Neuroprotective effect of ginger in the brain of streptozotocin-induced diabetic rats

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RESEARCH ARTICLE

Neuroprotective effect of ginger in the brain of streptozotocin-induced diabetic rats Gehan El-Akabawy ∗ , Wael El-Kholy Menoufia University, Department of Anatomy and Embryology, Faculty of Medicine, Menoufia, Egypt

a r t i c l e

i n f o

Article history: Received 3 August 2013 Received in revised form 17 January 2014 Accepted 31 January 2014 Available online xxx Keywords: Ginger Diabetes Brain Histology Immunohistochemistry

s u m m a r y Diabetes mellitus results in neuronal damage caused by increased intracellular glucose leading to oxidative stress. Recent evidence revealed the potential of ginger for reducing diabetes-induced oxidative stress markers. The aim of this study is to investigate, for the first time, whether the antioxidant properties of ginger has beneficial effects on the structural brain damage associated with diabetes. We investigated the observable neurodegenerative changes in the frontal cortex, dentate gyrus, and cerebellum after 4, 6, and 8 weeks of streptozotocin (STZ)-induced diabetes in rats and the effect(s) of ginger (500 mg/kg/day). Sections of frontal cortex, dentate gyrus, and cerebellum were stained with hematoxylin and eosin and examined using light microscopy. In addition, quantitative immunohistochemical assessments of the expression of inducible NO synthase (iNOS), tumor necrosis factor (TNF)-␣, caspase-3, glial fibrillary acidic protein (GFAP), acetylcholinesterase (AChE), and Ki67 were performed. Our results revealed a protective role of ginger on the diabetic brain via reducing oxidative stress, apoptosis, and inflammation. In addition, this study revealed that the beneficial effect of ginger was also mediated by modulating the astroglial response to the injury, reducing AChE expression, and improving neurogenesis. These results represent a new insight into the beneficial effects of ginger on the structural alterations of diabetic brain and suggest that ginger might be a potential therapeutic strategy for the treatment of diabetic-induced damage in brain. © 2014 Elsevier GmbH. All rights reserved.

1. Introduction Diabetes mellitus (DM) is one of the most common chronic metabolic disorders leading to complications in multiple organs and systems. These complications often result in either morbidity or mortality (Perkins and Bril, 2005). Diabetic encephalopathy is one of the complications that occur due to gradually developing end-organ damage in the central nervous system (Northam and Cameron, 2013). The damage could be as a result of chronically increased intracellular glucose concentration leading to several structural, neurochemical, and neurodegenerative changes in different regions in the brain including the frontal cortex (Kumar et al., 2008), hippocampus (Pamidi and Satheesha Nayak, 2012), and cerebellum (Hernandez-Fonseca et al., 2009). These pathological changes underpin different cognitive, motor, and neuroendocrine disturbances characterizing diabetic encephalopathy (Rajashree et al., 2011). For instance, diabetes-induced cerebellar dysfunction

∗ Corresponding author. Tel.: +20 1015406365. E-mail addresses: [email protected] (G. El-Akabawy), wael [email protected] (W. El-Kholy).

is associated with seizure generation, motor deficits, and memory impairment (Anu et al., 2010). Defects in hippocampal synaptic plasticity and transmission result in deterioration of learning and memory in diabetic patients (Shingo et al., 2012). Interestingly, accumulating recent evidence indicates that diabetes mellitus is an important risk factor for sporadic Alzheimer’s disease (AD) (Vignini et al., 2013). One of the underlying mechanisms of diabetic neuronal injury is the excessive free radical generation from the auto-oxidation of elevated intracellular glucose levels (Gradinaru et al., 2013). Indeed, several in vitro, experimental, and clinical studies have implicated oxidative stress in the pathogenesis of diabetic complications. Oxidative stress results in depolarization of the inner mitochondrial membrane, release of cytochrome c into the cytosol, and ultimately, induction of caspase mediated apoptosis (Gurpinar et al., 2012). Ginger (Zingiber officinale) is widely consumed as a spice for the flavoring of foods. Ginger is reported to have several beneficial pharmacological effects (hypoglycemic, insulinotropic, and hypolipidemic) in experimental animals (Shanmugam et al., 2011) and in humans (Huang et al., 2004). It has been documented that ginger or its extracts possess some pharmacological activities including analgesic (Young et al., 2005), anti-tumor (Habib et al.,

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Please cite this article in press as: El-Akabawy, G., El-Kholy, W., Neuroprotective effect of ginger in the brain of streptozotocin-induced diabetic rats. Ann. Anatomy (2014), http://dx.doi.org/10.1016/j.aanat.2014.01.003

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2008) and anti-oxidant effects (Shanmugam et al., 2010). Antioxidants in ginger include gingerols, shogaols and some phenolic ketone derivatives. Anti-oxidant therapy has proved to be remarkably beneficial to remedy reactive oxygen species (ROS)-induced injury in the CNS (Tsakanova et al., 2011). Therefore, antioxidants such as ginger could be promising candidates in ameliorating the pathological sequelae of diabetic encephalopathy. In this study, we set out to investigate whether ginger has a protective effects on neuropathological changes associated with streptozotocin (STZ)-diabetic brains in rats. Quantified immunohistochemical assessments were conducted to elucidate mechanism(s) of the potential protective effect of ginger. Our results have shown that ginger ameliorated the morphological and neuropathological changes induced by diabetes. Oxidative stress, apoptosis, and inflammation were reduced in the ginger-treated group. In addition, ginger treatment down-regulated astrogliosis, decreased acetylcholinesterase (AChE) expression, and enhanced neurogenesis. Up to the best of our knowledge, this study is the first to demonstrate the protective role of ginger on neuropathological alterations associated with diabetic brain. 2. Methodology 2.1. Animals Male albino rats aged 8–10 weeks at an average weight 150–200 g were maintained in the animal house of the Faculty of Medicine, Menoufia University and were subjected to a 12:12-h daylight/darkness and allowed unlimited access to chow and water. All the ethical protocols for animal treatment were followed and supervised by the animal facilities, Faculty of Medicine, Menoufia University. All studies were approved by The Animal Care and Use Committee of Faculty of Medicine, Menoufia University. 2.2. Experimental design and diabetes induction Animals were randomly divided into four groups: control, control + ginger, diabetic, and diabetic + ginger. Rats of each of these groups were subdivided into another three subgroups in which rats were sacrificed after 4, 6, and 8 weeks after starting the experiment. Eight rats were used in each group at each time point. In diabetic and diabetic + ginger groups, diabetes was induced by intraperitoneal injections of streptozotocin (STZ, Sigma, St. Louis, MO, USA) administrated at a dose of 60 mg/kg. STZ-induced diabetes is a widely used model of type I diabetes characterized by hyperglycemia. STZ was dissolved in a freshly prepared 0.1 M citrate buffer (pH 4.5). Rats were fasted for 12 h before STZ injection. Fasting blood glucose levels were measured 72 h after STZ induction using blood drawn from the tail plexus of conscious rats. Blood glucose concentrations were tested using the Span Diagnostic kit with Jinque test strips. Rats with blood glucose above 250 mg/dl were considered diabetic. Control and diabetic groups were provided with standard chow, while control + ginger and diabetic + ginger groups rats were gavage-fed with 500 mg/kg/day of ginger. There was no significant difference between control and control + ginger rats in all the outcomes (fasting blood glucose levels and immunohistological assessments of different markers) at each time point used in the study; therefore, these two groups were pooled in one group (control). 2.3. Histological and immunohistological (IHC) assessments At the end of each time point studied, each rat was deeply anaesthetized using ketamine (90 mg/kg) and xylazine (15 mg/kg) (i.p.)

and decapitated. Each brain was fixed in 10% neutral buffered formalin and embedded in paraffin wax for histological examination. Semi-serial 5 ␮m-coronal sections (1-in-20 series) were prepared from the frontal cortex, hippocampus, and cerebellar cortex and were dehydrated using ethanol and stained with hematoxylin & eosin (H&E). For immunohistological staining, paraffin sections (5 ␮m thick) were deparaffinized in xylene for 1–2 min and then rehydrated in descending grades of ethanol (100%, 95%, and 70% ethanol) two changes 5 min each, then brought to distilled water for another 5 min. Sections were rinsed with PBS, blocked for 30 min in 0.1% H2 O2 as inhibitor for endogenous peroxidase activity. After rinsing in PBS, sections were incubated for 60 min in blocking solution (10% normal goat serum) at room temperature (RT, 21 ◦ C). The sections were then incubated with the primary antibody (inducible NO synthase (iNOS), 1:500, Labvision; Caspase-3, 1:500, Labvision; tumor necrosis factor (TNF)-␣, 1:1000, Labvision; glial fibrillary acidic protein (GFAP), 1:300, Labvision, and Ki67, 1:500, Labvision, and acetylcholinesterase (AChE), 1:300, Novus) at RT for an hour. Sections were rinsed with PBS, followed by 20 min of incubation at RT with secondary biotinylated antibody. After rinsing the sections in PBS, enzyme conjugate “Streptavidin-Horseradish peroxidase” solution was applied to the sections for 10 min. Secondary antibody binding was visualized using 3,3 -diaminobenzoic acid (DAB) dissolved in PBS with the addition of H2 O2 to a concentration of 0.03% immediately before use. Finally, sections were PBS rinsed and counterstaining of slides was done using two drops or 100 ␮l of hemotoxylin. Slides were washed in distilled water until the sections turned blue. Finally, slides were dehydrated in ascending grades of ethanol (70%, 95%, and 100%) for 5 min each and were cleared in xylene and finally coverslipped using histomount mounting solution. For immunohistological quantitative assessment, five nonoverlapping fields (400×) per section were randomly captured by a digital camera (Olympus) in the frontal cortex and the cerebellum, whereas the entire dentate gyral area was analyzed for each brain section for each marker. The number of immunopositive cells in the fields taken from at least three anatomically comparable sections/animal was counted using imageJ software and averaged per field for each animal. The numbers calculated for at least five animals/experimental group were considered for comparison and statistical analyses.

2.4. Statistical analysis Results were expressed as mean ± SEM and significant differences between groups were evaluated using One way-ANOVA followed by a post hoc Bonferroni test. A level of significance of P < 0.05 was considered to be statistically significant.

3. Results 3.1. Effects of ginger on glucose blood level STZ injection resulted in a diabetic syndrome verified by the presence of polydypsia, polyuria, and hyperglycemia in the diabetic animals. Over the 8 weeks after the injection, mean blood glucose levels in STZ-injected rats were significantly higher than the control group (P < 0.001). Ginger treatment significantly lowered blood glucose levels in comparison with rats of the diabetic group at all experimental time points. Blood glucose levels are presented in Table 1.

Please cite this article in press as: El-Akabawy, G., El-Kholy, W., Neuroprotective effect of ginger in the brain of streptozotocin-induced diabetic rats. Ann. Anatomy (2014), http://dx.doi.org/10.1016/j.aanat.2014.01.003

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Fig. 1. Representative H&E staining of rat frontal cortex of different groups. In control groups, frontal cortex neurons appeared with vesicular open face nuclei (arrows, A–C) and showed scattered glia (black arrow heads, A–C). Mostly all the neurons of diabetic frontal cortex had pyknotic nuclei (arrows, D–F), some were surrounded by halos (black arrow heads, D–F), others showed acidophilic necrosis (stars, D and E). Diabetic neutrophil showed vacuolization (green arrow heads, D and F). Ginger-treated frontal cortex showed pyramidal cells with almost normal morphology (arrows, G–I); however, few apoptotic pyramidal cells (arrow heads, G–I) and congested blood vessels were detected (star, G). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

3.2. Effects of ginger on the frontal cerebral cortex of diabetic brain In control groups, the pyramidal cells of layer III showed open face nuclei and basophilic cytoplasm, while the smaller glia cells and blood capillaries were distributed in between neurons. In diabetic brains, almost all the neurons appeared as shrunken dark cells. Cells with pyknotic nuclei, condensed cytoplasm, and surrounded halos suggesting apoptotic cells were also observed. In addition, neurophil of the cerebral cortex showed vacuolization. Some of the cells showed acidophilic neuronal necrosis with nuclear pyknosis, perikaryal contraction of acidophilic cytoplasm, and vacuolization of adjacent neutrophil. In the treated group, pyramidal cells appeared with almost normal morphology showing basophilic vesicular nuclei and prominent nucleoli. However, some apoptotic pyramidal cells were still seen. Congested and dilated blood vessels were also observed in this group (Fig. 1). These results suggest that ginger attenuates the morphological and histopathological changes in diabetic frontal cortex. A significant up-regulation of iNOS expression, a marker of oxidative distress (Gurpinar et al., 2012; Celik et al., 2007; Liu et al., 2013b), was detected in the diabetic group (P < 0.001; Fig. 2). In the ginger-treated group, iNOS expression was dramatically

down-regulated compared to diabetic rats (P < 0.001; Fig. 2). The elevated expression of iNOS was associated with a significant increase in the number of caspase-3 positive cells in the diabetic group (P < 0.001; Fig. 2), which was dramatically reduced in the ginger-treated group (P < 0.001; Fig. 2). These results suggest that the antioxidant capacity of ginger ameliorates oxidative distress and apoptosis induced in the frontal cerebral cortex of diabetic rats. The expression of the inflammatory marker TNF-␣ was significantly increased in diabetic group compared to control (P < 0.001; Fig. 2), this increase showed dramatic decrease in the ginger-treated group (P < 0.001; Fig. 2). These results suggest that ginger reduces inflammation in diabetic frontal cortex. In diabetic brains, the frontal cortex showed a significant increase in GFAP-positive cells compared to control (P < 0.001; Fig. 2), this increase was dramatically decreased in diabetic + ginger brains (P < 0.001; Fig. 2), indicating that ginger modulates the astrogliosis induced in the frontal cerebral cortex of the diabetic group. Acetylcholinesterase (AChE) activity has been regarded as a marker of cholinergic function which is involved in the pathogenesis of diabetes. There was a significant increase in AChE activity in STZ-treated rats compared to control rats (P < 0.001; Fig. 2). Interestingly, in the ginger-treated group, the increased AChE expression was significantly decreased (P < 0.001; Fig. 2). However, Ki67 immunohistochemical staining showed no significant difference among different experimental groups (Fig. 2).

Table 1 Fasting blood glucose levels. Fasting blood glucose

Control

3 days 4 weeks 6 weeks 8 weeks

94.8 92.8 89.6 91.8

± ± ± ±

Diabetic 2.5 5.3 3.2 4.1

334.5 357.5 359.5 368.5

± ± ± ±

Diabetic + ginger 18.6* 17.2* 19.5* 15.2*

346.5 173.7 169.5 167.5

± ± ± ±

17.1* 11.20 12.60 13.30

All the values are mean ± SEM of five individual observations. Values are significant compared to control of the same time points (*P < 0.001) and diabetic group of the same time points (0 P < 0.001).

3.3. Effects of ginger on the hippocampus of diabetic brain The granular cell layers of control dentate gyri contained densely arranged neurons with rounded pale vesicular nuclei. In diabetic brains, many granular cells with darkly stained pyknotic nuclei appeared, suggesting apoptosis. Others showed cytoplasmic vacuolizations. In ginger-treated groups, most of the granular neurons appeared normal with pale basophilic vesicular nuclei; however,

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Fig. 2. Representative immunostaining of rat frontal cortex of different groups after 8 weeks: iNOS and caspase-3 immunoreactivity were dramatically increased in diabetic frontal cortex (A, B, D, and E). These increases were significantly reduced in the diabetic + ginger group (C and F). Ginger treatment attenuated the diabetes-induced increase in TNF-␣ (G–I), GFAP (J–L), and AChE (M–O) expression. However, there was no significant difference in the number of Ki67 cells (P–R). Similar findings were seen after 4 and 6 weeks of diabetes. Inserts show a higher magnification of the boxed regions. ***P < 0.001, compared with the diabetic group; 000 P < 0.001, compared with control. N = 8 for each group. Scale bar 100 ␮M, ×400.

few cells appeared apoptotic (Fig. 3). These results indicate that ginger ameliorated the histopathological alterations in the dentate gyri of diabetic brains. iNOS and caspase-3 immunoreactivity, markers of oxidative distress and apoptosis respectively, were increased in the granular layer of the diabetic dentate gyrus (P < 0.001; Fig. 4). This increase was significantly decreased in the gingertreated group (P < 0.001; Fig. 4). The expression of the inflammatory marker, TNF-␣, was increased in the diabetic group compared to control (P < 0.001; Fig. 4), this increase showed dramatic decrease in the ginger-treated rats (P < 0.05; Fig. 4). These results suggest that ginger significantly prevents the oxidative distress, apoptosis, and inflammation induced in the dentate gyrus of the diabetic group.

Dentate gyri of diabetic brains showed a significant increase in the number of GFAP positive cells (P < 0.001; Fig. 4). In the gingertreated group, this number was dramatically decreased (P < 0.001; Fig. 4). These results suggest that ginger reduces astrogliosis induced in the dentate gyri of diabetic brains. The immunoreactivity of AChE was increased in the diabetic dentate gyri compared to control ones (P < 0.001; Fig. 4). Ginger treatment protected the dentate gyrus and significantly reduced AChE activity (P < 0.001; Fig. 4). Ki67 immunostaining revealed a dramatic decrease in the number of Ki67 positive cells among granular cells suggesting suppression in neurogenesis (P < 0.001; Fig. 4). This down-regulation in the Ki67 cells was reversed significantly in the ginger-treated group, in

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Fig. 3. Representative H&E staining of rat hippocampus (dentate gyrus) of different groups. The control granular neurons presented clear and evident nuclei and nucleoli (arrows, A–C). Many of granular neurons of diabetic rat dentate gyri showed a densely stained shrunken appearance with minimal or no cytoplasm (arrows, D–F), some neurons showed vacuolization (arrow head, D). Granular neurons in ginger-treated group showed vesicular basophilic nuclei (arrows, G–I), only a few shrunken dark neurons were seen (arrow heads, G–I). Scale bar 100 ␮M, ×400.

which the Ki67 was significantly increased (P < 0.001; Fig. 4). This indicates that ginger enhances the suppressed neurogenesis in the diabetic dentate gyrus. 3.4. Effects of ginger on the cerebellar cortex of diabetic brain We focused on Purkinje cells as they were the most affected cells in the diabetic group. Purkinje cells of the control group appeared morphologically normal with rounded to flask shaped cells having rounded vesicular nuclei with darkly stained prominent nucleoli. Many Purkinje cells of the diabetic group were shrunken with darkly stained pyknotic nuclei and surrounded by perineural spaces (halos) suggesting apoptosis. Some of the Purkinje cells were necrotic with missing nuclei and ill-defined borders. At 8 weeks, Purkinje cells showed excessive vacuolization of their cytoplasm, multiple areas of vacuolization affected the neutrophil of the molecular layer at this time point. In the treated group, many Purkinje cells had nearly normal appearance similar to the control group; however, few were distorted with ghost shape appearance or darkly stained with pyknotic nuclei (Fig. 5). This indicates that, akin to the frontal cortex and dentate gyrus, ginger was able to reverse the morphological and pathological changes in the diabetic cerebellum. Akin to frontal cortex and dentate gyrus regions, immunohistochemical staining for iNOS and caspase-3 revealed a significant increase in their number in the diabetic cerebellar cortex (P < 0.001; Fig. 6); this increase was significantly reduced in ginger-treated group (P < 0.001; Fig. 6), indicating that antioxidant capability of ginger was able to protect diabetic cerebellar cortex from diabeticinduced oxidative distress and apoptosis. The expression of TNF-␣ was significantly increased in the cerebellum of diabetic group (P < 0.001; Fig. 6). Ginger treatment also reduced diabetic inflammation as suggested by the significant down-regulation of TNF-␣ in the ginger-treated group compared to the diabetic one (P < 0.05; Fig. 6). The number of cells expressing GFAP was dramatically increased in the diabetic group (P < 0.001; Fig. 6). This increase was significantly decreased in ginger-treated rats (P < 0.001; Fig. 6),

suggesting that ginger modulates astrogiosis induced in the cerebellar cortex of diabetic rats. Furthermore, AChE activity was up-regulated in the diabetic cerebellum; this was dramatically reduced in ginger-treated group (P < 0.001; Fig. 6). However, similar to the frontal cortex, the number of Ki67 positive cells showed no significant difference between different experimental groups (Fig. 6). 4. Discussion Diabetes mellitus, a major endocrine disorder, has become a serious health problem worldwide. Several studies have revealed that hyperglycemia has deleterious effects on the central nervous system (Gebel, 2012). So far, available therapy regimens for controlling diabetes have certain serious side effects; therefore, the demand for natural products with anti-hyperglycemic activity and fewer side effects is increasing. Hyperglycemia induced-oxidative stress plays a central role in neuronal damage in diabetic brains (Simmons, 2012; Zhao et al., 2013). Generation of excessive free radicals from auto-oxidation of elevated intracellular glucose levels is involved in neuronal injury. Anti-oxidant therapy involving the use of herbs and spices has been shown to protect the tissues against such damage (Birben et al., 2012; Bajaj and Khan, 2012). Ginger has been shown to dramatically increase anti-oxidant marker enzymes in diabetic brains (Shanmugam et al., 2011; Li et al., 2012). In this study, we set out to investigate, for the first time, whether ginger induced modulation at the biochemical level will be reflected in the morphological and neuropathological alterations in different regions of diabetic brains. Our results show that ginger improved the brain morphological and structural changes associated with diabetes. This beneficial effect was mediated by ginger ability to down-regulate diabetes-induced oxidative distress, apoptosis, inflammation, gliosis, and AChE expression in different regions of diabetic brain, in addition to its ability to improve diabetic brain suppressed neurogenesis. Our studies show that ginger treatment significantly lowered the increased blood glucose level of diabetic rats. The increased

Please cite this article in press as: El-Akabawy, G., El-Kholy, W., Neuroprotective effect of ginger in the brain of streptozotocin-induced diabetic rats. Ann. Anatomy (2014), http://dx.doi.org/10.1016/j.aanat.2014.01.003

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Fig. 4. Representative immunostaining of rat dentate gyri of different groups after 8 weeks: iNOS and Caspase-3 immunoreactivity were dramatically increased in diabetic dentate gyri (A, B, D, E). These increases were significantly reduced in the diabetic + ginger group (C and F). Ginger treatment attenuated the diabetes-induced increase of TNF-␣ (G–I), GFAP (J–L), and AChE (M–O) expression. The number of KI67 positive cells was decreased in dentate gyri of diabetic brains; this decrease was significantly increased in the diabetic + ginger dentate gyri (P–R). Similar findings were seen after 4 and 6 weeks of diabetes. Inserts show a higher magnification of the boxed regions. *P < 0.05 and ***P < 0.001, compared with the diabetic group; 000 P < 0.001, compared with control. N = 8 for each group. Scale bar 100 ␮M, ×400.

blood glucose level observed during STZ-induced diabetes is similar to previous reports. Diabetes results from irreversible destruction of pancreatic beta cells, causing degranulation and reduction of insulin secretion (Zhang and Tan, 2000; Kavalali et al., 2003). Previous studies showed that short-term treatment of ginger significantly decreased fasting blood glucose level after 1 h treatment in an STZ-type 1 diabetic rat model (Akhani et al., 2004). Long-term treatment with ginger not only affects blood glucose levels, but also decreases serum triglyceride and total cholesterol, increased insulin, and effectively prevented body weight loss in type 1 (Abdulrazaq et al., 2012) and in type 2 diabetic animals (Ray et al., 2009) and patients (Jolad et al., 2005). Ginger showed a protective

effect on pancreatic ␤-cells which led to modulation and increase in insulin release (Chakraborty et al., 2012). The mechanism underlying this action of ginger may also involve its interaction with the 5-HT3 (5-OH-tryptamine 3) receptor, a member of the serotonin receptor family known to be involved in modulating insulin release and inducing hypoglycemic effect (Heimes et al., 2009). Oxidative stress is proposed to be the main cause of neuropathy in diabetic subjects. The increased intracellular glucose induces oxidation stress that results in ROS and NO (nitric oxide) overproduction (Vincent et al., 2004). Indeed, increasing evidence indicates that oxidative and nitrosative stress is increased in diabetic patients. The excess generation of ROS and inducible NO

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Fig. 5. Representative H&E staining of rat cerebellar cortex of different groups. Normal rounded to flask shaped Purkinje cells appeared in control groups with vesicular nuclei and darkly stained prominent nucleoli (arrows, A–C). Many Purkinje cells of the diabetic groups appeared shrunken with darkly stained pyknotic nuclei and surrounded by halos (arrows, D and F), others seemed necrotic with missing nuclei and distorted borders (black head arrow, D). Purkinje cells of the 8 week diabetic group showed severe vacuolization of their cytoplasm (arrows, F), neutrophil vacuolization was also evident (green arrow heads, E and F). Many Purkinje cells in the treated groups show nearly normal appearance (arrows, G–I). However, few of the Purkinje cells in the treated group appear pyknotic (blue arrow heads, G&I) and others showed ghost appearance (black arrow heads, G&H). Scale bar 100 ␮M, ×400. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

synthase (iNOS) causes oxidative damage to cellular proteins, lipids, or DNA and subsequently inhibits their normal functions and disturbs homeostatics within the neuron, ultimately resulting in cell death via apoptosis (Fukudome et al., 2008; Nishikawa et al., 2000). In our study, the frontal cortex, hippocampus, and cerebellum of the diabetic rats showed an increase in the expression of iNOS, a marker of oxidative stress (Gurpinar et al., 2012; Celik et al., 2007; Liu et al., 2013b). In addition, we detected an increase in the number of apoptotic cells; this was confirmed by up-regulation of caspase-3 activities as detected by IHC. In line with our study, several studies have shown that diabetic brains demonstrate elevation in the apoptotic rate accompanied by an increase in oxidative stress markers in the cerebral cortex (Suge et al., 2012; Kolacek et al., 2010), hippocampus (Pamidi and Satheesha Nayak, 2012; Ahmadpour and Haghir, 2011), and cerebellum (Kamboj and Sandhir, 2011), indicating that diabetes enhances oxidative stress-induced apoptosis in these brain regions. Antioxidants have been shown to protect neurons against a variety of experimental neurodegenerative conditions (Baluchnejadmojarad and Roghani, 2011; Bhutada et al., 2011; Suge et al., 2012; Liu et al., 2013a). In the present work, ginger treatment significantly down-regulated the expression of iNOS and dramatically reduced the number of apoptotic cells, as detected by H&E evaluation, and also significantly decreased the number of caspase-3 positive cells as detected by IHC. Our results showed that the antioxidant properties assigned to ginger is able to histomorphologically ameliorate diabetic-induced oxidative stress and apoptosis, confirming what others have found biochemically (Shanmugam et al., 2011; Li et al., 2012). The role of pro-inflammatory cytokines, particularly tumor necrosis factor (TNF)-␣ in diabetic neuropathy has been wellestablished (Shi et al., 2013). The free oxygen radicals induced by hyperglycemia can enhance the production of TNF-␣ (Brownlee, 2001). Moreover, some inflammatory plasma markers (mainly related to TNF-␣ system) have been involved in diabetic

retinopathy and nephropathy (Schram et al., 2005). Our study showed that TNF-␣ increased in diabetic brains. Consistent with our result, many studies have indicated that diabetes has induced IFN␣ synthesis in diabetic subjects supporting the role of inflammation in the pathogenesis of diabetic complications (Jing et al., 2013 and Black, 2006). In our study, the increased TNF-␣ expression in in diabetic group was reversed in ginger-treated group in different brain regions studied. It is possible that the anti-inflammatory role of ginger is mediated by inhibiting diabetes induced ROS production. This result further support the antioxidant properties assigned to ginger. Our results show that diabetes induces a significant increase in the number of GFAP positive cells in all studied brain regions. The present findings are in agreement with previous reports that show that reactive gliosis occurs in diabetes, possibly as a result of oxidative stress. Glial cells respond to the oxidative insult by producing GFAP and S100B (Kaneko et al., 2002; Pekny and Nilsson, 2005). GFAP and S-100B may provide a surrogate marker for studying neurodegenerative changes in experimental diabetes mellitus. Our data show ginger treatment ameliorated diabetes-induced gliosis (Baydas et al., 2003a). This is consistent with previous studies in diabetic rats in which the beneficial effects of antioxidants against reactive gliosis were attributed to their free radical scavenging properties (Baydas et al., 2003b, 2005Duarte et al., 2012). This suggests that the gliosis that occurs in diabetes mellitus is likely mediated by oxidative distress and that antioxidants prevent reactive gliosis possibly by reducing damaging effects of reactive oxygen species in the central nervous system. Injecting 5 -bromo-2-deoxyuridine (BrdU) intraperitoneally followed by immunohistochemical detection has been used as the principal method of studying neurogenesis. However, such exogenous markers may produce toxic effects. Ki-67 is an effective mitotic marker and has been shown to have most of the benefits of BrdU and none of the costs (Kee et al., 2002). Using Ki67 staining, our

Please cite this article in press as: El-Akabawy, G., El-Kholy, W., Neuroprotective effect of ginger in the brain of streptozotocin-induced diabetic rats. Ann. Anatomy (2014), http://dx.doi.org/10.1016/j.aanat.2014.01.003

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Fig. 6. Representative immunostaining of rat cerebellar cortex of different groups after 8 weeks: iNOS and Caspase-3 immunoreactivity were dramatically increased in diabetic cerebelli (A, B, D, and E). These increases were significantly reduced in the diabetic + ginger group (C and F). Ginger treatment attenuated diabetes-induced increase in TNF-␣ (G–I), GFAP (J–L), and AChE (M–O) expression. However, there was no significant difference in the number of Ki67 cells (P–R). Similar findings were seen after 4 and 6 weeks of diabetes. Inserts show a higher magnification of the boxed regions. *P < 0.05 and ***P < 0.001, compared with diabetic group; 000 P < 0.001, compared with control. N = 8 for each group. Scale bar 100 ␮M, ×400.

results reproduced the results of previous studies, which showed that the cell proliferation rate in diabetic dentate gyri is lower compared with non-diabetic rats (Jackson-Guilford et al., 2000). The mechanism of reduced neuronal production is not yet known, but evidence suggests that lack of insulin and/or hyperglycemia within the brain may be contributing factors (Zhang et al., 2008). Our finding showed that ginger is able to antagonize the diabetes-induced suppression of neurogenesis, which indicates that oxidative stress is likely to be directly or indirectly implicated in suppression of neurogenesis in diabetic dentate gyri (Venturini et al., 2010; Hamilton et al., 2011).

Several studies have reported an increase in choline esterase (ChE) activity in the diabetic brain that was associated with cognitive deficits (Kuhad et al., 2008; Schmatz et al., 2009; Sherin et al., 2012). The activity of the acetylcholine esterase (AChE) has been reported to be affected by oxidative stress-induced perturbation in membrane fluidity (Sandhir et al., 1994). One of the most important mechanisms responsible for improving cholinergic function is accomplished by modulating the activity of AChE enzyme (Appleyard, 1994). In this study, treatment with ginger was able to prevent the increase in AChE activity in all cerebral structures evaluated in diabetic rats. These results are similar to those shown

Please cite this article in press as: El-Akabawy, G., El-Kholy, W., Neuroprotective effect of ginger in the brain of streptozotocin-induced diabetic rats. Ann. Anatomy (2014), http://dx.doi.org/10.1016/j.aanat.2014.01.003

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in studies with other antioxidants such as curcumin (Kumar et al., 2013), luteolin (Liu et al., 2013a), and vitamin E (Comin et al., 2010) that also prevented the rise in AChE activity in several cerebral structures in diabetic rats. In line with these findings, it is likely that the observed effect of ginger in the present study could be a subsequent to its protective role against oxidative damage in the brain areas evaluated. A substantial body of evidence suggests that ameliorating oxidative damage significantly corrects the cholinergic function in diabetic rats (Bhutada et al., 2011; Thakur et al., 2013). Changes in AChE activity and the acetylcholine neurotransmitter level are neurophysiologically associated with cognitive deficits observed in patients and in animal models of diabetes mellitus (Liapi et al., 2010; Sherin et al., 2012). Several antioxidants have been reported to restore the cognitive deficits via reducing AChE activity (Schmatz et al., 2009; Peeyush et al., 2011). Based on these finding, we can suggest that ginger might be useful for improving the cognitive deterioration by increasing cholinergic communication. In this study, intracytoplasmic vacuolization of cerebral pyramidal and cerebellar Purkinje neurons was detected. In addition, neutrophil showed various degrees of vacuolization. Brain edema is the most common serious complication of diabetic ketoacidosis in children, where mechanisms of rapid change in serum osmolality during therapy and others such as brain ischemia have been suggested (Gebara, 2001). In these experiments, hyperglycemia may cause brain acidosis and dehydration, both involved in diminished cerebral blood flow and ischemic-related edema (Edge, 2000; Hernandez-Fonseca et al., 2009). The correction of hyperglycemia by ginger treatment might be attributed in some morphological amelioration observed in this study including reduction of vacuolization (Hoffman et al., 2006). The beneficial effects of ginger on the neuropathological changes of diabetic brains were not only evident after short-term application, but also were detected after long-term administration. This indicates that these effects were not transient but were sustainable over long-term administration. As our results showed that there was no time-dependent effect of ginger, it would be interesting to investigate whether the short-term beneficial effect is irreversible. Proving that short-term supplementation of ginger will lead to long-term sustainable amelioration of diabetic manifestation and morphological alterations will introduce ginger as an effective relatively cheap therapeutic agent of great value in treating diabetic brains.

Author’s contribution Both authors have contributed equally to this work.

Conflict of interest The authors do not have any conflict of interest with the content of the paper.

Acknowledgment The authors would like to deeply thank Dr. Manar Farid for her generous assistance during conducting the experiments.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.aanat.2014.01.003.

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Please cite this article in press as: El-Akabawy, G., El-Kholy, W., Neuroprotective effect of ginger in the brain of streptozotocin-induced diabetic rats. Ann. Anatomy (2014), http://dx.doi.org/10.1016/j.aanat.2014.01.003