Secondary Logo

Share this article on:

Neuroprotective effect of melatonin in a rat model of streptozotocin-induced diabetic neuropathy: Light and electron microscopic study

Afifi, Noha M.

The Egyptian Journal of Histology: June 2013 - Volume 36 - Issue 2 - p 321–335
doi: 10.1097/01.EHX.0000428963.44300.63
Original articles

Introduction Diabetes mellitus (DM) is the most common cause of peripheral neuropathy in most countries. Oxidative stress appears to be the most important pathogenic factor in underlying diabetic complications, including neuropathy.

Aim of the work The present study aimed to investigate the possible neuroprotective effects of melatonin (MLT) in a rat model of streptozotocin (STZ)-induced diabetic neuropathy.

Materials and methods Thirty-six (15 weeks old) adult male albino rats were divided into three groups. Group I (n=6) served as the control group. In group II (n=15), DM was induced by a single intraperitoneal injection of STZ at a dose of 60 mg/kg body weight and rats were sacrificed after 6 weeks. Rats in group III (n=15) were rendered diabetic by a single intraperitoneal injection of STZ, and immediately after confirmation of DM, that is, 48 h after STZ (random blood sugar > 200 mg/dl), rats received MLT at a dose of 10 mg/kg/day by intraperitoneal injection for 6 weeks. Body weight and random blood sugar were measured for all groups. Sciatic nerves of all the sacrificed animals were subjected to light microscopic, electron microscopic, and morphometric studies.

Results In group II, DM induction was associated with the occurrence of neuropathy manifested by marked thickening of the epineurium and perineurium. Nerve fibers exhibited marked axonal atrophy, axonal shrinkage, axon–myelin separation, and in some sections total axonal destruction. Severe demyelination with evidence of myelin destruction was observed in the form of splitting and decompaction of myelin sheath lamellae, as well as vacuolization of the myelin sheath, forming fermentation chambers. In the MLT-treated group, vacuolization of the myelin sheath decreased remarkably and mild local axon separation from myelin sheaths was detected. Morphometric analysis revealed a significant increase in the number of total and apparently normal fibers and decrease in the number of apparently degenerated fibers in the nerve sections of MLT-treated rats, compared with nontreated diabetic rats.

Conclusion This study showed that MLT, in early stages of DM induction, decreased the destructive progress of DM and provided neuroprotection against damage resulting from STZ-induced hyperglycemia. Thus, it is recommended to start MLT therapy as soon as diagnosis of DM is established and even earlier in individuals at high risk for developing DM.

Department of Histology, Faculty of Medicine, Cairo University, Cairo, Egypt

Correspondence to Noha M. Afifi, MD. Department of Histology, Faculty of Medicine, Cairo University, Cairo, Egypt Tel: +20 106 962 6557 e-mail: noha_afifi@windowslive.com

Received September 18, 2012

Accepted October 3, 2012

Back to Top | Article Outline

Introduction

Diabetes mellitus (DM) is the most common cause of diabetic peripheral neuropathy (DPN) in most countries. Approximately 50–70% of diabetic patients exhibit diabetic neuropathy 1.

At the point of clinical detection, significant impairments in nerve function may have already handicapped patients. Sensory, motor, and autonomic fibers can all be targeted by chronic hyperglycemia typically occurring in a distal-to-proximal gradient, starting in the lower extremities in a ‘glove and stocking pattern’ 2.

Patients may experience painful burning and tingling sensations, followed by loss of fine touch and temperature sensations. Loss of sensation can predispose individuals to foot lesions, ulcerations, and lower extremity amputation secondary to gangrene. Peripheral neuropathy (PN) is globally recognized as the most common risk factor for foot disease in the diabetic population 3.

Although controlling blood glucose and glycated hemoglobin (HbA1C) levels using insulin therapy remains the most effective method for preventing DPN, this approach fails for many patients as it reduces the incidence of DPN by only 34% over a 9-year period. Further, insulin treatment has nearly no value for patients who already exhibit neuropathy when diagnosed with DM 4.

By far, several therapeutic interventions and various herbal and chemical medications have been suggested for the prevention of DM complications. However, their exact effectiveness as reliable preventive medications for diabetic neuropathy has not been considered seriously yet 5.

The underlying cause of diabetic neuropathy remains controversial. It likely includes a number of mechanisms involving microangiopathy with ischemia, excessive protein glycosylation, and oxidative stress, leading to structural changes on a genetically susceptible background 1.

Oxidative stress appears to be the most important pathogenic factor underlying diabetic complications including neuropathy. Lower endogeneous antioxidants and elevated lipid peroxidation levels are risk factors for the development of macrovascular and microvascular diabetic complications such as retinopathy, neuropathy, nephropathy, and atherosclerosis 6.

Mechanisms that contribute to increased oxidative stress in DM may include not only increased nonenzymatic glycosylation but also changes in the status of antioxidant defense systems, resulting in the development of complications and localized tissue damage 7.

As oxidative stress is the main cause of diabetic complications, administration of antioxidants appears to be one of the most reasonable therapeutic approaches. Some studies showed that diabetic complications may be reduced by antioxidant therapies, including supplementation with vitamins C and E, lipoic acid, and l-carnitin, in a variety of experimental animal models of DM 8.

Melatonin (N-acetyl-5-methoxytryptamine) (MLT) is an endogenous neurohormone derived from tryptophan. It is mainly released from the pineal gland. MLT participates in a number of physiological processes such as reproduction regulation and circadian rhythms; at the same time is a well-known potent antioxidant 9. MLT is an effective scavenger of different reactive oxygen species (ROS), such as hydroxyl and peroxyl radicals, and thus can prevent tissue damage 10.

Moreover, it has been found in some behavioral studies that MLT has a prolonged and severe antinociceptive effect in neuropathic pains by interaction with opioidergic and GABAergic system receptors 11.

Although some studies have cited the role of MLT in the treatment of neuropathy due to DM 12, there are few studies on its effect in prevention of diabetic neuropathy.

In many studies streptozotocin (STZ) has been regarded as the agent of choice for the induction of DM in animals. Being cytotoxic to pancreatic β-cells, it can be conveniently used to induce experimental DM in rats 13.

Aim of the work

The present study aimed to investigate the possible neuroprotective effect of MLT in a rat model of STZ-induced diabetic neuropathy, monitored by light and electron microscopy.

Back to Top | Article Outline

Materials and methods

Materials

Drugs used

  • (a) STZ was purchased from Sigma Chemical Company (St Louis, Missouri, USA) in the form of powder.
  • (b) MLT was purchased from Sigma Chemical Company (Puritan Pride Inc., USA) in the form of tablets, each containing 5 mg MLT.
Back to Top | Article Outline

Animals

The study was conducted at the Animal House of Kasr-Al Aini, Faculty of Medicine, according to the Ethical Guidelines for the Care and Use of Laboratory Animals. All experimental procedures conformed to the principles laid down by the National Research Council Guide for the Care and Use of Laboratory Animals.

Thirty-six (15 weeks old) adult male albino rats were included in the present study. Their weights ranged from 200 to 250±20 g (mean±SD, 210±3.24) at the beginning of the experiment. They were housed in a temperature and light-controlled room (12-h light/dark cycle) with free access to food and water.

The animals were divided into the following groups:

  • Group I (the control group): This group included six rats. They were injected intraperitoneally with citrate buffer (equivalent volumes of the drug) and were sacrificed with the corresponding experimental groups. One rat belonging to this group died.
  • Group II (the diabetic nontreated group): This group included 15 rats. Type I DM was induced in these animals by a single intraperitoneal injection of STZ at a dose of 60 mg/kg body weight. STZ was freshly dissolved in 0.05 mol/l citrate buffer (pH 4.5) 14. Diabetic rats were sacrificed 6 weeks after induction of DM (three rats belonging to this group died).
  • Group III (MLT-treated diabetic group): This group included 15 rats that were rendered diabetic by a single intraperitoneal injection of STZ at a dose of 60 mg/kg body weight. Immediately after confirmation of DM (by measuring blood glucose, 48 h after STZ injection), rats were administered MLT in a saline vehicle at a dose of 10 mg/kg/day by intraperitoneal injection for 6 weeks 15 (two rats belonging to this group died).
Back to Top | Article Outline

Methods

Body weight measurement

Body weight was recorded at the beginning of the experiment and at the end of the study period for all groups.

Back to Top | Article Outline

Laboratory investigations

Random blood sugar was measured for all groups. It was done in the Biochemistry Department, Faculty of Medicine, Cairo University. Forty-eight hours after STZ injection, development of DM was confirmed by measuring blood glucose levels in blood samples taken from the retro-orbital veins. Rats with blood glucose levels higher than 200 mg/dl were considered diabetic 14 (normal blood glucose<150 mg/dl) 4.

Back to Top | Article Outline

Light and electron microscopic studies

Animals were sacrificed using isoflurane inhalation. Sacral nerves were dissected from a standard site from the superior angle of the popliteal fossa. Small tissue pieces were fixed in fresh 2.5% glutaraldehyde in sodium phosphate buffer (pH 7.4). Specimens, each of 1 mm in length, were cut from the fixed sciatic nerve and washed in 0.5 mol/l phosphate buffer (pH 7.4) for 2 h (two changes) and postfixed in 1% osmium tetroxide in the same buffer, dehydrated, and embedded in Epon resin. For light microscopy, serial semithin 1 µm sections were cut using a Zeiss 6M ultramicrotome (Carl Zeiss AG, Germany, Oberkochen and Munich), stained with 1% toluidine blue, and examined. For electron microscopy, ultrathin sections (50–80 nm) were prepared and stained with uranyl acetate and lead citrate 16. The sections were examined using a Zeiss 100S transmission electron microscope.

Back to Top | Article Outline

Morphometric studies

Using a Leica Qwin 500 LTD image analyzer computer system (Cambridge, UK), the following parameters were measured:

  • Mean area% of myelinated nerve fibers, myelin sheaths, and endoneurial space. For each group, five slides of five different specimens were examined. From each slide, 10 nonoverlapping fields were measured at a magnification of ×400. The image analyzer was used to measure the area of nerve fibers, myelin sheaths, and endoneurial space, which was expressed as area percentage in relation to the area of the standard measuring frame.
  • The mean number of nerve fibers in the sciatic nerve was counted. The numbers of total, apparently normal, and apparently degenerated nerve fibers were counted. For each group, five slides of five different specimens were examined. For each slide, 10 nonoverlapping fields were measured at a magnification of ×400.
Back to Top | Article Outline

Statistical analysis

The mean values of the data obtained from the image analyzer were calculated and statistically compared using Statistical Package for Social Sciences (SPSS, Windows version 9; SPSS Inc., Chicago, Illinois, USA). Differences between the studied groups were examined for statistical significance as regards the various parameters using the analysis of variance test. This test is used to find a significant difference between more than two groups. A P value of less than 0.05 was considered significant. Data were tabulated and represented graphically 17.

Back to Top | Article Outline

Results

Body weight measurements

At baseline, no significant differences in body weight were detected between any of the study groups. A significant decrease in body weight was recorded in STZ-treated animals (P<0.05) when compared with the control group. After treatment with MLT for 6 weeks, there was no statistically significant difference in body weight compared with the nontreated diabetic group (Table 1).

Table 1

Table 1

Back to Top | Article Outline

Blood glucose changes

Treatment of rats with STZ resulted in a significant increase in serum glucose level when compared with the control values (P<0.05) and it remained elevated 6 weeks after STZ. After treatment with MLT, the blood glucose level showed a significant decrease compared with the nontreated diabetic group (Table 2, Histogram 1).

Table 2

Table 2

Histogram 1

Histogram 1

Back to Top | Article Outline

Light microscopic results

Examination of sciatic nerve sections from rats of the control group showed the normal histological architecture of nerve fascicles containing myelinated and unmyelinated fibers. Variation was detected in the diameter of the nerve fibers. Nerve fibers were grouped in fasciculi, each of which was surrounded by perineurium, and the whole nerve was surrounded by the epineurium. Axons appeared clear with a dark ring of myelin around them. Epineurial and endoneurial connective tissue (CT) with blood vessels were observed having flat endothelial lining (Figs 1 and 2).

Figure 1

Figure 1

Figure 2

Figure 2

Examination of semithin sections of sciatic nerves in the diabetic group revealed infiltration of the CT surrounding the nerve with several mast cells in addition to lipid droplets. Blood vessels in the surrounding CT as well as endoneurial vessels exhibited high endothelial lining, hypertrophy, and vacuolation of smooth muscles of the media with narrowing and sometimes obliteration of their lumina (Figs 3 and 4).

Figure 3

Figure 3

Figure 4

Figure 4

Marked thickening and irregularity of the epineurium surrounding the whole nerve was detected, as well as thickening of the perineurium surrounding the nerve fascicles. In some bundles, markedly separated nerve fibers were detected (Fig. 4).

Marked changes in the majority of the nerve fibers were demonstrated in the diabetic rats, ranging from axonal atrophy to total axonal destruction and loss. Axonal shrinkage and axon–myelin separation were noted. Onion-bulb formation with layers of Schwann cell processes enwrapping the axon was evident. Excessive deposition of collagen fibers and fibrosis were encountered between the nerve fascicles (Fig. 5).

Figure 5

Figure 5

Changes in the myelin sheath revealed severe demyelination with evidence of myelin destruction in the form of dark degeneration of myelinated fibers. Splitting, decompaction, and degradation of myelin sheath lamellae were observed, as well as vacuolization of the myelin sheath with formation of fermentation chambers (Figs 5 and 6).

Figure 6

Figure 6

Examination of semithin sections of sciatic nerves in the MLT-treated group revealed a better histological picture as compared with that of diabetic rats. Endoneurial blood vessels exhibited a flat endothelial lining and continuous basement membrane. Some of the nerve fibers exhibited a regular outline. Axonal changes and vacuolization of myelin were less obvious. Several Schwann cells exhibiting pale nuclei were observed enclosing the nerve fibers (Figs 7 and 8).

Figure 7

Figure 7

Figure 8

Figure 8

Back to Top | Article Outline

Electron microscopic results

Ultrathin sections of sciatic nerve from the control group revealed the usual ultrastructural configuration of small-diameter and large-diameter nerve fibers. The majority of nerve fibers were myelinated with some unmyelinated fibers in-between. Each nerve bundle was surrounded by perineurium and the whole nerve was surrounded by CT epineurium. Schwann cells showed normal ultrastructural features (Fig. 9). The myelin sheath appeared as compact electron-dense material. The axoplasm contained neurotubules and neurofilaments (Figs 10 and 11).

Figure 9

Figure 9

Figure 10

Figure 10

Figure 11

Figure 11

Examination of ultrathin nerve sections of the diabetic group demonstrated perineurial thickening. Demyelination was noted in the majority of nerve fibers in the form of vacuolation in myelin sheaths and splitting and decompaction of myelin sheath lamellae, with the formation of fermentation chambers. Axonal damage and total axonal destruction were seen in some fibers, with accumulation of neurofilaments and electron-dense axonal vesicles and inclusions in the axoplasm. Some myelinated fibers showed retraction of their axoplasm, which ranged from mild to severe retraction. The spaces between the axolemma and myelin sheaths were electrolucent, suggesting the accumulation of periaxonal edema (Figs 12–14). Small myelinated axons exhibited irregular contour and redundant myelin sheath (Fig. 15). Macrophages were detected engulfing the myelin debris (Fig. 16).

Figure 12

Figure 12

Figure 13

Figure 13

Figure 14

Figure 14

Figure 15

Figure 15

Figure 16

Figure 16

Degenerative changes were also observed in Schwann cells in the form of multiple cytoplasmic electrolucent vacuoles. Cytoplasmic lysis was noted and the mitochondria appeared with disrupted cristae. Schwann cell nuclei exhibited karyorrhexis (Fig. 17). Axoplasmic vacuolization of some unmyelinated fibers was noted as well (Fig. 18).

Figure 17

Figure 17

Figure 18

Figure 18

Endoneurial blood vessels exhibited interrupted basal laminae with absence of endothelial cells at some sites. Hypertrophy and vacuolation of smooth muscles of the media were observed. Wide separation of collagen fibers was observed in the endoneurium (Fig. 19). Other vessels were congested and showed high endothelial lining (Fig. 20).

Figure 19

Figure 19

Figure 20

Figure 20

Ultrastructural examination of nerve sections of rats treated with MLT showed fewer morphological alterations, compared with those from nontreated diabetic rats. Myelin breakdown was significantly diminished and the ultrastructural features of axons showed minimal degenerative changes. Vacuolation and lamellar separation of myelin were less obvious. Further, the fine structure of Schwann cells appeared normal. No vacuoles were detected in the axoplasm of unmyelinated fibers and they seemed to be normal (Figs 21 and 22).

Figure 21

Figure 21

Figure 22

Figure 22

Back to Top | Article Outline

Morphometric and statistical results

Mean area% of myelinated nerve fibers, myelin sheaths, and endoneurium in sciatic nerve sections

The mean area% of myelinated nerve fibers and myelin sheath showed a significant decrease in the diabetic group, compared with the control group. MLT treatment resulted in a significant increase in the mean area%, compared with the nontreated diabetic group.

The mean area% of endoneurium showed a significant increase in the diabetic group, compared with the control group. In the MLT-treated group, significant decrease in the endoneurial area% was reported, compared with the nontreated diabetic group (Table 3, and Histogram 2).

Table 3

Table 3

Histogram 2

Histogram 2

Back to Top | Article Outline

Mean number of nerve fibers in the sciatic nerve sections

Statistical analysis of the mean number of nerve fibers showed significant decrease in the number of total and apparently normal nerve fibers in the diabetic group, relative to the control group. Meanwhile, the number of apparently degenerated fibers significantly increased in the diabetic group.

In the MLT-treated group, significant increase in the number of total and apparently normal nerve fibers was seen in comparison with the nontreated diabetic group. Meanwhile, the number of apparently degenerated fibers decreased significantly (Table 4, and Histogram 3).

Table 4

Table 4

Histogram 3

Histogram 3

Back to Top | Article Outline

Discussion

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.

Back to Top | Article Outline

Conclusion

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.

Back to Top | Article Outline

Recommendations

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.

Table

Table

Back to Top | Article Outline

Acknowledgements

Conflicts of interest

There is no conflict of interest to declare.

Back to Top | Article Outline

References

1. Harati Y.Diabetic neuropathies: unanswered questions.Neurol Clin2007;25:303–317.
2. Oguejiofor OC, Odenigbo CU, Oguejiofor CBN.Evaluation of the effect of duration of diabetes mellitus on peripheral neuropathy using the United Kingdom Screening Test Scoring System, Biothesiometry and Aesthesiometry.Niger J Clin Pract2010;13:240–247.
3. Moretti B, Notarnicola A, Maggio G, Moretti L, Pascone M, Tafuri S, Patella V.The management of neuropathic ulcers of the foot in diabetes by shock wave therapy.BMC Musculoskeletal Disord2009;10:54.
4. Lennertz RC, Medler KA, Bain JL, Wright DE, Stucky CL.Impaired sensory nerve function and axon morphology in mice with diabetic neuropathy.J Neurophysiol2011;106:905–914.
5. Babaei-Balderlou F, Zare S, Heidari R, Farrokhi F.Effects of melatonin and vitamin E on peripheral neuropathic pain in streptozotocin-induced diabetic rats.Iranian J Basic Med Sci2010;132 Spring1–8.
6. Fox CS, Coady S, Sorlie PD, Levy D, Meigs JB, D’Agostino RB Sr, et al..Trends in cardiovascular complications of diabetes.J Am Med Assoc2004;292:2495–2499.
7. Gürpinar T, Ekerbiçer N, Uysal N, Barut T, Tarakçi F, Tuglu MI.The effects of the melatonin treatment on the oxidative stress and apoptosis in diabetic eye and brain.Scientific World J2012;2012:498489.
8. Golbidi S, Ebadi SA, Laher I.Antioxidants in the treatment of diabetes.Curr Diabetes Rev2011;7:106–125.
9. Reiter RJ, Tan D-X, Osuna C, Gitto E.Actions of melatonin in the reduction of oxidative stress: a review.J Biomed Sci2000;7:444–458.
10. Nishida S.Metabolic effects of melatonin on oxidative stress and diabetes mellitus.Endocrine2005;27:131–135.
11. Mantovani M, Pértile R, Calixto JB, Santos ARS, Rodrigues ALS.Melatonin exerts an antidepressant-like effect in the tail suspension test in mice: evidence for involvement of N-methyl-d-aspartate receptors and the l-arginine-nitric oxide pathway.Neurosci Let2003;343:1–4.
12. Negi G, Kumar A, Kaundal RK, Gulati A, Sharma SS.Functional and biochemical evidence indicating beneficial effect of melatonin and nicotinamide alone and in combination in experimental diabetic neuropathy.Neuropharmacology2010;58:585–592.
13. Lenzen S.The mechanisms of alloxan- and streptozotocin-induced diabetes.Diabetologia2008;51:216–226.
14. Wu KK, Huan Y.Streptozotocin-induced diabetic models in mice and rats.Curr Protoc Pharmacol2008Suppl 405.47.1–5.47.14.
15. Zangiabadi N, Vahid Sheibani V, Asadi-Shekaari M, Shabani M, Jafari M, Asadi A, et al..Effects of melatonin in prevention of neuropathy in STZ-induced diabetic rats.Am J Pharmacol Toxicol2011;6:59–67.
16. Hayat MA.Principals and techniques of electron microscopy: biological application.2000.4th ed.Edinburgh, UK:Cambridge University Press;37–59.
17. Armitage P, Berry G.Armitage P, Berry G.Medical research. Statistical methods.Medical research.1994.3rd ed.London:Blackwell Scientific Publications;12–48.
18. Yagihashi S, Yamagishi S-I, Wada R.Pathology and pathogenetic mechanisms of diabetic neuropathy: correlation with clinical signs and symptoms.Diabet Res Clin Pract2007;773 SupplS184–S189.
19. Veiga S, Leonelli E, Beelke M, Garcia-Segura LM, Melcangi RC.Neuroactive steroids prevent peripheral myelin alterations induced by diabetes.Neurosci Lett2006;402:150–153.
20. Morley JE, Thomas DR, Wilson M-MG.Cachexia: pathophysiology and clinical relevance.Am J Clin Nutr2006;83:735–743.
21. Brussee V, Guo G, Dong Y, Cheng C, Martinez JA, Smith D, et al..Distal degenerative sensory neuropathy in a long-term type 2 diabetes rat model.Diabetes2008;57:1664–1673.
22. LaPoint SF, Powers JM, Woodruff JM, MacCollin M, Jacoby LB, Vortmeyer AO, et al..Schwann cell-onion bulb tumor of the trigeminal nerve: hyperplasia, dysplasia or neoplasia?Acta Neuropathol2000;99:67–72.
23. Coste TC, Gerbi A, Vague P, Maixent JM, Pieroni G, Raccah D.Peripheral diabetic neuropathy and polyunsaturated fatty acid supplementations: natural sources or biotechnological needs?Cell Mol Biol (Noisy-le-grand)2004;50:845–853.
24. Kennedy JM, Zochodne DW.Experimental diabetic neuropathy with spontaneous recovery: is there irreparable damage?Diabetes2005;54:830–837.
25. Vargel I.Impact of vascularization type on peripheral nerve microstructure.J Reconstr Microsurg2009;25:243–253.
26. Sharma KR, Cross J, Farronay O, Ayyar DR, Shebert RT, Bradley WG.Demyelinating neuropathy in diabetes mellitus.Arch Neurol2002;59:758–765.
27. Cermenati G, Abbiati F, Cermenati S, Brioschi E, Volonterio A, Cavaletti G, et al..Diabetes-induced myelin abnormalities are associated with an altered lipid pattern: protective effects of LXR activation.J Lipid Res2012;53:300–310.
28. Altaf FM.Ultrastructural changes in the myelinated nerve fibers of the sciatic nerve in galactose intoxication in rats.Pak J Nutr2012;11:391–400.
29. Leon J, Acuña-Castroviejo D, Sainz RM, Mayo JC, Tan D-X, Reiter RJ.Melatonin and mitochondrial function.Life Sci2004;75:765–790.
30. Gumy LF, Bampton ETW, Tolkovsky AM.Hyperglycaemia inhibits Schwann cell proliferation and migration and restricts regeneration of axons and Schwann cells from adult murine DRG.Mol Cell Neurosci2008;37:298–311.
31. Cameron NE, Cotter MA, Low PA.Nerve blood flow in early experimental diabetes in rats: relation to conduction deficits.Am J Physiol1991;261:E1–E8.
32. Fazan VPS, de Vasconcelos CAC, Valença MM, Nessler R, Moore KC.Diabetic peripheral neuropathies: a morphometric overview.Int J Morphol2010;28:51–64.
33. Lu J, Wu DM, Hu B, Cheng W, Zheng YL, Zhang ZF, et al..Chronic administration of troxerutin protects mouse brain against d-galactose-induced impairment of cholinergic system.Neurobiol Learn Mem2010;93:157–164.
34. Rösen P, Nawroth PP, King G, Möller W, Tritschler H-J, Packer L.The role of oxidative stress in the onset and progression of diabetes and its complications: a summary of a congress series sponsored by UNESCO-MCBN, the American Diabetes Association and the German Diabetes Society.Diabetes Metab Res Rev2001;17:189–212.
35. Turkoglu E, Serbes G, Dolgun H, Oztuna S, Bagdatoglu OT, Yilmaz N, et al..Effects of α-MSH on ischemia/reperfusion injury in the rat sciatic nerve.Surg Neurol Int2012;3:98501.
36. Eckersley L, Ansselin AD, Tomlinson DR.Effects of experimental diabetes on axonal and Schwann cell changes in sciatic nerve isografts.Mol Brain Res2001;92:128–137.
37. Maritim AC, Sanders RA, Watkins JB III.Diabetes, oxidative stress, and antioxidants: a review.J Biochem Mol Toxicol2003;17:24–38.
38. Bozza PT, Viola JPB.Lipid droplets in inflammation and cancer.Prostaglandins Leukot Essent Fatty Acids2010;82:243–250.
39. Shi MA, Shi G-P.Different roles of mast cells in obesity and diabetes: lessons from experimental animals and humans.Front Immunol2012;3:7.
40. Korkmaz A, Topal T, Tan D-X, Reiter RJ.Role of melatonin in metabolic regulation.Rev Endocr Metab Disord2009;10:261–270.
41. Peschke E.Melatonin, endocrine pancreas and diabetes.J Pineal Res2008;44:26–40.
42. Dominguez-Rodriguez A, Abreu-Gonzalez P, Reiter RJ.Melatonin and cardiovascular disease: myth or reality?Rev Esp Cardiol2012;65:215–218.
43. Jaworek J, Brzozowski T, Konturek SJ.Melatonin as an organoprotector in the stomach and the pancreas.J Pineal Res2005;38:73–83.
Keywords:

diabetes mellitus; electron microscopy; melatonin; oxidative stress; peripheral neuropathy

© 2013 The Egyptian Journal of Histology