Diabetes mellitus is a chronic metabolic disorder that leads to long-term complications affecting some tissues such as heart, kidney, retina, and peripheral nerves. Peripheral neuropathy is one of the most frequent and potentially severe complications of diabetes, leading to pain, skin ulcers, muscle weakness, loss of independence, and overall impairment of the patient’s quality of life. Diabetic neuropathy is a multifaceted complication of both type I and II diabetes, with an estimated prevalence between 50 and 90% depending on the duration of diabetes and involves a spectrum of functional and structural changes in peripheral nerves [
At the pathological level, the decrease of intraepidermal nerve fiber density in diabetics with symptoms of neuropathy correlates with electrophysiological and psychophysical Abnormalities [
Long-term hyperglycaemia leads to subsequent enhanced oxidative stress, increased aldose reductase activity, accumulation of advanced glycation endproducts, and decreased Na+, K+-ATPase activity [
DFEs might be interesting since they have been recently identified as promising neuroprotective agents in several models of neurodegeneration [
According to recent studies, by Asadi-Shekaari et al. and Panahi et al., DFE significantly inhibited neuronal damage induced by cerebral ischemia [
We have assessed whether the use of DFE has neuroprotective effects against STZ-induced diabetic neuropathy at the neurophysiological, behavioral, and neuropathological levels. STZ, which induces type 1 diabetes mellitus when delivered to rats, is a useful etiological model for studying complications caused by diabetic hyperglycemia [
We were interested in investigating the effects of DFE-enriched diets on the development of diabetic neuropathy in STZ-induced diabetes in rats. We decided to investigate the effect of DFE-enriched diets specifically on MNCV, behavioral activities, and pathology in STZ-induced diabetes in rats.
To prepare DFE, fresh date fruit was prepared from Bam town orchards, Kerman province (South East of Iran). The kernel was removed and 100 gr of date was immersed in 1000 mL/distilled water for 48 hours. It was then mixed completely in a mixer and then the mixture was centrifuged at 4000 rpm at 4°C for 20 minutes. After sedimentation, the supernatant part was used for gavages. Streptozotocin was purchased from Sigma-Aldrich. Blood glucose kits were purchased from the Roche Company, Germany.
The care of laboratory animals followed the guiding principles for care and use of laboratory animals of the Neuroscience Research Center of Kerman Medical University and the study protocol was approved by the animal ethics committee of this institution (Code: EC/KNRC/88-15). This study was carried out on male rats of Wistar breed weighing
Diabetes was induced by intraperitoneal injection of 45 mg/kg freshly prepared STZ dissolved in 0.1 mol/L Citrate buffer (pH 4.4). At the end of the first week after STZ injection, blood sugar measurements were taken for diagnosing diabetes induction using tail’s vein blood (by ACCU-CHEK-ACTIVE kit made by Roche, Germany). Rats with fasting blood glucose levels over 200 mg/dL were used in the experiments [
Six weeks after starting intervention, the horizontal and vertical activities of the male rats from each group were measured in an open field with a computerized Ethovision software (version 3.1), a video tracking system for automation of behavioral experiments (Noldus Information Technology, The Netherlands). Black wooden box (
Slowing of sensory nerve conduction velocity (SNCV) and motor nerve conduction velocity (MNCV) was shown to develop within the first month of the onset of hyperglycemia in STZ-diabetic rats [
In order to study morphological alterations resulting from diabetic neuropathy, sciatic nerve was isolated and divided into 2 mm segments. They were fixed immediately with glutaraldehyde solution 2.5% in 0.1 M Phosphate Buffer Solution (PBS) for 24 hr. The specimens were washed with PBS for 3 times and post fixed for 2 hr in 1% tetroxide osmium and dehydrated in graded concentration of ethanol and finally embedded in Epon 812 resin. Semithin sections (300 nm) were stained with 1% toluidin blue and examined by light microscopy. Ten fields of transverse sections were morphometrically analyzed by computerized image analysis system (Motic Images China e-kup Co.,Ltd). MFD, AD and MSD were measured for each section. For ultrastructural study, ultrathin sections (70 nm) were stained with 1% uranyl acetae and 2% lead citrate before viewing in Philips EM300 (Philips, Eindhoven, Netherlands).
The parametric data obtained by the experiments have been analyzed by using SPSS version 17.0. The quantitative data are expressed as the mean ± SEM. Multiple comparisons were performed among experimental groups by one-way analysis of variance (ANOVA) followed by Tukey-Kramer post hoc test.
In the diabetic groups, the plasma glucose concentrations at the end of the first week were 296.19% greater than those of control group (
Body weight and blood glucose levels of all groups.
Animal group | Body weight (g) | Blood glucose (mg dL−1) | |
Before STZ injection | End experiment | Before sacrifice | |
Control (8) | |||
Sham (9) | |||
Vehicle (8) | |||
DFE (8) |
Mean ± SEM (
In the assessment of diabetic neuropathy on the explorative activity of rats in an open field behavioral test, TDM, mobility, rearing and grooming frequency were significantly decreased in sham group compared with the control group, but there were no significant differences between control, sham, and DFE groups in time spent either in the centre (the most anxiogenic area) or in the perimeter of the open field. Analysis of the TDM and mobility showed that DFE group rats travelled significantly more than rats in the sham group (
Effect of Date fruit extract on explorative behavior of rats in open field test. (a) Rearing frequency:
Seven weeks after STZ injection, the sciatic MNCV of rats in the sham group (
Effect of Date fruit extract on motor nerve conductive velocity (MNCV) in rats. Data are the Mean ± SEM.
Light microscopy study of sciatic nerves sections showed the normal structure and morphology of myelinated fibers in the control group. In the sham and vehicle groups, some abnormalities including increased numbers of mast cells, edema, and myelin sheath splitting were seen (Figure
The effect of DFE on histomorphometric parameters of rat sciatic nerve.
Groups | MMFD ( | AD ( | MSD ( | |
---|---|---|---|---|
Control | 4 | |||
DFE | 5 | |||
Sham | 4 |
Light micrograph of transverse semithin sections of rat sciatic nerves. (a) Control group: myelinated nerve fibers are in normal structure and morphology. (b) Sham group: nerve fibers show some abnormalitities such as myelin splitting (black arrow), mast cell infiltration (M), and edema. (c) The DFE-treated group; the proportion of nerve fibers with abnormalities was reduced
Ultrastructural evaluation of sciatic nerves confirmed the light microscopy findings. In addition, some evidences of axonal degeneration such as mitochondrial swelling and disintegration of neurofilaments were observed under transmission electron microscope (Figure
Electron micrograph of rat sciatic nerves. (a) Control group: myelinated fiber with normal structure and morphology. Axon is in normal condition too. (b) Sham group: Myelinated fiber shows myelin splitting (white arrow). Loosening of myelin lamellae may account for paler staining in the outer myelin sheath. Axonal degeneration was revealed with enlarged mitochondria (M) and neurofilament disintegration ((a) ×8900, (b) ×11500).
In the present study, we evaluated the neuroprotective effects of a DFE diet on the development of experimental neuropathy. According to our data, DFE (4 mL/kg/day, orally) has a beneficial effect in the prevention of experimental neuropathy in male rats. In fact, we found a positive effect of DFE on electrophysiological marker of diabetic neuropathy (MNCV) and open field behavioural test. Furthermore, we showed that DFE partially prevented the deficit in the pathological morphology of myelinated sciatic nerve fibers in diabetic rats.
Patterns of diabetes-related peripheral nerve abnormalities can be categorised as distal symmetric sensory motor neuropathy, autonomic neuropathy, and focal asymmetric neuropathy. The most common pattern of injury is the symmetric neuropathy that involves distal sensory and motor nerves; small sensory fiber neuropathy is often prominently affected in the form of peripheral neuropathy of diabetic mellitus. With increased duration of the disease, diabetic subjects developed decreased sensations in the distal extremities that will result later in the loss of pain sensation and ulcers. Treatment of diabetic neuropathy is a major goal, but despite multiple attempts, no satisfactory management is yet available. Moreover, clinical data have shown that diabetic patients suffering peripheral neuropathy manifest electrophysiological conduction abnormalities [
Abnormal fibers undergoing axonal degenerative changes and myelin breakdown are reported to appear in the sciatic nerve of STZ-induced diabetic rats [
Diabetes-induced slowing of conduction of large-myelinated fibers (LMFs) was accompanied by a decrease in the proportion of the largest fibers present in the sciatic nerve. There is no marked fiber loss in short-term diabetic rats, and the reduction in the proportion of large fibers was accompanied by a parallel increase in the proportion of medium-sized fibers, suggesting either delayed radial growth of axons or atrophy of large axons. As axonal caliber is a major determinant of conduction velocity, the capacity of DFE to restore or maintain the proportion of large myelinated fibers in the sciatic nerve of diabetic rats reveals a potential mechanism that could explain the effect of DFE on conduction. Physiopathology of diabetic neuropathy includes activation of the polyol pathway, increases in oxidative stress and advanced glycosylation end products, perturbation in neurotrophism, and abnormalities in essential fatty acids metabolism [
Oxidative stress causes vascular impairment leading to decrease in nerve blood flow, resulting in endoneurial hypoxia and impaired neural function which may cause MNCV slowing [
Our findings, in accordance with earlier studies, indicate that treatment with STZ increases morphological alteration in the MFs of the sciatic nerve. The most abundant myelin abnormality observed in our study was myelin infolding in the axoplasm and fiber irregular shapes (Figure
Electrophysiological and morphologic observations have been confirmed by the explorative behavior of animals observed in the open field test. In this test, TDM, rearing, and mobility are signs of alterations in the animal’s spontaneous movements and explorative behaviour that showed a significant decrease in diabetes due to diabetic neuropathy. In agreement with our results, significantly decreased locomotor and exploratory activity (number of crossed fields, rearings, and bar approaches) have been reported in STZ-diabetic rats [
According to the results of the open field test, in the group that received DFE, as opposed to the sham group, TDM, mobility, and rearing showed a significant increase and became close to the control group. Therefore, it can be concluded that the neuroprotective effect of DFE in the prevention of diabetic neuropathy and consequently the prevention of defects in explorative behaviour due to diabetes is significant.
As mentioned above, the pathology of diabetic neuropathy also involves polyol pathway flux, oxidative stress, accumulation of advanced glycation endproducts, and microvascular injuries. Oxidative stress is the cause as well as sequel of almost all major pathophysiological pathways in diabetic neuropathy. Growing attention has been paid to the pathogenic roles of oxidative stress in diabetic neurological complications. Reactive oxygen species (ROSs) are generated by nonenzymatic protein glycation through a complex series of chemical and cellular intermediates [
Recent studies showed that the Iranian dates are strong radical scavengers and can be considered as a good source of natural antioxidants for medicinal and commercial uses [
Although the animals in the nondiabetic control group consumed the diet as much as diabetic groups, the difference of weight gain among them was significant (Table
To sum up, our study shows that treatment with DFE can decrease behavioral, neurophysiologic and pathological alterations induced by diabetes in the peripheral nerves of rats and that an antioxidant-related mechanism contributed to the improvement of diabetic neuropathy.
We found that DFE-diet intake in STZ-induced diabetic rats provided protection against deterioration of the peripheral nerve. We conclude that antioxidative property is one of the major profiles that is implicated in neuron protection. Thus our findings suggest that this compound may be considered as a potential preventative approach for peripheral diabetic neuropathy.
This work was supported by a project grant from Neuroscience Research Center of Kerman Medical University.