PN is one of the major complaints among diabetic patients and is associated with several problems such as cardiovascular defects, retinopathy, and muscular pain or weakness. As these defects affect the quality of life, treatment of diabetic neuropathy or prevention of its accompanying symptoms has been considered a major goal 18.
PN comprises functional and structural changes in the peripheral nerves. Some of the morphological alterations in the myelinated fibers of the peripheral nerves associated with hyperglycemia are also seen in rat models of STZ-induced diabetic neuropathy 19.
In the present study, STZ injection successfully induced DM, manifested by a significant increase in serum glucose concentration, compared with the control group. Diabetic rats also presented a significant decrease in body weight relative to controls. It has been previously reported that elevated glucose level and diminished insulin level in DM trigger the release of triglycerides from adipose tissue and catabolism of amino acids in muscular tissue. This results in a loss of both fat and lean mass, leading to a significant reduction in total body weight 20.
At the morphological level, development of neuropathy was manifested in the current study by damage to myelinated fibers, including altered myelin/axon integrity, retraction of axoplasm with subsequent periaxonal edema, axonal atrophy, and damage of myelin sheaths.
It was previously hypothesized that axonal changes might be the ‘primum movens’ of diabetic neuropathy. Axonal shrinkage and separation from the myelin sheath and periaxonal edema could explain the marked degenerative changes in the nerve fibers with consequent reduction in nerve conduction of STZ models 21. Further, onion-bulb formation was detected in the nerve sections of diabetic rats in this study. Onion bulbs are concentric lamellar structures formed by Schwann cell processes, which may be seen in several generalized or localized diseases of the peripheral nerve, including diabetic neuropathy 22.
In the current work, neurofilaments and electron-dense inclusions accumulated in the axoplasm of most degenerating axons, probably because of stagnation of axoplasmic flow. These dense inclusions were previously described by other authors who postulated that tissue lysosomal phospholipid content increased in DM, forming intralysosomal inclusion bodies that were indigestible by phospholipases 23.
Later events in diabetic neuropathy may center around impaired axonal transport of neurofilaments or other cytoskeletal structures. Limited cytoskeletal support by perikarya may result in flawed axons or axons that are incapable of dynamic restructuring, probably resulting from rapid glycosylation of proteins 24.
Degenerative nerve changes reported in the current work were proved by statistical analysis, revealing a statistically significant decrease in the number of total and apparently normal nerve fibers with concomitant increase in the number of apparently degenerated fibers in the diabetic group, when compared with controls.
In the present study, severe demyelination was detected with evidence of myelin destruction in the form of splitting and decompaction of myelin lamellae. Vacuolization of myelin sheaths was evident with formation of fermentation chambers. Honey-comb degeneration of myelin was seen. This was previously described by other investigators as grade 3 degeneration of myelin comprising separation of myelin and disruption of myelin configuration 25. As in any neuropathy, macrophages engulfing the myelin debris were observed. Loss of salutatory conduction of nerve impulses that results from demyelination leads to decrease in conduction velocity and conduction block 26.
It has been reported that rats with DPN display an altered myelin lipid composition pattern and blunted expression of key genes in the fatty acid biosynthetic pathway. These defects are associated with increased myelin abnormalities in the peripheral nerve of diabetic rats 27.
As myelin is produced by Schwann cells, these data suggest that the degeneration of myelinated nerve fibers in DM might be due to Schwann cell abnormalities. In the current study, vacuolation was evident in Schwann cell cytoplasm, together with cytoplasmic lysis, mitochondria with distorted and completely absent cristae, and karyorrhexis of Schwann cell nuclei. These vacuoles might be electrolucent fat vacuoles. The accumulation of several fat vacuoles in Schwann cell cytoplasm could be regarded as the cumulative effect of increased myelin degeneration and catabolism 28. Mitochondrial dysfunction has been regarded as one of the pathophysiological causes of neurodegenerative diseases such as Alzheimer, Parkinson, and DM-induced PN. Hypoxia, hyperglycemia, and increased oxidative stress contribute directly and indirectly to Schwann cell dysfunction 29.
It has been postulated that poorly controlled hyperglycemia reduces peripheral nerve regeneration in DM, possibly by inhibiting proliferation of Schwann cells, which might exacerbate nerve injury-related diabetic neuropathy 30. Therefore, the degenerative changes detected in Schwann cells in the present study could explain the marked affection of nerve axons.
In the current work, a significant decrease in the area% of myelinated fibers and myelin sheath in diabetic nerve sections was reported. Thus, diminution of the fiber area was predominantly the result of reduction of the myelin sheath area.
In the present study, decreased area% of fibers was compensated by a significant increase in the area% of the endoneural space, and this explains the endoneurial edema and wide separation of collagen fibers in diabetic sections. Further, reduction in the area% of myelin sheath is related to the demyelinating process with subsequent catabolism. Perineurial lamellae were separated, and loss of the perineurial barrier was suggested by endoneurial edema, which explains the marked myelinated fiber changes.
Endoneurial edema observed in the current work probably plays a role in reducing the endoneurial blood flow. Consequently, it can play a major part in the degenerative changes in Schwann cells and myelinated nerve axons recorded in the present study. It can also be an important factor in the pathogenesis of neuropathy in diabetic rats, which supports the reported conclusion of other authors that ischemia is characteristic of hyperglycemic models 31.
Decrease in nerve blood flow in diabetic neuropathy leads to nerve metabolic abnormality and consequently to defects in ATP-sensitive ion exchanger pumps like the Na–K pump. Defects in the Na–K pump finally lead to membrane inability to preserve the resting potential and consequently disturbs nerve conductivity 3.
Different mechanisms for the pathogenesis of diabetic complications have been described but none has achieved general acceptance. These mechanisms have been divided into two major subgroups: abnormalities that suggest a metabolic etiology and abnormalities that suggest a vascular etiology 32.
The formation of advanced glycation end products may explain many of the diabetic complications. In terms of PN, the protein glycation cascade may lead either to demyelination or to axonal atrophy. Glycation of the myelin proteins would account for myelin destruction and consequent demyelination. In contrast, glycation of collagen could lead to a reduction in nerve growth factor, leading to axonal atrophy 1.
Diabetic neuropathy has been attributed to accumulation of ROS, especially superoxide radicals, and hydrogen peroxide release 33. Oxidative stress results from an imbalance between radical-generating and radical-scavenging systems – that is, increased free radical production or reduced activity of antioxidant defenses or both 34.
The vascular theory assumes that hyperglycemia and metabolic derangement affect the structure and function of endoneurial microvessels, which then induce fiber changes by altering the blood–nerve barrier, inducing hypoxia or ischemia 35.
Frequent vascular abnormalities were observed in the present study, including high endothelial lining of endoneurial blood vessels, thickening of their walls due to hypertrophy of smooth muscles of the media, and narrowing and sometimes complete obliteration of their lumina. Other vessels appeared congested. Thickening of the perineurial cell basement membrane might result in compression of microvessels traversing the perineurium, thus causing ischemia. Such endoneurial vascular damage and the resultant ischemia in turn result in the neurodegeneration seen in DM. Further, electron microscopy revealed interrupted basal lamina of endoneurial blood vessels and loss of some of the lining endothelial cells, which again contribute to dysfunctional blood–nerve barrier with consequent neurodegenerative complications.
Luminal narrowing and mural thickening of these vessels were compounded by basal laminar thickening of the perineurium. These morphological findings emphasize the impact of diabetic microangiopathy on specialized endothelium and suggest that local anatomic factors in the perineurial sheath render the nerve vulnerable to chronic ischemia 36.
Further, the vacuoles observed in the media of some blood vessels in the current study might be lipid deposition. This represents part of the atherosclerotic process, a major complication of DM. DM produces disturbances in lipid profiles, especially an increased susceptibility to lipid peroxidation, which is responsible for the increased incidence of atherosclerosis 37.
The detection of accumulated lipid droplets in the CT surrounding the diabetic nerves was an interesting observation in the current work. The role of lipid droplets outside of lipid and cholesterol storage has recently begun to be elucidated and includes a close association with inflammatory responses through the synthesis and metabolism of ‘eicosanoids’, signaling molecules that exert complex control on several body systems mainly in inflammation. Lipid droplets are also involved in metabolic disorders such as DM and obesity 38.
Similarly, increased numbers of mast cells were detected surrounding the diabetic nerves. Recent studies of experimental animals and humans have suggested that mast cells are involved in obesity and DM. Mast cell functions in DM are very complicated and depend on the type of DM and on different diabetic complications. Mast cell activation is a significant risk factor for human pre-DM and DM. Mast cell stabilization prevents diet-induced DM and improves pre-established DM in experimental animals. Observations from animal and human studies have suggested beneficial effects of treating diabetic patients with mast cell stabilizers 39.
As oxidative stress plays an important role in the development of complications in DM, potent antioxidants are now being investigated. Antioxidant therapy has been thought to decrease oxidative stress. MLT has attracted increased attention in recent years and is known to reduce oxidative stress 7. MLT is considered to be one of the most potent antioxidant agents that has negligible toxicity even in very high doses 10.
In the present study, MLT therapy induced a significant decrease in blood glucose level, compared with diabetic rats. There is favorable evidence that the circadian rhythm of MLT influences insulin secretion by the endocrine pancreas and reduces blood glucose levels in diabetic rats. Insulin levels are also adapted to day/night changes through MLT-dependent synchronization. Diabetic patients show a reduced diurnal serum MLT level and increased pancreatic MLT receptors 40.
MLT influences insulin secretion both in vivo and in vitro. The effects are mediated by specific high-affinity membrane receptors MT1 and MT2, which are present in both the pancreatic tissue and islets of rats and humans, resulting in an increase in insulin release. Such action would be expected to reduce the incidence of DM 41. It was recently reported that MLT enhanced insulin receptor kinase phosphorylation, suggesting the potential existence of signaling pathway cross-talk between MLT and insulin 42.
In the present study, treatment with MLT showed protective effects against peripheral nerve injury as it could prevent most of the degenerative nerve abnormalities detected in the diabetic group. Vacuolization of the myelin sheath decreased remarkably. Axonal shrinkage and vacuolization of Schwann cells were evident only in a few fibers. This was confirmed morphometrically by a significant increase in the number of total and apparently normal nerve fibers in the MLT-treated group compared with the diabetic group.
MLT counteracts the increase in ROS-induced lipid peroxidation. It is a direct scavenger of free radicals and has indirect antioxidant effects because of its stimulation of the expression and activity of antioxidative enzymes such as glutathione peroxidase, superoxide dismutase, and catalase 43.
Diabetic neuropathy was evident in our rat models 6 weeks after induction of DM by STZ. Morphological abnormalities targeted the nerve fibers, myelin sheath, and Schwann cells.
MLT administration in early stages of DM induction, before neurotic damage and occurrence of diabetic neuropathy, could decrease the destructive progress of DM and cause neuroprotection against damages resulting from diabetic hyperglycemia.
The neuroprotective effect of MLT is attributed to its antioxidant properties and its hypoglycemic effect.
The beneficial effects of the antioxidative treatment support the hypothesis that oxidative stress and free radicals have an important role in neuronal pathology in DM.
Individuals who are considered to be at high risk for development of DM, especially when DM runs in families, should be identified.
Diabetic outpatients should be screened for the prevalence of PN.
In view of our findings on experimental rats, MLT is recommended as a promising agent for the prevention of diabetic neuropathy if future human studies also prove the same neuroprotective effects of MLT.
Further molecular investigations are needed to elucidate the exact mechanism of action and examine the potential therapeutic effects of MLT on diabetic tissue damage, particularly in humans.
Conflicts of interest
There is no conflict of interest to declare.
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Keywords:© 2013 The Egyptian Journal of Histology
diabetes mellitus; electron microscopy; melatonin; oxidative stress; peripheral neuropathy