brain research 1602 (2015) 153–159
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Research Report
Protective effect of rhGLP-1 (7–36) on brain ischemia/reperfusion damage in diabetic rats Libo Zhaoa, Jia Xub,1, Qian Wanga, Zhonglian Qianc, Wanyu Fenga, Xiaoxing Yind, Yi Fanga,n a
Department of Pharmacy, Peking University People’s Hospital, Beijing 100044, China Department of Pharmacy, Mawangdui Hospital, Changsha, 410016, China c Department of Pharmacy, The First Hospital, Huhhot, 010010, China d Department of Pharmacy, Xuzhou Medical College, Xuzhou 221004, China b
art i cle i nfo
ab st rac t
Article history:
In recent years, GLP-1 and its analogs have been developed for the treatment of type 2
Accepted 9 January 2015
diabetes. It has been reported that stimulating the GLP-1 receptor can protect neurons
Available online 16 January 2015
against metabolic and oxidative insults, and therefore can be used in the treatment of
Keywords:
stroke and Parkinson's disease. The present study aimed to examine the neuroprotective
GLP-1
effects of rhGLP-1 (7–36) and its possible mechanisms against acute ischemia/reperfusion
Brain ischemia/reperfusion
injuries induced by middle cerebral artery occlusion (MCAO) in diabetic rats. The type 2
Diabetic rats
diabetic rat model was established by a combination of a high-fat diet and low-dose
Oxidative stress
streptozotocin (STZ). RhGLP-1 (7–36) (20, 40, 80 μg/kg) was given intraperitoneally before reperfusion. The neuroprotective effects of rhGLP-1 (7–36) were evaluated by changes in neurological deficit scores and 2,3,5-Triphenyltetrazolium chloride (TTC) staining. Changes in blood glucose were used to assess hypoglycemic effects. The content of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), inducible nitric oxide syntheses (iNOS) and endothelial nitric oxide syntheses (eNOS) after MCAO/R administration (2 h and 46 h) were examined to investigate the possible mechanisms of RhGLP-1 (7–36). Haematoxylin and eosin (H&E) staining was used for histopathological observation. Compared with the control group, rhGLP-1 (7–36)-treated groups decreased nerve function deficiency scores; significantly reduced infarction volume percentage, MDA, iNOS and blood glucose; and significantly increased SOD, GSH-PX and eNOS. In addition, rhGLP-1 (7–36) groups enhanced the density of surviving neurons and increased vascular proliferation. The current study suggests a neuroprotective effect of rhGLP-1 (7–36) in diabetic MCAO/R rats since anti-oxidative and anti-nitrosative stress effects can contribute to beneficial effects against ischemia/reperfusion injury. & 2015 Elsevier B.V. All rights reserved.
n
Corresponding author. Tel.: þ86 10 66583834. E-mail address: [email protected] (Y. Fang). 1 This author contributed equally to this work and should be considered as co-first author.
http://dx.doi.org/10.1016/j.brainres.2015.01.014 0006-8993/& 2015 Elsevier B.V. All rights reserved.
154
1.
brain research 1602 (2015) 153–159
Introduction
Stroke is the third leading cause of death and paralysis worldwide and remains a serious challenge to public health due to its high incidence and life threatening nature (WHO, 2004; Feigin et al., 2014). Close to 60–70% of strokes are ischemic strokes (Silvestrelli et al., 2002). In addition to hypertension and hypercholesterolemia, diabetes is another important risk factor for strokes, especially in patients less than 65 years of age (Kissela et al., 2005; Lawes et al., 2004; Towfighi et al., 2012). Despite the fact that many mechanisms leading to cell death during ischemia/reperfusion brain injury have been identified in the past decades such as excitotoxicity, calcium dysregulation, oxidative and nitrosative stress, and inflammation (Moskowitz et al., 2010), the underlying mechanisms are not yet well understood. Recombinant tissue plasminogen activator rt-PA (alteplase) is the only treatment approved by the FDA since it has been shown to improve functional outcomes in randomized trials of patients treated soon after acute ischemic stroke. However, due to its short therapeutic window, it is not widely used (Wardlaw et al., 2012). As a result, developing new treatments and drugs for ischemia stroke patients is increasingly important. The glucagon-like peptide-1 (7–36) amide (GLP-1) is a member of the proglucagon family of incretin hormones secreted mainly by the enteroendocrine cells of the intestine in response to the presence of nutrients. GLP-1 and its analogs facilitate glucose-induced insulin release, suppress glucagon secretion, slow gastric emptying and suppress appetite by activating the GLP-1 receptor (GLP-1R), which is widely expressed in islet cells, the kidney, lungs, brain and gastrointestinal tract (Dunphy et al., 1998; Lund et al., 2014). As a result, analogs of GLP-1 have been developed for the treatment of type 2 diabetes. Endogenous GLP-1 is rapidly inactivated by cleavage of the NH2-terminal dipeptide by the widely expressed serine protease dipeptydil peptidase-IV (DPP-IV) (Meier et al., 2006). Thus, long-acting GLP-1 analogs have been developed to prolong the effect by changing the structure of the peptide. Exenatide is the first GLP-1 analog approved by the FDA for the treatment of type 2 diabetes (Diamant et al., 2014). The absence of amidation of the NH2terminal is the main difference between recombinant human glucagon-like peptide-1 (7–36) amide rhGLP-1 (7–36) and endogenous GLP-1. It has been reported that stimulating the GLP-1 receptor can protect neurons against metabolic and oxidative insults; as a result, it can be used for the treatment of stroke and Parkinson's disease (Li et al., 2009; Perry et al., 2007). In addition, previous studies have suggested that after ischemia/reperfusion, the expression of GLP-1R protein significantly increases, and GLP-1R agonists protect against ischemia-induced neuronal death possibly by increasing GLP-1R expression and attenuating microglia activation against transient cerebral ischemic damage (Lee et al., 2011). Furthermore, experiments have shown that GLP-1R agonists have neuroprotective effects on cerebral ischemia models, possibly through anti-oxidative effects and increased intracellular cAMP levels (Sato et al., 2013; Teramoto et al., 2011). The aim of the current study was to investigate the protective effects of rhGLP-1 (7–36) on diabetic rats with
ischemia/reperfusion damage to the brain and to explore the mechanisms responsible.
2.
Results
2.1.
Effects on neurological deficits
After MCAO for 2 h and then followed 46 h reperfusion, the diabetic rats showed characteristics of circling and rotating to the left when crawling, falling to the left side when standing, and even not walking spontaneously. The neurological status was assessed using a 5-point scale. Sham-operated diabetic rats showed no neurological symptoms, as indicated by a neurological status score of zero. As shown in Fig. 1, the nimodipine and rhGLP-1 (7–36) groups had higher neurological status scores and showed improvements in neurological deficiencies compared to the model group receiving a high dose of rhGLP-1 (7–36): the best results, although no statistically significant different, were found in the low and middle rhGLP-1 (7–36) groups. These results indicate that both nimodipine and rhGLP-1 (7–36) reduced neurological dysfunction following diabetes combined with MCAO/R and showed dose-related alleviation in the rhGLP-1 (7–36) groups.
2.2.
Effects on cerebral infracted volume
As shown in Fig. 2A, normal brain tissue was deep stained red and the infarcted brain tissue was stained white, large infarction areas were presented in the model group whereas no infarctions were observed in the sham group. As seen in Fig. 2B, we know that compared with the model group, the nimodipine and rhGLP-1(7–36) groups showed significantly reduced infarction volume percentages during MCAO/R injury and the rhGLP-1 (7–36) high group showed the best protective effects of all groups.
Fig. 1 – Changes in neurological deficiency scores in different groups: the rhGLP-1 (7–36) high group significantly reduced neurological deficiency compared with the model group (nmeans vs. model group, Po0.05; △ means vs. sham group, Po0.05).
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Fig. 2 – Effects of rhGLP-1 (7–36) on the infracted volume of diabetic rats that were subjected to an occlusion of the middle cerebral artery for 2 h followed by 46 h reperfusion. A) Representative photographs of TTC staining of coronal brain slices: (a) Sham group; (b) model group; (c) nimodipine group; (d) rhGLP-1 (7–36) low group; (e) rhGLP-1 (7–36) middle group; (f) rhGLP1 (7–36) high group. B) Compared with the model group, the infarction volume percentage of nimodipine and rhGLP-1 (7–36) groups were significantly reduced (nmeans vs. model group Po0.05; nnmeans vs. model group Po0.01; △△ means vs. sham group, Po0.01, n ¼6).
2.3.
Biochemical observations
2.3.1.
Blood glucose observations
As presented in Table 1, there were no statistically significant differences in the various groups prior to injection of the drugs. Thirty minutes following injection, blood glucose levels varied in the reduced rhGLP-1 (7–36) groups: the rhGLP-1 (7–36) middle group showed a decreasing trend with
no statistical significance and the rhGLP-1 (7–36) high group showed a statistical significance reduction (Po0.05) compared with the model group, while blood glucose levels were elevated in the nimodipine group.
2.3.2.
Effects on levels of MDA, SOD and GSH-PX
Table 2 shows a significantly increased presence of MDA and reduced activity of SOD and GSH-PX in the model group
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compared with the sham group. Treatment with nimodipine and rhGLP-1 (7–36) significantly reduced MDA content and increased activity of SOD and GSH-PX compared with the model group after MCAO/R.
of the cerebral cortex, thereby enhancing the density of the surviving neurons and increasing vascular proliferation (Fig. 3c, d, e, and f).
2.3.3.
3.
Effects on levels of iNOS and eNOS
The data in Table 3 indicate that the activity of iNOS was significantly higher and the activity of eNOS was significantly lower in the other groups compared with the sham group. Nimodipine significantly lowered iNOS activity compared with the model group, and the rhGLP-1(7–36) dose required to suppress iNOS activity in the high group was the best. Meanwhile, levels of eNOS were significantly increased in the nimodipine group, and rhGLP-1 (7–36) at doses of 40 and 80 μg/kg significantly upregulated levels of eNOS compared with the model group (Po0.01).
2.4.
Histological observations
H&E staining was used to observe histological features and cerebral ischemia/reperfusion injury regions mainly located in hippocampus, frontoparietal cortex, striatum and preoptic areas. Representative histological photographs are shown in Fig. 3. As shown, no histological abnormalities were observed in the sham group, while the model group showed that most neurons in the infarct core appeared shrunken and contained eosinophilic cytoplasm and triangulated pyknotic nuclei with many vacuolars in the neuron space. The infarct core was surrounded by necrotic neurons that exhibited pyknotic shapes and condensed nuclei in the peri-infarct zones (Fig. 3b). Compared with the model group, the nimodipine and rhGLP-1 (7–36) groups were able to improve the structure
Discussion
GLP-1 is secreted by the L-cells of the intestine in response to food intake. Many studies have shown that GLP-1 can stimulate the release of insulin in a glucose-dependent manner (Choi and Lee, 2011; D'Alessio et al., 1995). GLP-1 is also thought to play a role in the proliferative and antiapoptotic action on pancreatic β-cells and intestinal epithelial cells, respectively; it also inhibits gastric emptying and acid secretion (Ma et al., 2014). Recently, GLP-1 analogs were found to have protective effects on heart ischemia/reperfusion damage (Hausenloy et al., 2013; Salling et al., 2012). In the current study, we demonstrated that rhGLP-1 (7–36) could induce neuroprotection by attenuating infarction volume and by improving neurologic function in a diabetic combined MCAO/R rat model. The protective effects of rhGLP-1 (7–36) at 80 μg/kg were more potent than those at 20 and 40 μg/kg, showing a dose response tendency. Oxidative stress is a key pathological factor in brain ischemic/reperfusion injuries. It is involved in the cytotoxic consequences of mismatches between the production of reactive oxygen species (ROS) and free radicals and the ability of cells to defend against them (Ying and Xiong, 2010). ROS's include oxygen-centered radicals with unpaired electrons Table 3 – Nitric oxide syntheses activity in different groups of MCAO/R rat brains (n ¼6) Groups
Nitric oxide syntheses activity (U/L)
Table 1 – Summary of blood glucose readings before and after injection of drugs for 30 min (n ¼6) Groups
0 min (mmol/L)
30 min (mmol/L)
Sham Model Nimodipine RhGLP-1(7–36) low RhGLP-1(7–36) middle RhGLP-1(7–36) high
25.475.5 25.874.2 24.176.5 25.973.0 26.073.7 25.975.5
25.374.9 24.974.1 25.377.2 25.474.2 23.673.4 22.974.9n
n
Means vs. model group Po0.05.
Sham Model Nimodipine RhGLP-1(7–36) low RhGLP-1(7–36) middle RhGLP-1(7–36) high
iNOS
eNOS
4.9270.21 6.0370.37△△ 5.2970.20nn 5.3370.19nn 5.2170.34nn 5.0970.24nn
29.6371.24 24.8171.18△△ 26.5870.94nn 24.0970.84 26.0070.96n 27.2270.92nn
n
Means vs. model group Po0.05. Means vs. model group Po0.01. △△ Means vs. sham group Po0.01. nn
Table 2 – Results of oxidative stress indicators in different groups of MCAO/R rat brains (n ¼6) Groups
Sham Model Nimodipine RhGLP-1(7–36) low RhGLP-1(7–36) middle RhGLP-1(7–36) high n
Means vs. model group Po0.05. Means vs. model group Po0.01. △△ Means vs. sham group Po0.01. nn
Oxidative stress indicators SOD (U/L)
MDA (nmol/L)
GSH-PX (pmol/mL)
126.7973.75 106.7375.53△△ 109.3872.23 107.4476.01 115.8475.52nn 116.4574.51nn
2.7270.14 3.8070.06△△ 3.2070.10nn 3.3270.13nn 3.1370.09nn 2.9170.09nn
30.5371.07 23.9970.96△△ 26.2570.91nn 25.0570.75n 27.7370.66nn 28.5570.72nn
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Fig. 3 – Representative histopathology photographs of brain tissue in diabetic rats after cerebral ischemia/reperfusion injury in different groups (H&E 100). (a) Sham group; (b) model group. Significant reversal of injury was observed in the nimodipine group (c), the rhGLP-1 (7–36) low group (d), the rhGLP-1 (7–36) middle group (e), and the rhGLP-1 (7–36) high group (f). such as the superoxide anion O2 and the hydroxyl radical OH or covalent molecules such as hydrogen peroxide (H2O2). SOD detoxifies O2 to H2O2, which is converted to H2O by catalase or GSH-PX (Chan, 2001). Oxidative stress can also be assessed by measuring the oxidized products of macromolecules such as lipids. Lipid peroxides are derived from unstable polyunsaturated fatty acids and decompose to form a complex series of compounds such as MDA. Cerebral ischemia can cause significant amounts of MDA formation in the ischemia hemisphere (Chen et al., 2009). Measuring the content of SOD, MDA and activity of GSH-PX in the brain cortex can show damage levels during the ischemia/reperfusion progress. In the current study, rhGLP-1 (7–36) significantly reduced MDA content and increased the activity of SOD and GSH-PX compared with the model group, indicating that rhGLP-1 (7–36) inhibited the oxidative stress response process. Nitric oxide (NO) is an important reactive nitrogen species that has also been demonstrated to play an important role in ischemic brain injury. Excessive NO can produce toxicity in nerve cells in the form of free radicals (Zhao et al., 2005). NO generated from neuronal nitric oxide syntheses (NOS) is known to exist in three isoforms: nNOS, iNOS and eNOS. The major regulator of vascular tone is the endothelium through endothelial NOS (eNOS). nNOS and inducible iNOS enzymes are expressed in neurons, glial cells and vascular myocytes, and can contribute to NO production in ischemic processes (Tajes et al., 2013). In the current research, dose-related rhGLP-1 (7–36) to suppress the activity of iNOS in the high group had the best outcomes, and levels of eNOS were significantly increased in rhGLP-1 middle and high groups compared with the model group. In addition, iNOS activity was significantly greater and eNOS activity was significantly lower in other groups compared with the sham group; this implies that although rhGLP1 showed benefits to brain ischemia/reperfusion injuries, it was still worse than the sham group. These results show that the effects of rhGLP-1 (7–36) on cerebral I/R injuries in diabetic rats could be involved in regulating iNOS and eNOS activities.
In conclusion, results of the above experiments indicate that rhGLP-1 (7–36) can decrease blood glucose and protect the brain from ischemia/reperfusion damage by anti-oxidative stress and anti-nitric oxide damage in diabetic/MCAO/R rats. Specifically, rhGLP-1 (7–36) can dose-dependently reduce blood glucose, thereby significantly improving neurological function and reduce infraction size two hours after ischemia followed by 46-hour reperfusion. Additionally, it improved the activities of SOD, GSH-PX, eNOS and enhanced the density of surviving neurons and increased vascular proliferation. Anti-oxidant and anti-nitric oxide damage appears to be a basic and essential mechanism of the neuroprotective effects of rhGLP1 (7–36). These results suggest that rhGLP-1 (7–36) could be a promising therapeutic drug for the treatment of diabetes combined with ischemic cerebrovascular disease. However, the mechanisms of cerebral ischemia/reperfusion injuries are varied. rhGLP-1 could also play a role in neuroprotection by other mechanisms. As such, further research is required.
4.
Experimental procedures
4.1.
Animals
Male pathogen free Sprague Dawleys rats (80–120 g) obtained from the Animal Center of the Academy of Military Medical Sciences, Beijing, China were housed in a light- (12 h light/ dark cycle) and temperature-controlled (21.5–22.5 1C) environment. The animals and experimental procedures used were approved by the Ethics Committee of the Peking University People's Hospital (NO: 2011-66).
4.2.
Reagents and chemicals
TTC and STZ were purchased from Sigma-Aldrich Co. (Saint Louis, USA), nimodipine injection was purchased from Byer Schering Pharma AG (Beijing, China), lyophilizing rhGLP-1
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(7–36) powder was manufactured by Shanghai Huayi BioTechnology Co., ltd., (Shanghai, China) and was diluted in saline to 0.2 mg/ml. The kits for biochemical analysis, including MDA, SOD, GSH-PX, iNOS and eNOS were purchased from Beijing Fangcheng Bio-Technology Co., Ltd (Beijing, China). The high-fat diet consisted of standard flour (17.5%), soy flour (10.0%), bran (7.0%), corn flour (12.0%), fishmeal (2.5%), salt (0.5%), yeast extract (0.5%), lard (15.0%), sucrose (10.0%), milk powder (5.0%), sesame oil (5.0%), eggs (10.0%) and peanuts (5.0%).
4.3.
Diabetic and brain ischemia/reperfusion model
After being fed a high-fat diet for four weeks, rats were given STZ (30 mg/kg) intraperitoneally. Tail vein blood samples were obtained to detect fasting blood glucose (FBG) levels; an FBG Z11.1 mmol/L confirmed diabetic rats (Srinivasan et al., 2005). In order to confirm the successful induction of type 2 diabetes-like symptomatology, we also detected the Lee's index, the HOMA insulin resistance index (HOMA IR), the HOMA insulin sensitivity index (HOMA ISI), the levels of total cholesterol (TC), free fatty acid (FFA) and triglyceride (TG) as reference indexes, more details was attached as supplementary data (Appendix 1). Next, the diabetic rats were monitored for blood glucose levels for at least two weeks to ensure the stability of the animal model. Then, the middle cerebral artery occlusion/reperfusion (MCAO/R, 2/ 46 h) model was induced using the intraluminal suture method. Body temperature was maintained at 3770.5 1C during and shortly after surgery with a heating lamp. First, diabetic rats were anaesthetized by chloral hydrate (10%, 0.3 ml/kg). Next, the carotid and external carotid arteries were ligatured and the internal carotid artery was temporarily closed. A fishing line (0.24–0.26 mm diameter) was advanced through the internal carotid artery to the origin of the middle cerebral artery. After that, the carotid artery was ligatured to prevent the nylon line from falling off. Finally, the wound was closed and the animal was placed in its cage. After two hours of occlusion, the line was withdrawn and the diabetic rats were injected with drugs intraperitoneally.
4.4.
Groups
Rats were randomly divided into the following groups: the sham-operated group (n¼ 6), the MCAO/R with saline injection group (n¼ 6), the MCAO/R with nimodipine treatment group (nimodipine, 1 mg/kg weight; n¼ 6), and MCAO/R groups with low (20 μg/kg), medium (40 μg/kg), and high (80 μg/kg) rhGLP-1 (7–36) (n¼6). It should be noted that the sham group was operated with the same surgical procedure except that the nylon line did not reach the origin of the middle cerebral artery.
4.5.
Assessment of neurological deficit scores
Neurological symptoms after the animals woke up (ischemia for 2 h) and after 46 h of reperfusion were assessed using neurological deficit scores (grades 0–4), which were modified as described previously (Longa et al., 1989). Neurological evaluation was scored on a 5-point scale: 0¼ no neurological deficit; 1 ¼failure to extend the left forepaw fully; 2 ¼circling
to the left; 3¼ inability to bear weight on the left; and 4 ¼no spontaneous walking with depressed level of consciousness. The neurological deficits were evaluated by two independent observers blinded to the surgery. Differences in neurological deficit scores between animal wake up and after 46 h reperfusion were used to evaluate the efficacy of different groups.
4.6.
Evaluation of infarct volume by TTC staining
To analyze infarct volume, rats were sacrificed after 46 h reperfusion. Brains were removed rapidly and frozen at 20 1C for 10 min. Then the brains were sliced into six coronal slices of about 2 mm thick. Brain slices were immediately stained with 2% TTC solution at 37 1C for 30 min. The staining images were recorded by a digital camera and quantified by ImageJ software. Total infract volumes were calculated by summation of the infarct area in six brain slices and integrated by thickness. The percentage of total infraction was calculated as total infarct volumes of six brain slices/total brain volume 100%. Both surgeon and image analyzer operator were blinded to the treatment given to each animal.
4.7.
Biochemical analysis
Before and after 30 min of injection of drugs, rat vein blood was used to detect blood glucose. Rats were sacrificed and their brains rapidly removed after being assessed for neurobehavior; the ischemia cortex was dissected and stored at 20 1C for analysis. Samples were homogenized with phosphate buffer (PBS, pH¼ 7.4) to a concentration of 10%. The content of MDA, GSH-PX and the activity of SOD, iNOS and eNOS were determined using commercial assay kits according to manufacturer instructions (Beijing Fangcheng Biotechnology Co., Ltd.) using the enzyme-linked immunosorbent assay (ELISA) method.
4.8.
Histopathology
The brains of the five groups were fixed with 10% formalin, embedded in paraffin wax and cut into longitudinal sections of 5 μm thickness. The sections were stained with haematoxylin and eosin (H&E) for histopathological observation.
4.9.
Statistical analysis
SPSS 16.0 was used to calculate the variance among groups. Data were expressed as mean7S.D. One-way analysis of variance (ANOVA) followed by Least Significant Difference tests were performed. Significant differences were accepted when P was o0.05 or o0.01.
Conflict of interest The authors have declared that there were no conflicts of interests.
brain research 1602 (2015) 153–159
Acknowledgments This project was supported by the Peking University People’s Hospital Research and Development Funds (No: RDB2013-22)
Appendix A.
Supporting information
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.brainres. 2015.01.014.
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receptor stimulation preserves primary cortical and dopaminergic neurons in cellular and rodent models of stroke and Parkinsonism. Proc. Natl. Acad. Sci. U. S. A. 106, 1285–1290. Longa, E.Z., Weinstein, P.R., Carlson, S., Cummins, R., 1989. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20, 84–91. Lund, A., Knop, F.K., Vilsboll, T., 2014. Glucagon-like peptide-1 receptor agonists for the treatment of type 2 diabetes: differences and similarities. Eur. J. Intern. Med. 25, 407–414. Ma, X., Guan, Y., Hua, X., 2014. Glucagon-like peptide 1potentiated insulin secretion and proliferation of pancreatic beta-cells. J. Diabetes. Meier, J.J., Gethmann, A., Nauck, M.A., Gotze, O., Schmitz, F., Deacon, C.F., Gallwitz, B., Schmidt, W.E., Holst, J.J., 2006. The glucagon-like peptide-1 metabolite GLP-1-(9-36) amide reduces postprandial glycemia independently of gastric emptying and insulin secretion in humans. Am. J. Physiol. Endocrinol. Metab. 290, E1118–E1123. Moskowitz, M.A., Lo, E.H., Iadecola, C., 2010. The science of stroke: mechanisms in search of treatments. Neuron. 67, 181–198. Perry, T., Holloway, H.W., Weerasuriya, A., Mouton, P.R., Duffy, K., Mattison, J.A., Greig, N.H., 2007. Evidence of GLP-1-mediated neuroprotection in an animal model of pyridoxine-induced peripheral sensory neuropathy. Exp. Neurol. 203, 293–301. Salling, H.K., Dohler, K.D., Engstrom, T., Treiman, M., 2012. Postconditioning with curaglutide, a novel GLP-1 analog, protects against heart ischemia-reperfusion injury in an isolated rat heart. Regul. Pept. 178, 51–55. Sato, K., Kameda, M., Yasuhara, T., Agari, T., Baba, T., Wang, F., Shinko, A., Wakamori, T., Toyoshima, A., Takeuchi, H., Sasaki, T., Sasada, S., Kondo, A., Borlongan, C.V., Matsumae, M., Date, I., 2013. Neuroprotective effects of liraglutide for stroke model of rats. Int. J. Mol. Sci. 14, 21513–21524. Silvestrelli, G., Corea, F., Paciaroni, M., Milia, P., Palmerini, F., Parnetti, L., Gallai, V., 2002. The Perugia hospital-based stroke registry: report of the 2nd year. Clin. Exp. Hypertens. 24, 485–491. Srinivasan, K., Viswanad, B., Asrat, L., Kaul, C.L., Ramarao, P., 2005. Combination of high-fat diet-fed and low-dose streptozotocin-treated rat: a model for type 2 diabetes and pharmacological screening. Pharmacol. Res. 52, 313–320. Tajes, M., Ill-Raga, G., Palomer, E., Ramos-Fernandez, E., Guix, F.X., Bosch-Morato, M., Guivernau, B., Jimenez-Conde, J., Ois, A., Perez-Asensio, F., Reyes-Navarro, M., Caballo, C., Galan, A.M., Alameda, F., Escolar, G., Opazo, C., Planas, A., Roquer, J., Valverde, M.A., Munoz, F.J., 2013. Nitro-oxidative stress after neuronal ischemia induces protein nitrotyrosination and cell death. Oxid Med. Cell Longev 2013, 826143. Teramoto, S., Miyamoto, N., Yatomi, K., Tanaka, Y., Oishi, H., Arai, H., Hattori, N., Urabe, T., 2011. Exendin-4, a glucagon-like peptide-1 receptor agonist, provides neuroprotection in mice transient focal cerebral ischemia. J. Cereb. Blood Flow Metab. 31, 1696–1705. Towfighi, A., Markovic, D., Ovbiagele, B., 2012. Current national patterns of comorbid diabetes among acute ischemic stroke patients. Cerebrovasc. Dis. 33, 411–418. Wardlaw, J.M., Murray, V., Berge, E., Del, Z.G., Sandercock, P., Lindley, R.L., Cohen, G., 2012. Recombinant tissue plasminogen activator for acute ischaemic stroke: an updated systematic review and meta-analysis. Lancet 379, 2364–2372. WHO publishes definitive atlas on global heart disease and stroke epidemic, 2004. Indian J. Med. Sci. 58, 405–406. Ying, W., Xiong, Z.G., 2010. Oxidative stress and NADþ in ischemic brain injury: current advances and future perspectives. Curr. Med. Chem. 17, 2152–2158. Zhao, L.R., Spellman, S., Kim, J., Duan, W.M., McCarthy, J.B., Low, W.C., 2005. Synthetic fibronectin peptide exerts neuroprotective effects on transient focal brain ischemia in rats. Brain Res. 1054, 1–8.
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Research Report
Protective effect of rhGLP-1 (7–36) on brain ischemia/reperfusion damage in diabetic rats Libo Zhaoa, Jia Xub,1, Qian Wanga, Zhonglian Qianc, Wanyu Fenga, Xiaoxing Yind, Yi Fanga,n a
Department of Pharmacy, Peking University People’s Hospital, Beijing 100044, China Department of Pharmacy, Mawangdui Hospital, Changsha, 410016, China c Department of Pharmacy, The First Hospital, Huhhot, 010010, China d Department of Pharmacy, Xuzhou Medical College, Xuzhou 221004, China b
art i cle i nfo
ab st rac t
Article history:
In recent years, GLP-1 and its analogs have been developed for the treatment of type 2
Accepted 9 January 2015
diabetes. It has been reported that stimulating the GLP-1 receptor can protect neurons
Available online 16 January 2015
against metabolic and oxidative insults, and therefore can be used in the treatment of
Keywords:
stroke and Parkinson's disease. The present study aimed to examine the neuroprotective
GLP-1
effects of rhGLP-1 (7–36) and its possible mechanisms against acute ischemia/reperfusion
Brain ischemia/reperfusion
injuries induced by middle cerebral artery occlusion (MCAO) in diabetic rats. The type 2
Diabetic rats
diabetic rat model was established by a combination of a high-fat diet and low-dose
Oxidative stress
streptozotocin (STZ). RhGLP-1 (7–36) (20, 40, 80 μg/kg) was given intraperitoneally before reperfusion. The neuroprotective effects of rhGLP-1 (7–36) were evaluated by changes in neurological deficit scores and 2,3,5-Triphenyltetrazolium chloride (TTC) staining. Changes in blood glucose were used to assess hypoglycemic effects. The content of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), inducible nitric oxide syntheses (iNOS) and endothelial nitric oxide syntheses (eNOS) after MCAO/R administration (2 h and 46 h) were examined to investigate the possible mechanisms of RhGLP-1 (7–36). Haematoxylin and eosin (H&E) staining was used for histopathological observation. Compared with the control group, rhGLP-1 (7–36)-treated groups decreased nerve function deficiency scores; significantly reduced infarction volume percentage, MDA, iNOS and blood glucose; and significantly increased SOD, GSH-PX and eNOS. In addition, rhGLP-1 (7–36) groups enhanced the density of surviving neurons and increased vascular proliferation. The current study suggests a neuroprotective effect of rhGLP-1 (7–36) in diabetic MCAO/R rats since anti-oxidative and anti-nitrosative stress effects can contribute to beneficial effects against ischemia/reperfusion injury. & 2015 Elsevier B.V. All rights reserved.
n
Corresponding author. Tel.: þ86 10 66583834. E-mail address: [email protected] (Y. Fang). 1 This author contributed equally to this work and should be considered as co-first author.
http://dx.doi.org/10.1016/j.brainres.2015.01.014 0006-8993/& 2015 Elsevier B.V. All rights reserved.
154
1.
brain research 1602 (2015) 153–159
Introduction
Stroke is the third leading cause of death and paralysis worldwide and remains a serious challenge to public health due to its high incidence and life threatening nature (WHO, 2004; Feigin et al., 2014). Close to 60–70% of strokes are ischemic strokes (Silvestrelli et al., 2002). In addition to hypertension and hypercholesterolemia, diabetes is another important risk factor for strokes, especially in patients less than 65 years of age (Kissela et al., 2005; Lawes et al., 2004; Towfighi et al., 2012). Despite the fact that many mechanisms leading to cell death during ischemia/reperfusion brain injury have been identified in the past decades such as excitotoxicity, calcium dysregulation, oxidative and nitrosative stress, and inflammation (Moskowitz et al., 2010), the underlying mechanisms are not yet well understood. Recombinant tissue plasminogen activator rt-PA (alteplase) is the only treatment approved by the FDA since it has been shown to improve functional outcomes in randomized trials of patients treated soon after acute ischemic stroke. However, due to its short therapeutic window, it is not widely used (Wardlaw et al., 2012). As a result, developing new treatments and drugs for ischemia stroke patients is increasingly important. The glucagon-like peptide-1 (7–36) amide (GLP-1) is a member of the proglucagon family of incretin hormones secreted mainly by the enteroendocrine cells of the intestine in response to the presence of nutrients. GLP-1 and its analogs facilitate glucose-induced insulin release, suppress glucagon secretion, slow gastric emptying and suppress appetite by activating the GLP-1 receptor (GLP-1R), which is widely expressed in islet cells, the kidney, lungs, brain and gastrointestinal tract (Dunphy et al., 1998; Lund et al., 2014). As a result, analogs of GLP-1 have been developed for the treatment of type 2 diabetes. Endogenous GLP-1 is rapidly inactivated by cleavage of the NH2-terminal dipeptide by the widely expressed serine protease dipeptydil peptidase-IV (DPP-IV) (Meier et al., 2006). Thus, long-acting GLP-1 analogs have been developed to prolong the effect by changing the structure of the peptide. Exenatide is the first GLP-1 analog approved by the FDA for the treatment of type 2 diabetes (Diamant et al., 2014). The absence of amidation of the NH2terminal is the main difference between recombinant human glucagon-like peptide-1 (7–36) amide rhGLP-1 (7–36) and endogenous GLP-1. It has been reported that stimulating the GLP-1 receptor can protect neurons against metabolic and oxidative insults; as a result, it can be used for the treatment of stroke and Parkinson's disease (Li et al., 2009; Perry et al., 2007). In addition, previous studies have suggested that after ischemia/reperfusion, the expression of GLP-1R protein significantly increases, and GLP-1R agonists protect against ischemia-induced neuronal death possibly by increasing GLP-1R expression and attenuating microglia activation against transient cerebral ischemic damage (Lee et al., 2011). Furthermore, experiments have shown that GLP-1R agonists have neuroprotective effects on cerebral ischemia models, possibly through anti-oxidative effects and increased intracellular cAMP levels (Sato et al., 2013; Teramoto et al., 2011). The aim of the current study was to investigate the protective effects of rhGLP-1 (7–36) on diabetic rats with
ischemia/reperfusion damage to the brain and to explore the mechanisms responsible.
2.
Results
2.1.
Effects on neurological deficits
After MCAO for 2 h and then followed 46 h reperfusion, the diabetic rats showed characteristics of circling and rotating to the left when crawling, falling to the left side when standing, and even not walking spontaneously. The neurological status was assessed using a 5-point scale. Sham-operated diabetic rats showed no neurological symptoms, as indicated by a neurological status score of zero. As shown in Fig. 1, the nimodipine and rhGLP-1 (7–36) groups had higher neurological status scores and showed improvements in neurological deficiencies compared to the model group receiving a high dose of rhGLP-1 (7–36): the best results, although no statistically significant different, were found in the low and middle rhGLP-1 (7–36) groups. These results indicate that both nimodipine and rhGLP-1 (7–36) reduced neurological dysfunction following diabetes combined with MCAO/R and showed dose-related alleviation in the rhGLP-1 (7–36) groups.
2.2.
Effects on cerebral infracted volume
As shown in Fig. 2A, normal brain tissue was deep stained red and the infarcted brain tissue was stained white, large infarction areas were presented in the model group whereas no infarctions were observed in the sham group. As seen in Fig. 2B, we know that compared with the model group, the nimodipine and rhGLP-1(7–36) groups showed significantly reduced infarction volume percentages during MCAO/R injury and the rhGLP-1 (7–36) high group showed the best protective effects of all groups.
Fig. 1 – Changes in neurological deficiency scores in different groups: the rhGLP-1 (7–36) high group significantly reduced neurological deficiency compared with the model group (nmeans vs. model group, Po0.05; △ means vs. sham group, Po0.05).
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Fig. 2 – Effects of rhGLP-1 (7–36) on the infracted volume of diabetic rats that were subjected to an occlusion of the middle cerebral artery for 2 h followed by 46 h reperfusion. A) Representative photographs of TTC staining of coronal brain slices: (a) Sham group; (b) model group; (c) nimodipine group; (d) rhGLP-1 (7–36) low group; (e) rhGLP-1 (7–36) middle group; (f) rhGLP1 (7–36) high group. B) Compared with the model group, the infarction volume percentage of nimodipine and rhGLP-1 (7–36) groups were significantly reduced (nmeans vs. model group Po0.05; nnmeans vs. model group Po0.01; △△ means vs. sham group, Po0.01, n ¼6).
2.3.
Biochemical observations
2.3.1.
Blood glucose observations
As presented in Table 1, there were no statistically significant differences in the various groups prior to injection of the drugs. Thirty minutes following injection, blood glucose levels varied in the reduced rhGLP-1 (7–36) groups: the rhGLP-1 (7–36) middle group showed a decreasing trend with
no statistical significance and the rhGLP-1 (7–36) high group showed a statistical significance reduction (Po0.05) compared with the model group, while blood glucose levels were elevated in the nimodipine group.
2.3.2.
Effects on levels of MDA, SOD and GSH-PX
Table 2 shows a significantly increased presence of MDA and reduced activity of SOD and GSH-PX in the model group
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compared with the sham group. Treatment with nimodipine and rhGLP-1 (7–36) significantly reduced MDA content and increased activity of SOD and GSH-PX compared with the model group after MCAO/R.
of the cerebral cortex, thereby enhancing the density of the surviving neurons and increasing vascular proliferation (Fig. 3c, d, e, and f).
2.3.3.
3.
Effects on levels of iNOS and eNOS
The data in Table 3 indicate that the activity of iNOS was significantly higher and the activity of eNOS was significantly lower in the other groups compared with the sham group. Nimodipine significantly lowered iNOS activity compared with the model group, and the rhGLP-1(7–36) dose required to suppress iNOS activity in the high group was the best. Meanwhile, levels of eNOS were significantly increased in the nimodipine group, and rhGLP-1 (7–36) at doses of 40 and 80 μg/kg significantly upregulated levels of eNOS compared with the model group (Po0.01).
2.4.
Histological observations
H&E staining was used to observe histological features and cerebral ischemia/reperfusion injury regions mainly located in hippocampus, frontoparietal cortex, striatum and preoptic areas. Representative histological photographs are shown in Fig. 3. As shown, no histological abnormalities were observed in the sham group, while the model group showed that most neurons in the infarct core appeared shrunken and contained eosinophilic cytoplasm and triangulated pyknotic nuclei with many vacuolars in the neuron space. The infarct core was surrounded by necrotic neurons that exhibited pyknotic shapes and condensed nuclei in the peri-infarct zones (Fig. 3b). Compared with the model group, the nimodipine and rhGLP-1 (7–36) groups were able to improve the structure
Discussion
GLP-1 is secreted by the L-cells of the intestine in response to food intake. Many studies have shown that GLP-1 can stimulate the release of insulin in a glucose-dependent manner (Choi and Lee, 2011; D'Alessio et al., 1995). GLP-1 is also thought to play a role in the proliferative and antiapoptotic action on pancreatic β-cells and intestinal epithelial cells, respectively; it also inhibits gastric emptying and acid secretion (Ma et al., 2014). Recently, GLP-1 analogs were found to have protective effects on heart ischemia/reperfusion damage (Hausenloy et al., 2013; Salling et al., 2012). In the current study, we demonstrated that rhGLP-1 (7–36) could induce neuroprotection by attenuating infarction volume and by improving neurologic function in a diabetic combined MCAO/R rat model. The protective effects of rhGLP-1 (7–36) at 80 μg/kg were more potent than those at 20 and 40 μg/kg, showing a dose response tendency. Oxidative stress is a key pathological factor in brain ischemic/reperfusion injuries. It is involved in the cytotoxic consequences of mismatches between the production of reactive oxygen species (ROS) and free radicals and the ability of cells to defend against them (Ying and Xiong, 2010). ROS's include oxygen-centered radicals with unpaired electrons Table 3 – Nitric oxide syntheses activity in different groups of MCAO/R rat brains (n ¼6) Groups
Nitric oxide syntheses activity (U/L)
Table 1 – Summary of blood glucose readings before and after injection of drugs for 30 min (n ¼6) Groups
0 min (mmol/L)
30 min (mmol/L)
Sham Model Nimodipine RhGLP-1(7–36) low RhGLP-1(7–36) middle RhGLP-1(7–36) high
25.475.5 25.874.2 24.176.5 25.973.0 26.073.7 25.975.5
25.374.9 24.974.1 25.377.2 25.474.2 23.673.4 22.974.9n
n
Means vs. model group Po0.05.
Sham Model Nimodipine RhGLP-1(7–36) low RhGLP-1(7–36) middle RhGLP-1(7–36) high
iNOS
eNOS
4.9270.21 6.0370.37△△ 5.2970.20nn 5.3370.19nn 5.2170.34nn 5.0970.24nn
29.6371.24 24.8171.18△△ 26.5870.94nn 24.0970.84 26.0070.96n 27.2270.92nn
n
Means vs. model group Po0.05. Means vs. model group Po0.01. △△ Means vs. sham group Po0.01. nn
Table 2 – Results of oxidative stress indicators in different groups of MCAO/R rat brains (n ¼6) Groups
Sham Model Nimodipine RhGLP-1(7–36) low RhGLP-1(7–36) middle RhGLP-1(7–36) high n
Means vs. model group Po0.05. Means vs. model group Po0.01. △△ Means vs. sham group Po0.01. nn
Oxidative stress indicators SOD (U/L)
MDA (nmol/L)
GSH-PX (pmol/mL)
126.7973.75 106.7375.53△△ 109.3872.23 107.4476.01 115.8475.52nn 116.4574.51nn
2.7270.14 3.8070.06△△ 3.2070.10nn 3.3270.13nn 3.1370.09nn 2.9170.09nn
30.5371.07 23.9970.96△△ 26.2570.91nn 25.0570.75n 27.7370.66nn 28.5570.72nn
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Fig. 3 – Representative histopathology photographs of brain tissue in diabetic rats after cerebral ischemia/reperfusion injury in different groups (H&E 100). (a) Sham group; (b) model group. Significant reversal of injury was observed in the nimodipine group (c), the rhGLP-1 (7–36) low group (d), the rhGLP-1 (7–36) middle group (e), and the rhGLP-1 (7–36) high group (f). such as the superoxide anion O2 and the hydroxyl radical OH or covalent molecules such as hydrogen peroxide (H2O2). SOD detoxifies O2 to H2O2, which is converted to H2O by catalase or GSH-PX (Chan, 2001). Oxidative stress can also be assessed by measuring the oxidized products of macromolecules such as lipids. Lipid peroxides are derived from unstable polyunsaturated fatty acids and decompose to form a complex series of compounds such as MDA. Cerebral ischemia can cause significant amounts of MDA formation in the ischemia hemisphere (Chen et al., 2009). Measuring the content of SOD, MDA and activity of GSH-PX in the brain cortex can show damage levels during the ischemia/reperfusion progress. In the current study, rhGLP-1 (7–36) significantly reduced MDA content and increased the activity of SOD and GSH-PX compared with the model group, indicating that rhGLP-1 (7–36) inhibited the oxidative stress response process. Nitric oxide (NO) is an important reactive nitrogen species that has also been demonstrated to play an important role in ischemic brain injury. Excessive NO can produce toxicity in nerve cells in the form of free radicals (Zhao et al., 2005). NO generated from neuronal nitric oxide syntheses (NOS) is known to exist in three isoforms: nNOS, iNOS and eNOS. The major regulator of vascular tone is the endothelium through endothelial NOS (eNOS). nNOS and inducible iNOS enzymes are expressed in neurons, glial cells and vascular myocytes, and can contribute to NO production in ischemic processes (Tajes et al., 2013). In the current research, dose-related rhGLP-1 (7–36) to suppress the activity of iNOS in the high group had the best outcomes, and levels of eNOS were significantly increased in rhGLP-1 middle and high groups compared with the model group. In addition, iNOS activity was significantly greater and eNOS activity was significantly lower in other groups compared with the sham group; this implies that although rhGLP1 showed benefits to brain ischemia/reperfusion injuries, it was still worse than the sham group. These results show that the effects of rhGLP-1 (7–36) on cerebral I/R injuries in diabetic rats could be involved in regulating iNOS and eNOS activities.
In conclusion, results of the above experiments indicate that rhGLP-1 (7–36) can decrease blood glucose and protect the brain from ischemia/reperfusion damage by anti-oxidative stress and anti-nitric oxide damage in diabetic/MCAO/R rats. Specifically, rhGLP-1 (7–36) can dose-dependently reduce blood glucose, thereby significantly improving neurological function and reduce infraction size two hours after ischemia followed by 46-hour reperfusion. Additionally, it improved the activities of SOD, GSH-PX, eNOS and enhanced the density of surviving neurons and increased vascular proliferation. Anti-oxidant and anti-nitric oxide damage appears to be a basic and essential mechanism of the neuroprotective effects of rhGLP1 (7–36). These results suggest that rhGLP-1 (7–36) could be a promising therapeutic drug for the treatment of diabetes combined with ischemic cerebrovascular disease. However, the mechanisms of cerebral ischemia/reperfusion injuries are varied. rhGLP-1 could also play a role in neuroprotection by other mechanisms. As such, further research is required.
4.
Experimental procedures
4.1.
Animals
Male pathogen free Sprague Dawleys rats (80–120 g) obtained from the Animal Center of the Academy of Military Medical Sciences, Beijing, China were housed in a light- (12 h light/ dark cycle) and temperature-controlled (21.5–22.5 1C) environment. The animals and experimental procedures used were approved by the Ethics Committee of the Peking University People's Hospital (NO: 2011-66).
4.2.
Reagents and chemicals
TTC and STZ were purchased from Sigma-Aldrich Co. (Saint Louis, USA), nimodipine injection was purchased from Byer Schering Pharma AG (Beijing, China), lyophilizing rhGLP-1
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(7–36) powder was manufactured by Shanghai Huayi BioTechnology Co., ltd., (Shanghai, China) and was diluted in saline to 0.2 mg/ml. The kits for biochemical analysis, including MDA, SOD, GSH-PX, iNOS and eNOS were purchased from Beijing Fangcheng Bio-Technology Co., Ltd (Beijing, China). The high-fat diet consisted of standard flour (17.5%), soy flour (10.0%), bran (7.0%), corn flour (12.0%), fishmeal (2.5%), salt (0.5%), yeast extract (0.5%), lard (15.0%), sucrose (10.0%), milk powder (5.0%), sesame oil (5.0%), eggs (10.0%) and peanuts (5.0%).
4.3.
Diabetic and brain ischemia/reperfusion model
After being fed a high-fat diet for four weeks, rats were given STZ (30 mg/kg) intraperitoneally. Tail vein blood samples were obtained to detect fasting blood glucose (FBG) levels; an FBG Z11.1 mmol/L confirmed diabetic rats (Srinivasan et al., 2005). In order to confirm the successful induction of type 2 diabetes-like symptomatology, we also detected the Lee's index, the HOMA insulin resistance index (HOMA IR), the HOMA insulin sensitivity index (HOMA ISI), the levels of total cholesterol (TC), free fatty acid (FFA) and triglyceride (TG) as reference indexes, more details was attached as supplementary data (Appendix 1). Next, the diabetic rats were monitored for blood glucose levels for at least two weeks to ensure the stability of the animal model. Then, the middle cerebral artery occlusion/reperfusion (MCAO/R, 2/ 46 h) model was induced using the intraluminal suture method. Body temperature was maintained at 3770.5 1C during and shortly after surgery with a heating lamp. First, diabetic rats were anaesthetized by chloral hydrate (10%, 0.3 ml/kg). Next, the carotid and external carotid arteries were ligatured and the internal carotid artery was temporarily closed. A fishing line (0.24–0.26 mm diameter) was advanced through the internal carotid artery to the origin of the middle cerebral artery. After that, the carotid artery was ligatured to prevent the nylon line from falling off. Finally, the wound was closed and the animal was placed in its cage. After two hours of occlusion, the line was withdrawn and the diabetic rats were injected with drugs intraperitoneally.
4.4.
Groups
Rats were randomly divided into the following groups: the sham-operated group (n¼ 6), the MCAO/R with saline injection group (n¼ 6), the MCAO/R with nimodipine treatment group (nimodipine, 1 mg/kg weight; n¼ 6), and MCAO/R groups with low (20 μg/kg), medium (40 μg/kg), and high (80 μg/kg) rhGLP-1 (7–36) (n¼6). It should be noted that the sham group was operated with the same surgical procedure except that the nylon line did not reach the origin of the middle cerebral artery.
4.5.
Assessment of neurological deficit scores
Neurological symptoms after the animals woke up (ischemia for 2 h) and after 46 h of reperfusion were assessed using neurological deficit scores (grades 0–4), which were modified as described previously (Longa et al., 1989). Neurological evaluation was scored on a 5-point scale: 0¼ no neurological deficit; 1 ¼failure to extend the left forepaw fully; 2 ¼circling
to the left; 3¼ inability to bear weight on the left; and 4 ¼no spontaneous walking with depressed level of consciousness. The neurological deficits were evaluated by two independent observers blinded to the surgery. Differences in neurological deficit scores between animal wake up and after 46 h reperfusion were used to evaluate the efficacy of different groups.
4.6.
Evaluation of infarct volume by TTC staining
To analyze infarct volume, rats were sacrificed after 46 h reperfusion. Brains were removed rapidly and frozen at 20 1C for 10 min. Then the brains were sliced into six coronal slices of about 2 mm thick. Brain slices were immediately stained with 2% TTC solution at 37 1C for 30 min. The staining images were recorded by a digital camera and quantified by ImageJ software. Total infract volumes were calculated by summation of the infarct area in six brain slices and integrated by thickness. The percentage of total infraction was calculated as total infarct volumes of six brain slices/total brain volume 100%. Both surgeon and image analyzer operator were blinded to the treatment given to each animal.
4.7.
Biochemical analysis
Before and after 30 min of injection of drugs, rat vein blood was used to detect blood glucose. Rats were sacrificed and their brains rapidly removed after being assessed for neurobehavior; the ischemia cortex was dissected and stored at 20 1C for analysis. Samples were homogenized with phosphate buffer (PBS, pH¼ 7.4) to a concentration of 10%. The content of MDA, GSH-PX and the activity of SOD, iNOS and eNOS were determined using commercial assay kits according to manufacturer instructions (Beijing Fangcheng Biotechnology Co., Ltd.) using the enzyme-linked immunosorbent assay (ELISA) method.
4.8.
Histopathology
The brains of the five groups were fixed with 10% formalin, embedded in paraffin wax and cut into longitudinal sections of 5 μm thickness. The sections were stained with haematoxylin and eosin (H&E) for histopathological observation.
4.9.
Statistical analysis
SPSS 16.0 was used to calculate the variance among groups. Data were expressed as mean7S.D. One-way analysis of variance (ANOVA) followed by Least Significant Difference tests were performed. Significant differences were accepted when P was o0.05 or o0.01.
Conflict of interest The authors have declared that there were no conflicts of interests.
brain research 1602 (2015) 153–159
Acknowledgments This project was supported by the Peking University People’s Hospital Research and Development Funds (No: RDB2013-22)
Appendix A.
Supporting information
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.brainres. 2015.01.014.
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