Background
Diabetes mellitus is a chronic metabolic disorder, characterized by disturbed glucose metabolism due to an absolute or relative insulin deficiency [
1]. The prevalence of diabetes in Egypt is high and it can be considered as a major clinical and public health problem [
2]. Neurological complications are among the central problems in diabetes mellitus. Over 60% of individuals with diabetes are affected by neurological disorders [
3]. Diabetic neuropathy is attributed to chronic hyperglycemia which may induce damage to nerve cells and decrease neurovascular flow especially during neuronal ischemia [
4]. Cerebral ischemia is a leading cause of death and disability worldwide and diabetes is a risk factor for ischemic cerebrovascular diseases [
5]. Reperfusion following cerebral ischemia leads to the generation of pro-oxidant species which cause neuronal damage by acting directly on macromolecules, including proteins, lipids and DNA, or indirectly by interfering with cell signaling pathways and gene expression regulation [
6]. Furthermore, several mechanisms are responsible for diabetic neuropathy including dyslipidemia, inflammation and apoptosis [
4,
7,
8]
.
The most proposed molecular mechanism by which hyperglycemia induces complications in diabetes is increased oxidative stress [
9]
. Oxidative stress is a relative overload of oxidants caused by increased free radical production and/or decreased antioxidant defense systems. Increased free radical production exerts toxic effects on membrane phospholipids, resulting in formation of toxic products such as MDA [
1,
10]
.
Diabetes is usually associated with inflammation [
8]
. When excess glucose is shunted through alternative metabolic pathways, this leads to increase in TGF-β1 and NF-κB (inflammatory mediators). COX-2 is an important enzyme that is upregulated by NF-κB, which is observed in peripheral nerves and vascular tissues in experimental diabetes. Pharmacological blockade or gene ablation of COX-2 prevents diabetes-induced changes in peripheral nerves [
4]
. Also, COX-2 enzyme is responsible for the production of prostaglandins, a family of powerful inflammatory mediators produced by activated microglia in the neuroinflammatory/neurodegenerative diseases, and not surprisingly, COX-2 has been considered a major therapeutic target [
11]
. IL-4 has been demonstrated to have anti-inflammatory activities specially on activated microglia, including inhibition of the expression of TNF-α, as well other pro-inflammatory cytokines [
12]
. Apoptosis, programmed cell death, can contribute to a variety of disease states in the nervous system such as diabetes, ischemia and Alzheimer’s disease [
13]
.
Resveratrol, naturally occurring polyphenol, is found in high concentration in the skin and seeds of grapes, peanuts and ground nuts and has been reported to have several biological effects, including a potent antioxidative effect via preventing lipid peroxidation, cardioprotective, anticancer, and anti-inflammatory activity attributed to COX-2 inhibition [
10,
14]
. Moreover, resveratrol may be helpful in preventing and treating some metabolic diseases, including diabetes through reduction of blood glucose, preservation of cells, and improvement in insulin action [
14]
. In addition, resveratrol can reduce the oxidative stress produced in STZ-diabetic rats. [
15]
. It has been reported that resveratrol administration to the hypercholesterimic rats attenuated the increase in serum lipid profile [
16]
.
The present study was designed to clarify the adverse effects of diabetes on cerebral outcomes, evaluate the effect of resveratrol on modulating cerebral complications in diabetic and diabetic cerebral ischemic-reperfused rats and investigate the histological changes in cerebral tissue of diabetic, diabetic cerebral ischemic-reperfused and treated rats.
Discussion
Administration of STZ (45 mg/kg) in adult male albino rats resulted in induction of diabetes that was confirmed by a remarkable increase in serum glucose level as compared to normal rats. Other symptoms such as weight loss, polyuria, polydepsia and polyphagia were also observed in diabetic rats (data not reported). These results are in agreement with other studies [
1,
2,
10,
36]
. Moreover, marked dyslipidemia, oxidative stress and inflammatory responses were observed in diabetic rats and these findings are in harmony with various reported studies [
2,
36‐
39]
.
In the present study, diabetic rats showed a picture of eosinophilic degeneration and strong Bax immunoreaction of pyramidal cells that were contracted with eosinophilic cytoplasm, small darkly stained nuclei and some of them were surrounded with halos and these findings are in agreement with
Amin et al. [
40]
. These results were confirmed with AgNOR stain which showed a significant decrease in the mean number of AgNOR dots in the diabetic group.
Bhatt et al. [
41] stated that Nucleolar Organiser Regions (NORs) are segments of DNA, closely associated with nucleoli of the cells on the short arms of the acrocentric chromosomes, 13, 14, 15, 21 and 22, containing coding gene for Ribosomal RNA and contribute to the regulation of the cellular synthesis. Recent modification of a silver staining technique allows the interphasic NORs to be visualized under LM in conventional histopathological sections, where they are called as “Argyrophilic Nucleolar Organizer Regions (AgNORs)”. Also, cresyl violet stained sections of the diabetic group showed a decrease (not significant) in the mean number of viable neurons.
Pamidi et al.[
32] found that the diabetic rats exhibited a decrease in the mean number of surviving neurons, counted using cresyl violet stained sections of cerebral cortex, when compared to the control group. These results indicated a decrease in the activity of the neuronal cells of the cerebral cortex which is most probably due to oxidative stress and apoptosis. It has been reported that neurons in hyperglycemic environment displayed signs of apoptosis due to hyperglycemia-induced oxidative stress [
4]
. Also,
Zhao et al. [
42] proved that diabetes upregulated the expression of Bax and caspase-3 which led to apoptosis of the pyramidal neurons in STZ induced diabetic rats.
The current study showed that cerebral ischemic-reperfusion for diabetic rats specifically exaggerated oxidative stress, inflammation and apoptosis including a significant increase of cerebral content of MDA, upregulation of COX-2 gene expression, a severe depletion of cerebral GSH and IL-4 contents and a significant increase in the apoptotic index and the optical density of Bax reaction. The major pathological mechanisms of cerebral ischemic injury include excitotoxicity, oxidative stress, inflammation, and apoptosis, which are associated with mitochondrial dysfunction and a rapid decrease of adenosine triphosphate (ATP). Depletion of GSH in cerebral ischemia leads to lipid peroxidation and neuronal cell apoptosis, in which the Bcl-2 family proteins (e.g. anti-apoptotic Bcl-2, pro-apoptotic Bax) are involved [
43,
44]
. Furthermore, there is evidence that elevated ROS levels within mitochondria generated by cerebral ischemic-reperfusion alters the expression of pro-apoptotic factor Bax, anti-apoptotic Bcl-2 and caspase-3 [
43,
45].
Histologically, the cerebral tissues of cerebral ischemic-reperfusion diabetic rats showed features of eosinophilic degeneration and some neurons were surrounded with halos. These findings are in agreement with
Levison Damr [
46] who stated that cerebral ischemia or anoxia led to eosinophilic degeneration, mostly of pyramidal cells of cerebral cortex as the whole cell shrinks, contracts, the cytoplasm loses its Nissl granules and becomes eosinophilic. The nucleus is basophilic, hyperchromatic, small and pyknotic and moves to more peripheral position and the nucleolus disappear. Also, AgNOR stained sections showed significant decrease in mean number of AgNOR dots. Furthermore, cresyl violet stain showed a significant decrease in the mean number of viable neurons.
Pamidi et al. [
32] supported our findings as they found that the untreated diabetes mellitus coupled with stress can induce highly significant damage in the neurons of rat cerebral which was shown by a significant decrease in the number of surviving neurons of cresyl violet stained sections.
In the present study, treatment of the diabetic and the diabetic ischemic-reperfused rats with resveratrol induced a remarkable reduction of plasma glucose level and corrected the diabetic dyslipidemia. These results are in harmony with other studies [
16,
47] which reported that resveratrol reduced blood TAG, TC and LDL-C and elevated HDL-C in hypercholesterolemic rats
. Moreover,
Gnoni and Paglialonga [
48] reported that resveratrol decreased fatty acid and TAG synthesis through inhibition of fatty acid synthase in isolated rat hepatocytes. This may represent a potential mechanism contributing to the reported hypolipidemic effect of resveratrol.
Administration of resveratrol significantly ameliorated diabetes-induced oxidative stress, inflammation and apoptosis. Various studies suggested the neuroprotective activity of resveratrol through its antioxidant and anti-inflammatory properities [
15,
49,
50]. Resveratrol was reported to inhibit lipid peroxidation and neuronal cell death induced by oxidative stress and enhance various antioxidant enzymes [
10,
51]
. These effects could be attributed to its property as a potent scavenger of ROS and RNS.
Zhang et al. [
49] demonstrated interesting anti-inflammatory activities for resveratrol. It can attenuate the activation of immune cells and the subsequent synthesis and release of pro-inflammatory mediators through the inhibition of the transcriptional factors such as NF-κB. In addition, it has been shown to inhibit the activation of microglia, cerebral microphages and reduce the production of pro-inflammatory mediators. Therefore, resveratrol may exert neuroprotection in neurodegenerative diseases accompanied by microglial activation. This hypothesis is best evidenced with the present study showing that resveratrol increased cerebral IL-4, anti-inflammatory cytokine targeting the microglia, in treated animals.
IL-4 was reported to suppress NF-κB which is a transcription factor that resides in the cytoplasm of every cell and translocates to the nucleus when activated. Its activation is induced by a wide variety of agents including stress, inflammatory stimuli and free radicals. Activation of NF-kB upregulates the expression of COX-2 (an inflammatory enzyme) [
52]
. The present study reported a significant upregulation of COX-2 gene expression in cerebral cortex of diabetic and diabetic cerebral ischemic-reperfused rats while resveratrol treatment downregulated COX-2 gene expression of treated animals. These results are in accordance with
Kumar and Sharma [
18] who stated that COX-2 enzyme is an inducible enzyme, becoming abundant in activated macrophages and other cells at sites of inflammation. This enzyme has been reported to be elevated in metabolic diseases as well as in diabetic condition.
The present study showed that treatment of the diabetic and the diabetic ischemic-reperfused groups with resveratrol reduced the extent of eosinophilic degeneration and apoptosis of neurons and showed moderate improvement of the cerebral tissues. Also, the mean number of AgNOR dots showed an increase (not significant) and the mean number of viable neurons stained with cresyl violet stain showed an increase (significant in the diabetic ischemic-reperfused group and not significant in the diabetic group) upon treatment with resveratrol. Therefore, these results showed that resveratrol has anti-apoptotic potency in consistent with previous reports [
44,
53,
54]
. Also, [
55] reported that resveratrol treatment attenuated rat cerebral damage after cerebral ischemia by downregulation of Bax expression.
The findings of the present study introduced new insights into the pathogenesis and treatment of neurodegenerative diseases, especially diabetic cerebral complications.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
HEM and SEE designed the research protocol, supervised the interpretation of the results and contributed to the revision of the manuscript. RAH and AAH performed the experimental work, carried out the statistical analysis, contributed to the interpretation of the results and drafted the manuscript. All authors read and approved the final manuscript.