nerve of severe hypoglycemic and also in moderate hypoglycemic animals but not in controls, untreated diabetic and normoglycemic groups. There was also a significant correlation between episodes of hypoglycemia (less than or equal to 2.0 mmol/l) and the prevalence of axonal degeneration and regeneration in CSII-treated diabetics. Motor nerve conduction velocity was significantly reduced in the moderate and severe hypoglycemic groups and also in untreated diabetic animals when compared with controls. However, it was significantly improved in the normoglycemic group over the untreated diabetic and severe hypoglycemic groups. In conclusion, this study has demonstrated that severe or even mild hypoglycemia produced a detrimental effect on peripheral nerve structure and function in experimental diabetes. Therefore, it may be desirable to avoid even asymptomatic hypoglycemia in the management of diabetes.     

The effect of pancreatic islet transplantation on experimental diabetic neuropathy.

Quantitative light and electronmicroscopical morphometric techniques were used to determine the effect of pancreatic islet transplantation on experimental diabetic neuropathy. Groups of STZ-diabetic rats were given islet transplants at 3 weeks after diabetes onset (prevention) and at 6 months after diabetes onset (reversal). Comparisons were made with onset controls, age-matched non-diabetic controls and untreated diabetic controls 6 months later (n = 8 for all groups). Euglycaemia and normal levels of glycosylated haemoglobin were achieved in both groups of diabetics after islet transplantation. Loss of body weight in diabetic animals was prevented by early islet transplantation, but was only partially reversed following delayed islet transplantation. Normal growth of myelinated fibres and axons during development was retarded in untreated diabetics, but was normal in rats given islet transplants soon after the onset of diabetes (cross-sectional perimeter and area). Diabetics transplanted with islets after a delay had myelinated fibres and axons with diminished calibre. Teased fibre preparations of nerves from diabetics which had received islet transplants showed no excess of abnormalities. This study has shown that the development of certain structural abnormalities of peripheral nerve fibres is prevented in diabetic rats which receive transplants of islets of Langerhans soon after the onset of diabetes. However, once established abnormal fibre morphology can not be completely ameliorated merely by achieving and sustaining euglycaemia through delayed islet transplantation.     

Influence of streptozotocin-induced diabetes on myelinated nerve fibre maturation and on body growth in the rat

Summary Observations were made between the ages of 2 and 12 months on rats made diabetic with streptozotocin at the age of 1 month, and compared with the findings in age-matched controls. Tibial length and body weight in the control animals increased progressively over the period examined, the growth rate being more rapid in the initial stages. Both of these parameters were consistently less in the diabetic animals over the whole of the observation period. Myelinated fibre numbers and diameters were measured in the tibial and plantar nerves. In the tibial nerve, fibre diameter did not differ between the diabetic and control animals up until 4 months of age; thereafter it changed little in the diabetic animals, but continued to increase in the controls. The findings in the medial plantar nerve were more difficult to analyse but showed comparable although less pronounced changes; fibre diameter may be have diminished in the diabetic nerves after 6 months. Teased fibre studies demonstrated few abnormalities in the tibial nerve, either in the control or the diabetic rats. In the lateral plantar nerves, there was a significant excess of axonal degeneration and regeneration in the diabetic nerves.It was concluded that diabetes impairs growth in nerve fibre diameter, but only after 4 months of age. Before then, no growth retardation is obvious, despite the fact that tibial length and body weight are less. This suggests that the peripheral nervous system may be protected against growth retardation during the early part of the postnatal growth period. The significance of the axonal degeneration in the plantar nerves is uncertain, but it may represent either an increased vulnerability of diabetic nerve to compression injury or, less probably, a distal axonopathy related to the diabetic state.     

Near nerve local insulin prevents conduction slowing in experimental diabetes

Insulin may have direct effects on axons through its actions on insulin receptors or through cross occupancy of insulin-like growth factor-1 receptors. We tested the hypothesis that insulin itself influences conduction of myelinated fibers independent of hyperglycemia in experimental diabetes. Low dose intermittent (0.2 units thrice weekly) Toronto (regular) insulin was injected at the sciatic notch and knee near the left sciatic nerve of rats rendered diabetic with citrate buffered streptozotocin or nondiabetic rats given citrate only. Identical volumes of normal saline were injected near the contralateral right sciatic nerve. The diabetic rats developed hyperglycemia, elevated glycosylated hemoglobin levels and had slowing of right (saline treated) sciatic tibial motor and caudal sensory conduction velocity. In contrast, local insulin treatment on the left side prevented conduction slowing, unilaterally increasing conduction velocity. In nondiabetic rats, conduction velocities were slightly higher on the insulin treated side, but the influence of insulin was less robust than in diabetics. The insulin treated sural branches of the sciatic nerves in diabetics had a higher percentage of small (≤9.0 μm diameter) myelinated fibers than the saline treated nerves. Local insulin has a trophic influence on myelinated fibers that is prominent in diabetic nerves and is independent of hyperglycemia.     

胰岛素治疗对糖尿病大鼠学习记忆功能障碍及大脑皮层额叶和海马神经肽含量变化的影响

目的观察胰岛素治疗对糖尿病大鼠学习记忆功能障碍及大脑皮层、海马内生长抑素(SS)、血管活性肠肽(VIP)含量变化的作用。方法采用链脲菌素制备雄性WiStar大鼠糖尿病模型，设正常对照组、胰岛素治疗糖尿病组、未治疗糖尿病组。3个月后采用T型水迷宫试验测定大鼠学习记忆功能，采用放免法测定皮层额叶、海马区脑组织SS、VIP含量。结果与正常对照组比较，未治疗糖尿病大鼠游迷宫时间显著延长、正确次数显著减少，额叶皮层、海马区脑组织SS含量显著降低，VIP含量无显著变化；胰岛素治疗组大鼠上述变化无显著意义。结论糖尿病引起大鼠学习记忆功能障碍和SS下降；SS下降与学习记忆功能障碍有关；早期胰岛素治疗可使学习记忆功能和SS含量恢复正常。     

Streptozotocin induced diabetes as a model of phrenic nerve neuropathy in rats

Phrenic neuropathies are increasingly recognized in peripheral neuropathies but reports on experimental models of the phrenic nerves diabetic neuropathy are scanty. In the present study, we investigated the phrenic nerve neuropathy, due to experimental diabetes induced by streptozotocin (STZ) and the evolution of this neuropathy in diabetic rats treated with insulin. Proximal and distal segments of the left and right phrenic nerves were morphologically and morphometrically evaluated, from rats rendered diabetic for 12 weeks, by injection of STZ. Control rats received vehicle. Treated rats received a single subcutaneous injection of insulin on a daily basis. The nerves were prepared for light microcopy study by means of conventional techniques. Morphometry was carried out with the aid of computer software. The phrenic nerves of diabetic rats showed smaller myelinated axon diameters compared to controls. The g ratio was significantly smaller for myelinated fibers from diabetic rats compared to controls. Insulin treatment prevented these alterations. Histograms of size distribution for myelinated fibers and axons from control rats were bimodal. For diabetic animals, the myelinated fiber histogram was bimodal while the axon distribution turned to be unimodal. Insulin treatment also prevented these alterations. Our results confirm the phrenic nerve neuropathy in this experimental model of diabetes and suggest that conventional insulin treatment was able to prevent and/or correct the myelinated axon commitment by diabetes.     

Peripheral nerve abnormalities related to galactose administration in rats.

Electrophysiological, biochemical, and morphometric observations were made on the peripheral nerves of rats after galactose feeding. Motor nerve conduction velocity was found to be reduced. This was associated with an accumulation of galactitol in the peripheral nerves and a diminution in their myoinositol content. An increased water content and fascicular area, taken in conjunction with a probable increase in the area of the endoneurial spaces, indicated overhydration of the peripheral nerves. Morphometric observations on the myelinated fibre population in the tibial nerve showed no loss of fibres and although both the maximal and the average diameter of the myelinated fibres was slightly less than in age-matched controls, this was insufficient to explain the reduction in conduction velocity. Segmental demyelination was not detected and the relationship between myelin thickness and axon circumference was not altered. Electron microscope observations revealed no ultrastructural changes in the myelinated fibres and, in particular, no abnormalities at the nodes of Ranvier or indication of abnormal hydration of the Schwann cells. The relevance of these findings to the peripheral nerve changes in human and experimental diabetes is discussed.     

Differential neuropathies in hyperglycemic and hypoglycemic diabetic rats

We investigated the effects of hyperglycemia and hypoglycemia on development of peripheral neuropathy in somatic motor and sensory nerves in type 1 diabetic BB/Wor rats. The animals were maintained in a hyper- or hypoglycemic state by treatment with insulin for 3 months. Nondiabetic siblings served as controls. Qualitative analysis of the gastrocnemius and sural nerves by light and electron microscopy revealed signs of Wallerian-type axonal degeneration and regeneration of large myelinated fibers in the hypoglycemic but not the hyperglycemic animals. Degeneration was more common in the gastrocnemius nerve than in the sural nerve. In hypoglycemic rats, myelinated fibers in both the gastrocnemius and sural nerves had significantly shorter internodes and smaller diameters. The decreased fiber diameter was related (r = -0.9) to the duration of severe hypoglycemia (相似文献   

Ultrastructural abnormalities of myelinated fibres in the tibial nerve of streptozotocin-diabetic rats

In this paper we have examined the ultrastructural changes in myelinated fibre structure after the administration of streptozotocin to Sprague-Dawley rats which had passed the rapid growth period. Myelinated fibre size in the tibial nerve was found to be less in diabetic animals 4 and 6 months after the induction of diabetes, when compared to age-matched controls, but not less than onset. The relative contributions of axon and myelin to this reduction in fibre dimensions were examined. When myelin area was plotted against axon area (derived from perimeter) it showed that the pathological insult of diabetes had a greater effect on the rate of myelin production. The incidence of axonal glycogenosomes was also assessed. These results are discussed in detail.     

Neuropathic changes associated with insulin treatment of diabetic rats: Electron microscopic and morphometric analysis

Tibial nerves from control, untreated alloxan diabetic, and 4-week insulin treated alloxan diabetic rats were examined with light microscopy and computerized morphometric analysis of axons. Teased fiber preparations and electron microscopy were utilized to evaluate nerve degeneration. The insulin treatment regimens included daily injections of protamine zinc insulin (PZI), daily injections of ultralente insulin, and continuously delivered insulin through osmotic minipumps. Evaluation of axon:myelin ratios, teased fiber profiles, and electron microscopic cross sections of nerves demonstrated different degrees of neuropathic changes within the treated groups. The control group and untreated diabetic group showed little or no degeneration, while all insulin-treated groups showed evidence of Wallerian degeneration. Among these insulin treated groups, the PZI-treated group showed the greatest number of degenerating profiles while the minipump group showed the least. These data suggest that insulin treatment of alloxan diabetes results in axonal degeneration which closely resembles findings in human diabetic neuropathies. The substantially diminished number of degenerating axons seen in the osmotic minipump insulin-treated rats suggests that continuous delivery of insulin may decrease the neuropathic changes seen with single injection insulin therapy. Since virtually all insulin-dependent diabetic patients receive daily administration of insulin, the possibility that peripheral neuropathies may in part result from the insulin treatment requires more extensive investigation in a variety of animal models to separate the neuropathic effects of diabetes from the neuropathic effects of insulin therapy.     

Hypoglycaemia causes degeneration of large myelinated nerve fibres in the vagus nerve of insulin-treated diabetic BB/Wor rats

The aim of this study was to find out whether dysglycaemia causes neuropathy in the vagus nerve of insulin-treated diabetic BB/Wor rats. Specimens were collected from the left vagus nerve proximal and distal to the level of recurrent laryngeal branch and from the recurrent branch itself in control rats and diabetic BB/Wor rats subjected to hyper- or hypoglycaemia. Myelinated and unmyelinated axons were counted and myelinated axon diameters were measured by electron microscopy. In controls, the vagus nerve proximal to the recurrent branch exhibited three regions in terms of fibre composition: part A was mainly composed of large myelinated axons, part B contained small myelinated and unmyelinated axons, and part C contained mainly unmyelinated axons. The distal level resembled part C at the proximal level and the recurrent branch resembled parts A and B. In hyperglycaemic rats, a normal picture was found at the proximal and distal levels of the vagus nerve and in the recurrent branch. In hypoglycaemic rats, signs of past and ongoing degeneration and regeneration of large myelinated axons were found at the proximal and distal levels and in the recurrent branch. We conclude that hypoglycaemia elicits degenerative alterations in large myelinated axons in the vagus and recurrent laryngeal nerves in diabetic BB/Wor rats. The absence of signs of neuropathy in unmyelinated and small myelinated axons suggests that the sensory and autonomic components of the nerve are less affected. In contrast, the hyperglycaemic rats examined here did not show obvious degenerative alterations.     

Pathogenesis of diabetic neuropathy

Samples of lumbosacral trunk, posterior tibial nerve, and sural nerve obtained at autopsy from diabetic and nondiabetic patients without mononeuropathy multiplex were evaluated using 1-mu-thick epoxy sections and teased nerve fiber preparations. Focal fascicular lesions characterized by reduced density of myelinated axons within fascicles were found predominantly in the specimens from diabetics, mainly in the posterior tibial nerve and lumbosacral trunk. In severe examples, the perineurium and even surrounding epineurium were damaged, stamping the lesions as ischemic. In addition, identical lesions were found in biopsies of nerves of nondiabetics with vasculitis. Density of myelinated fibers at the three sites demonstrated a proximal-distal graded loss that was significantly greater in the diabetic samples. The loss from the lumbosacral trunk to the posterior tibial nerve was correlated with the density of focal lesions in the lumbosacral trunk in the diabetic (p = 0.025), indicating that distal fiber loss was partly due to the focal lesions. Teased nerve fiber abnormalities were common only in sural nerves of diabetics, suggesting that they are secondary. We conclude that beyond the possible metabolic abnormalities involved in the genesis of diabetic polyneuropathy, focal fascicular lesions, likely due to diabetic microangiopathy, are also important in the development of diabetic neuropathy.     

Distal neuropathy in experimental diabetes mellitus

Although nerve conduction slowing is a well-accepted abnormality in rats with acute experimental diabetes, reports of neuropathological changes in diabetic rat nerves have been inconsistent. To examine this further, we studied electrophysiological and morphological features of posterior tibial nerves and their distal branches from four-week streptozotocin-induced diabetic rats and matched controls. Diabetic rat posterior tibial motor conduction was slowed (mean ± 1 SD, 34.8 ± 3.1 m/sec; controls, 41.2 ± 2.5 m/sec), and evoked muscle response amplitudes were only half of control values. Using quantitative techniques, we documented a diminution in number of the largest myelinated fibers in otherwise normal mid posterior tibial nerves, with an increase in the smaller sizes, indicating either a degree of axonal atrophy or impaired fiber growth during development. The principal pathological finding was active breakdown of myelinated fibers in the most distal motor twigs of hind foot muscles supplied by posterior tibial branches, with preservation of fibers in more proximal segments of these nerves. This anatomical lesion in diabetic nerves could account for both observed conduction slowing and lowered muscle response amplitudes. A consistent feature in both diabetic and control mid lateral plantar nerves was a zone of demyelination that apparently occurs at this natural site of nerve entrapment in rats. Taken together, the pathological abnormalities of peripheral nerve in acute experimental diabetes are best explained as resulting from a distal axonopathy.     

A pathologic study of the vagus nerves in diabetes mellitus

A systematic morphometric examination at different levels of the vagus nerves obtained at autopsy in 4 control subjects and 3 diabetics was carried out. Morphometric studies on myelinated fibres were performed on the nerve at mid-cervical, lung hilum and diaphragmatic levels. In 2 insulin-dependent diabetics the density of myelinated fibres was below the lower limit of the control range at all levels. In all the diabetic subjects there was significant reduction in the density of fibres at the most distal level in the nerves. The teased fibre studies showed predominantly changes of axonal degeneration. They are consistent with the findings of abnormal vagal function in diabetics.     

Autonomic Neuropathy In Sorbitol Dehydrogenase Inhibitor (Sdi)-Treated Streptozotocin-Diabetic Rats

We have developed an animal model of diabetic autonomic neuropathy characterized by neuroaxonal dystrophy (NAD) involving ileal mesenteric nerves and prevertebral sympathetic superior mesenteric ganglia (SMG) in chronic streptozotocin-diabetic rats. We have examined the effect of SDI-158, which interrupts the conversion of sorbitol to fructose (and reactions dependent on the second step of the polyol pathway), on NAD in control and diabetic rats. SDI-treatment (SDI-Rx) did not produce NAD in control animals despite the fact that sciatic nerve sorbitol levels reached the same levels as untreated diabetic animals. SDI-Rx resulted in a dramatically increased frequency of NAD in ileal mesenteric nerves and SMG. SDI-Rx diabetic rats developed lesions prematurely, after only one month of diabetes, and in greater numbers than untreated diabetics. SDI-Rx for 1,2,3 and 6 months showed a statistically significant but progressively smaller effect on NAD. The ultrastructural appearance of SDI-Rx ganglia and mesenteric nerves were identical to that previously reported in long-term untreated diabetics; however, short term SDI-Rx resulted in less compacted tubulovesicular elements in the SMG. Three month SDI-Rx superior cervical ganglia failed to develop NAD in the same animals in which large numbers of lesions were found in the SMG. Dystrophic axons involved ileal mesenteric nerves while sparing those to the jejunum. Dystrophic tyrosine hydroxylase immunoreactive sympathetic axons were distributed to myenteric and submucosal ganglia within the gut wall but not to the mesenteric vasculature. Therefore, although SDI-Rx diabetic rats show an exaggerated severity and accelerated time course of NAD compared to untreated diabetics, lesion appearance, immunoreactivity and distribution are comparable to those found in untreated STZ-diabetic rats of much longer durations.     

Peripheral nerve structure and function in experimental diabetes

Observations have been made on the peripheral nerves of rats in which diabetes had been induced by streptozotocin or alloxan. Motor nerve conduction velocity was found to become reduced, the reduction developing within a few days in severely diabetic animals. Conduction velocity remained diminished during survival times of up to 1 year.Histometric studies of the myelinated fibre population of the sural and tibial nerves showed no loss of fibres or reduction in their calibre. Evidence of segmental demyelination was not detected and myelin/axon ratios were normal. Cation binding at the nodes of Ranvier was unaltered. No explanation for the reduced nerve conduction velocity in morphological terms was therefore obtained.Observations on the abdominal vagus nerve revealed no loss of unmyelinated axons or reduction in their calibre. Measurements of Schwann cell and endoneurial capillary basal laminal thickness showed no differences between diabetic and control animals. Ultrastructural examination of other features of the peripheral nerves failed to define any pathological alterations in the diabetic animals. It is therefore concluded that the extrapolation of biochemical findings in experimental diabetes in an attempt to explain the origin of human diabetic neuropathy, where there are associated structural changes, should be made with caution.     

Ultrastructural axonal pathology in experimentally diabetic and aging control rats

Electron microscopic examination of tibial nerves from streptozotocin-diabetic, alloxan-diabetic and age-matched control rats was undertaken at two weeks and two, four, eight, and twelve months following the induction of diabetes. Many myelinated axons of both diabetic and control rats contained glycogen-like granules, axon-Schwann cell networks and fingerlike intrusions of myelin. These axonal changes were observed more frequently with advancing age and duration of diabetes, suggesting that they are related to aging or repeated injury. A larger proportion of diabetic axons than control axons were affected at early time periods, but by eight and twelve months the control axons were as frequently (or more frequently) involved as diabetic axons. Thus, experimental diabetes may confer upon peripheral myelinated axons an increased susceptability to aging or repeated injury. Specific morphologic abnormalities in peripheral myelinated axons associated uniquely with streptozotocin or alloxan diabetes in the rat were not noted.