Investigating effects of grape seed extract on neuropathic pain in the streptozotocin-induced diabetic mice.

Diabetes mellitus is a complicated and serious health problem involving peripheral neuropathy. This situation causes to loss of senses, tingle and pain. Diabetic peripheral neuropathy (DPN) affects 236 million people around the World. Hence there is a need to investigate alternative ways of cure focusing on DPN. Flavonoids have potential on pain due to their permeability characteristic in the capillary microcirculation system and in lowering blood pressure. Flavonoids are common in grape seed. The main flavonoids of grape seed involve proanthocyanidins which might be an effective agent in cure of pain in DPN. The purpose of this study is to investigate effects of grape seed extract on neuropathic pain in the Streptozotocin-induced diabetic mice. In the study, 50 eight-week old BALB-C strain mice in five different groups (Control, Diabetic Control, Control+25 mg/kg, Diabetes+25 mg/kg and Diabetes+50 mg/kg) were used. To induce diabetes in thirty of these animals, single dose Streptozotocin (180 mg/kg) was administered intraperitoneally. After diabetes was observed, grape seed extract (25 mg/kg and 50 mg/kg body weight) was administered by oral gavage in three groups (Cont­rol+25 mg/kg, Diabetes+25 mg/kg and Diabetes+50 mg/kg) during six weeks. At the end of the second and sixth weeks, pain threshold measurements in hot plate test were performed in line with a predetermined thermal pain model. Also the tissues of the sciatic nerve and abdominal aorta from the animals were histologically investigated. As a result hot plate measurements, pain threshold values of the animals in Diabetes+50mg/kg group significantly differed from the measurements of the animals in control group in the first measurements and from the animals in Diabetes+25 mg/kg group in the second measurements (p<0.05). However pain threshold values of the animals in Diabetes+25 mg/kg group differed significantly from the values of the animals in control+25 mg/kg group and control group. It means pain threshold values of the animals in Diabetes+50 mg/kg and Diabetes+25 mg/kg groups were significantly lower than the values of the animals in the other groups. The results of histological investigations showed that degenerations of myelin sheet and axons in diabetic control group were decreased significantly in   Diabetes+50 mg/kg and Diabetes+25 mg/kg groups. Moreover degenerations of aorta tissues of animals in diabetic group were not seen in the animals of Dia­betes+50 mg/kg except for tunica adventitia inflammation. It can be said that grape seed extract decreased threshold of neuro­pathic pain in the Streptozotocin-induced diabetic mice and prevented degenerations of myelin sheet and axons, and aorta tissues.

Diabetes mellitus is a metabolic disease affecting 382 million people around the World [1,2]. Diabetes involves either insufficient insulin secretion or deficiency in tissue response to insulin [3]. Based on the deficiency of insulin metabolism, diabetes causes different complications involving hypoglycemia [4,5], ketoacidosis [6] and peripheral neuropathy [7]. Neuropathy is characterized by loss of sense, tingle and pain [7]. In neuropathy deficiency of distal axons, loss of axons and loss of myelin in neuron fibers are observed [8]. Moreover the neuropathic patients feel depression and pain [9]. Neuropathic pain affects 6-8% of the World population and its prevalence increases with diabetes mellitus [10]. Neuropathic pain frequently causes to burning sensations, to sleep disturbances and to tiredness [11,12]. These symptoms prevent the patients from working and social life.
In neuropathy, production of free oxygen radicals and increased load of mitochondrial electron transfer cause damage to cells. Since accumulation of free oxygen radicals lead to increased levels of lipid, DNA and protein peroxidation which induce cell death and decrease blood flow in nervous system [13,14]. Hence prevention of peroxidation caused by free oxygen radicals might be a beginning point in decreasing neuropathic pain in diabetic patients. Flavonoids provide a way to investigate this claim, since content of flavonoids involves antioxidant activity [15], especially proantocyanidin which is a solvable short chain polymer found in grape seed is a strong anti-oxidant (free radical scavenger) [16]. Proantocyanidin's entioxidant activity is predicted by its phenolic hydrogen content [17,18]. However for defining a proanthocyanidin as an antioxidant two basic conditions must be provided: the first is that when its concentrations is low relative to the substrate, it must prevent oxidative injury while the second is that the product following the scavenging must be stable [19].
Proantocyanidin scavenges peroxyl and hydroxyl radicals and prevents lipid peroxidation [20,21]. Moreover a recent study showed preventive effect of grape seed extract on DNA damage [22]. Grape seed extract also contributes to prevention of capillary blood vessels by decreasing the effect of free radical harms on the walls of the vessels; hence it decreases the risk of platelet aggregations and arteriosclerosis [23,24]. Therefore the purpose of this study to investigate effects of grape seed extract on neuropathic pain in the Streptozotocin-induced diabetic mice

MATERIALS AND METHODS OF RESEARCH
Experimental Animals: In the study 50 eightweek BALB-C strain female mice were used (30±5 g). The mice were hold in a room at 22-25°C 19/ Том XXIV / 4 and they were exposured to 12 dark and 12 light cycle. The animals were in cages for five mice and they were fed with tap water and standard pellet food for mice. Then, the animals were divided into five different groups (10 mice per group): I. Group: Control (n=10) II. Group: Diabetic Control (n=10) III. Group: Control + 25 mg/kg grape seed extract (n=10) IV. Group: Diabetes+25 mg/kg grape seed extract (n=10) V. Group: Diabetes +50 mg/kg grape seed extract (n=10) Inducing Diabetes: In three groups of the study, Streptozotocin (STZ) (Sigma-Aldrich) (180 mg/kg) solved in 0.4 ml (0.1 M) sodium citrate (pH=4.5) were intraperitonally injected to the animals as a single dose. Blood glucose levels were measured by taking blood from tails of the animals and using glucose meter. Animals having satiety blood glucose levels over 200 mg/dl were accepted as diabetics.
Application of Grape Seed Extract: The extracts (25 ve 50 mg/kg) were prepared daily and fresh by solving them in distillated water. The extracts were given by oral gavage way provided by plastic part of the grey vascular access tool (16G). During the gavage process, the animals were anesthetized by ether and the extracts were given at the same time of days.
Hot Plate Test: In the test the animals were put on a hot platform and time of their reactions were recorded for determining pain threshold value. For the mice appropriate Hot Plate Analgesiameter (Harvard, Edenbridge, UK) was used. Before the experiments all of the animals were tested in hot plate test during 5 days to provide accommodation to the system (147). Then the animals were rested for one day and they were tested two times in sixth day. During the experimental Hot Plate test the temperature of the platform was adjusted to 50±0.5 °C, the mice could not go out of the system due to its barriers and they were tested for pain threshold. After the animals were put into the platform, a chronometer was started and their retract times of extremities and licking them were recorded in a silent environment. If the animals did not retract the extremities in 60 sec., they were taken from the system to prevent tissue harms and they were not included in the study. The test was applied to the animals after the oral gavage process.
Histological Applications Sciatic Nerve Tissue: Sciatic nerve tissues of the animals were fixed in form aldehyde (%10). Then the tissues were embedded into the paraffin after washing them in tap water, dehydration and poli-shing. Samples in 4 µm thickness were taken from the paraffin blocks and, deparafinisation and rehydration processes were applied to the samples. Then the samples were stained by hematoksilen-eozin (H-E) method and they were observed by Leica DFC-280 research microscope.
Sciatic Nerve: Changes in nerve cells (axonal changes) were analyzed by Leica Q Win image analysis system. During the analysis each slice of the samples stained by H-E method were observed by using 100:1 (100X) magnifying power. Axonal changes were scored as none (0), slight (1), mild (2) and strong (3).
Aorta Tissue: Aorta tissues of the animals were fixed in form aldehyde (%10). Similar to sciatic nerve tissues, aorta tissues were embedded into the paraffin after washing them in tap water, dehydration and polishing. Then slices from the samples in 4 µm thickness were taken from the paraffin blocks. The samples were stained by hematoksileneozin (H-E) and orsein methods, and then Leica DFC-280 research microscope was used to observe stained samples.
Abdominal Aorta: For the analysis of abdominal aorta, tunica media thicknesses of randomly selected six samples stained by hematoksilen-eozin (H-E) method were measured by using 40X magnifying power and Leica Q Win image analysis system. Elastic fibers stained by orsein were analyzed by considering loss of elastic fibers and defects in undulated-waved structure. Defects in elastic fibers were scored as strong (0), slight (1), mild (2) and severe (3) by observing them in 40X magnifying power.
Statistical Analysis: For analysis of quantitative data, descriptive (mean±standart deviation) values were represented. In following inferential statistics Wilcoxon Signed Ranks Test was used to compare first and second scores on Hot Plate Test while paired samples t-test was used to compare total first and second scores on Hot Plate Test. Between-group comparisons of Hot Plate scores and histological data were made by Mann Whitney U test. For all of the analyses, 0.05 was accepted as significance threshold. Findings

Findings on Blood Glucose Levels and Hot Plate Test
Blood glucose levels of the animals before and after grape seed extract application showed that all of the diabetes groups showed significant rise in their blood glucose levels relative to control group animals in both before and after the application. Table 1 represents results of analysis on blood glucose levels of the animals before and after grape seed extract application. In terms of blood glucose levels, just diabetic control animals represented a significant increase in their blood glucose levels between two measurements. After determining blood glucose levels, pain threshold data were analyzed and the results showed that pain threshold values of the all groups were significantly increased except for the control group. Table 2 represents results of statistical analysis of hot plate test data. When the groups were compared in terms of their scores on two hot plate test applications, it was seen that there were significant differences in hot plate scores of the experimental groups and control groups. Results on comparisons of the groups in terms of hot plate scores are seen in table 3. Table 3 showed that there was statistically significant difference between hot plate test I scores of the animals in Control and Diabetes+50 mg/kg groups. Similarly hot plate test I scores of the animals in Control group were significantly higher than the scores of the animals in Control+25 mg/kg group. When looked at the hot plate test II scores, it was seen that the scores of the animals in Diabetes+25 mg/kg were significantly higher than the scores of the control group animals, animals in Control+25 mg/kg group and the animals in Diabetes+50 mg/kg group.

Findings Histolopathological Parameters
Histological analyses were done in two ways as statistical comparison and microscope observation. Statistical analyses of two tissues showed that histopathological scores of the animal tissues in diabetes control group were significantly higher than control group animals in terms of sciatic nerve damage and elastic fiber defects. The results are represented in table 4.

Groups
Histopathological score (Sciatic Nerve Tissue) Sciatic nerve tissue damages of the animals in diabetic control group involved thick perineuriums, shrinkage in axons and degenerations in myelin sheet structure. Figure 1 represents the damages of sciatic nerve tissue.
На умовах ліцензії CC BY 4.0 Moreover aorta tissue damages of the animals in diabetic control group involved loss of elastic fibers and defects in undulated-waved structure. Figure 2 represents the damages of aorta tissue.
As seen in the figures, the animals in diabetic control group had damages and deficits in their tissues of aorta and sciatic nerve. However there were no significant damages and deficits in the tissues of the animals in the other groups.

RESULTS AND DISCUSSION
Findings of the study showed that content of the grape seed extract prevented significant increases in blood glucose levels of diabetic animals. This situation might be related to effect of flavonoids in the extract on increasing plasma insulin levels in rats. One of the study reported that narigine flavonoid induced increase of plasma insulin levels in STZ-induced diabetic rats [25]. In another study, the grape seed extract showed antihyperglisemic feature and it prevented increase in blood glucose levels of the animals [26]. Similarly Wu et al. applied the grape seed extract to the rats and their findings showed that the grape seed extract prevented abnormal elevation of blood glucose in diabetic rats [27]. In following analyses, pain thresholds were also in focus and the findings showed that the thresholds increased significantly except for control group animals. Also the threshold value of the diabetes group taking the grape seed extract (50 mg/kg) differed significantly in the first measurement while the other diabetes group taking the grape seed extracts (25 mg/kg) represented significantly higher threshold value than the control group animals in the second measurement. Actually the threshold values of the animals in diabetic control in two measurements were not different from the values of control group animals. This means change in pain threshold occurs during diabetes because diabetic neuropathy involves thermal hipo and hiperalgesia, allodini and degenerations in nerve fibers [28]. However contribution of the grape seed extract depends on dose and time. Hence similarity of pain threshold values of group taking the grape seed extract with those for control group animals might be related to dose and time factors [29].
When looked at the histopathological findings, it is seen that the animals in diabetic control group 19/ Том XXIV / 4 represent thick perineuriums in sciatic nerve tissue, shrinkage in axons, degenerations in myelin sheet structure, loss of elastic fibers and defects in undulated-waved structure in aorta tissue. The results led us to think that applying grape seed extract contributed to prevent histological damages and defects of diabetes, since the animal tissues in the groups taking grape seed extract did not significantly differ from control group. In a previous study, it was shown that the grape seed extract contributed to healing of Schwan cell damage, decrease of demyelinisations, and prevention of structural abnormalities in peripheral neurons [30]. Also the extract was shown to be having protective feature on nerve tissue function [31]. For vessel function it was also shown that the grape seed extract contributed to microcirculation of blood and to prevention of endothelial dysfunction [32]. These contributions of the grape seed extract are thought to be related to its anti-oxidant [33], anti-proliferative [34] and neuroprotective [35] features.

CONCLUSION
In sum the findings of this study indicate that the grape seed extract is an effective agent in healing of damage in sciatic nerve tissues in diabetic neuropathy. Also it is partially effective on healing of damage in vascular tissues. In spite of these contributions, dose and application time are the determinants of the effectiveness. In following studies, dose and application time should be studied to see the picture clearly. As another point, analgesic effects of the extract was not determined in this study, this parameter should be studied by considering dose and application time.
Acknowledgement. This study was supported by Inonu University Scientific Research Project Fund with the project number; 2013/178. Portions of this work were presented at the TFBD 41. National Physiology Congress (Çanakkale, Turkey, 2015). This research did not receive any specific funding.
Conflict Statement. No potential conflict of interest was reported by the authors.