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Ellagic acid-induced thrombotic focal cerebral ischemic model in rats
JPM-06164; No of Pages 6 Journal of Pharmacological and Toxicological Methods xxx (2014) xxx–xxx
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Original article
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Ellagic acid-induced thrombotic focal cerebral ischemic model in rats
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Xiaoming Pang 1, Tianxia Li 1, Liuxin Feng, Jingjing Zhao, Xiaolu Zhang, Juntian Liu ⁎
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Department of Pharmacology, Xi'an Jiaotong University School of Medicine, Xi'an, China
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Introduction: Ischemic stroke is a common cause of human disability and death. Animal models of focal cerebral ischemia are widely utilized to mimic human ischemic stroke. Although models of focal cerebral ischemia have been well established, very few evidence is based on triggering the intrinsic coagulation system to induce focal cerebral ischemia. Ellagic acid (EA) has been identified to trigger the intrinsic coagulation system via activating coagulation factor XII. However, it remains unknown whether EA can serve as a novel pharmacological approach to induce a new model of focal cerebral ischemia in rats. Methods: EA was used for inducing focal cerebral ischemia in adult rats. The dose- and time-dependent effects of EA were characterized. The cerebral infarction ratio was determined with triphenyltetrazolium chloride staining, and the histopathological analysis of the brain tissue was performed under light microscopy. The neurological deficit score was evaluated by a modified method of Bederson. Malondialdehyde (MDA) level and lactate dehydrogenase (LDH) and superoxide dismutase (SOD) activities in serum were determined by spectrophotometry. Results: Injection of EA into the middle cerebral artery of rats was able to generate focal cerebral infarction and increased the neurological deficit score and the brain weight to body weight ratio in dose- and time-dependent manners. Furthermore, EA raised serum LDH activity and MDA level and decreased serum SOD activity in a dose-related fashion. Discussion: This is the first evidence to show that EA induces focal cerebral ischemia in rats, which is similar to human ischemia stroke in pathogenesis. This model holds promise for pathological, pharmacological and clinical studies of ischemic stroke. © 2014 Published by Elsevier Inc.
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Article history: Received 18 August 2013 Accepted 2 January 2014 Available online xxxx
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Keywords: Animal model Ellagic acid Focal cerebral ischemia Stroke
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1. Introduction
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Stroke is a heterogeneous disease with a complex pathophysiology and causes death and disability of patients as well as a significantly economic burden. With the fact that life expectancy increases, stroke is projected to be a more challenging disease in the future (Mukherjee & Patil, 2011). Ischemic stroke which is caused by an embolic or thrombotic occlusion of an artery supplying a specific territory of brain accounts for 80% of all strokes. Animal models of focal cerebral ischemia have been developed to mimic human stroke and play a major role in the experimental study of stroke (Durukan & Tatlisumak, 2007). Over the past years, animal models have largely advanced our knowledge on the pathophysiology and etiology of ischemic stroke (Mergenthaler, Dirnagl, & Meisel, 2004). However, due to the heterogeneity of human stroke, most translational stroke trials that aim to introduce basic experimental findings into clinical therapeutic strategies have failed, which is mainly attributed to ischemic stroke models not simulating pathogenesis of human stroke.
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Abbreviations: MCAO, middle cerebral artery occlusion; EA, ellagic acid; TTC, 2,3,5triphenyltetrazolium chloride; MDA, malondialdehyde; LDH, lactate dehydrogenase; SOD, superoxide dismutase; MCA, middle cerebral artery; BBB, blood brain barrier. ⁎ Corresponding author at: Postbox 58, Xi'an Jiaotong University School of Medicine, 76 West Yanta Road, Xi'an 710061, China. Tel./fax: +86 29 82655188. E-mail address: [email protected] (J. Liu). 1 Co-first author.
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The focal cerebral ischemia model commonly used in the animal experiment is the middle cerebral artery occlusion (MCAO) model via intraluminal suture, which is developed in rats by Koizumi et al. and, is subsequently modified (Belayev, Alonso, Busto, Zhao, & Ginsberg, 1996; Longa, Weinstein, Carlson, & Cummins, 1989). By controlling the suture, both transient ischemia and permanent ischemia are performed using this model. However, the intraluminal suture in MCAO model varies largely from most human focal cerebral ischemia because it is actually a mechanical occlusion model. Thromboembolism is one of the most frequent causes of ischemic stroke in humans. Therefore, development of thromboembolic focal cerebral ischemia models will serve as a unique approach to mimic human stroke, which exhibits an advantage over other stroke models. Although thromboembolic models have previously been reported (Kudo, Aoyama, Ichimori, & Fukunaga, 1982; Zhang et al., 1997), few models are developed based on triggering the intrinsic coagulation system to induce thrombosis in vivo. Ellagic acid (EA), 2,3,7,8-tetrahydroxy-chromeno [5,4,3-cde] chromene-5, 10-dione, is a plant phenol found in various fruits. It has been confirmed that EA triggers the intrinsic coagulation via activating coagulation factor XII, and promotes platelet aggregation by thrombin (Damas, Remacle-Volon, & Adam, 1989; Ratnoff & Crum, 1964), which have been applied in inducing the hypercoagulable state (Botti, 1966; Girolami, Cella, Burul, & Zucchetto, 1976; Iomhair & Lavelle, 1996; Ratnoff & Saito, 1982; Shiozaki et al., 1994). Hara et al. first established the global cerebral ischemia by injection of EA suspension into the common carotid artery of rats, and conducted a primary evaluation
1056-8719/$ – see front matter © 2014 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.vascn.2014.01.001
Please cite this article as: Pang, X., et al., Ellagic acid-induced thrombotic focal cerebral ischemic model in rats, Journal of Pharmacological and Toxicological Methods (2014), http://dx.doi.org/10.1016/j.vascn.2014.01.001
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2.1. Chemical reagents
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EA was purchased from Sigma (St. Louis, MO, USA), and dissolved in 1 M NaOH, and then pH in the solution was adjusted to 7.4 with 1 M HCl. 2,3,5-Triphenyltetrazolium chloride (TTC) was obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Assay kits for detecting malondialdehyde (MDA), lactate dehydrogenase (LDH) and superoxide dismutase (SOD) were provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
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2.2. Animals
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Male Sprague–Dawley rats weighing 280–320 g were purchased from the Experimental Animal Center of Xi'an Jiaotong University School of Medicine, Xi'an, China. Animals were housed at 20–25 °C with a relative humidity of 50%–60% and a 12 h light/dark cycle. Food and water were provided ad libitum. Rats were fasted for 24 h before the experiment. All the experimental procedures carried out in this study were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Xi'an Jiaotong University, and approved by the Institutional Animal Care Committee of Xi'an Jiaotong University.
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2.3. Establishment of the focal cerebral ischemia model in rats
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2.4. Experimental protocol
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In order to present a comprehensive evaluation on the model, we examined dose- and time-dependent characteristics of EA-induced focal cerebral ischemia. In the dose–effect experiment, rats were randomly divided into the sham group and three EA-treated groups (0.5, 1.0, 1.5 mg/mL; n = 8–10). The neurological deficit score, the cerebral infarction size, the brain weight to body weight ratio, LDH, SOD, MDA in serum and morphological changes were evaluated at 24 h after the injection of EA or saline. In the time-effect experiment, rats were randomly divided into the sham group and EA-treated group
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The surgical process of the focal cerebral ischemia model induced by EA was modified according to Zhang's thrombotic focal cerebral ischemia model (Zhang et al., 1997) and Longa's MCAO model (Longa et al., 1989). Rats were anesthetized with intraperitoneal injection of chloral hydrate (300 mg/kg). The left common carotid artery was exposed at the level of the external and internal carotid artery bifurcation under the sterile condition. Catheter (0.3 mm, O.D.) filled with EA or saline was inserted into the left common carotid artery and gently advanced into the internal carotid artery for a length of about 20–21 mm until a slight resistance was felt. Such resistance indicated that the catheter just passed over the proximal segment of the anterior cerebral artery. Then, the catheter was pulled back 2 mm and at this point, the intraluminal catheter was about 2 mm to the origin of the middle cerebral artery (MCA). Next, 0.1 mL EA was slowly injected into MCA of rats through the catheter in the model group (EA-treated group), while 0.1 mL normal saline was given to rats in the sham group in an identical fashion to the EA-treated group instead. Finally, the catheter was pulled out 5 min after the injection. During the whole process of animal surgery, the rat rectal temperature was maintained at 37.0 °C with a heat lamp.
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2.6. Assessment of the brain weight to body weight ratio
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In the end of each experiment, rats were sacrificed by decapitation under the deep anesthesia with chloral hydrate. Brains were removed, and weighed immediately. The brain weight to body weight ratio was calculated according to the following formula:
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The neurological deficit score was recorded 6 h after the injection of EA in the dose-effect experiment, and at the indicated time after the injection of EA in the time-effect experiment. The neurological status of each rat was carefully evaluated by an observer who was blinded to the experimental procedure following a modified method of Bederson (Bederson et al., 1986). ① Rats were gently held by the tail, suspended one meter above the floor, and observed for forelimb flexion. Normal rats extended both forelimbs toward the floor. Rats that consistently flexed the forelimb contralateral to the injured hemisphere were scored as 4, otherwise scored as 0. ② Rats were pushed in the contralateral direction and scored as: 0 (resistance to lateral push), 1 (initially reduced but progressive resistance), 2 (reduced resistance), or 3 (lateral down fall). ③ Rats were then allowed to move freely to observe circling behavior. The movement was scored as: 0 (straight movement), 1 (movement to the right), 2 (circling movement), or 3 (no movement). The scores of three tests were summed and represented the neurological deficit score (0 to 10).
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2. Materials and methods
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Brain weight to body weight ratio ð%Þ ¼ ðbrain weight=body weightÞ 100
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2.5. Evaluation of neurological deficit
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(1.5 mg/mL; n = 8–10). The above-described measurements were 140 analyzed 3, 6, 12, 24 h after the injection of EA or saline. 141
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(Hara, Iwamoto, Ishihara, & Tomikawa, 1994). However, there is no study to identify whether EA induces focal cerebral ischemia and the comprehensive evaluation. The aim of the present study was to develop a stable EA-induced focal cerebral ischemia model similar to human thrombotic ischemia stroke in rats.
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2.7. Detection of cerebral infarction size
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Brains were removed and sliced into six 2-mm-thick coronal slices (first to sixth from rostral to caudal). The brain slices were incubated in 2% phosphate buffer solution of TTC for 30 min at 37 ºC, and immersed in 10% formalin overnight. The unstained area (pale) was the infarcted area, while the stained area (red) was the normal area. The slices were photographed with a digital camera and the infarction volume was calculated by summing the infarction volume of sequential sections with a commercial image processing software program (Photoshop, Adobe Systems; Mountain View, CA, USA). The investigator who analyzed the image was blinded to the experimental groups. The cerebral infarction size was presented as the infarction ratio, which was calculated according to the following formula:
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Infarction ratio ð%Þ ¼ ðinfarction volume=whole brain volumeÞ 100 181 180
2.8. Determination of MDA, LDH and SOD
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Blood was collected via the abdominal aorta in the end of the experiment. Then, the separated serum was used to spectrophotometrically determine LDH activity at 450 nm, SOD activity at 550 nm and MDA concentration at 532 nm following the manufacturer's instructions.
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2.9. Histopathological analysis
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Brains were removed and fixed in 10% formalin in the end of the experiment. Then, the brain tissue was embedded in paraffin and sectioned at 4 μm thickness in the coronal plane. The sections were stained with haematoxylin and eosin, and examined under light microscopy.
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Please cite this article as: Pang, X., et al., Ellagic acid-induced thrombotic focal cerebral ischemic model in rats, Journal of Pharmacological and Toxicological Methods (2014), http://dx.doi.org/10.1016/j.vascn.2014.01.001
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3.1. Neurological deficit of rats with focal cerebral ischemia induced by EA
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The result in Fig. 1A showed that the neurological deficit score of rats was increased in a dose-dependent manner 24 h after the injection of EA. Compared with the sham group, there is a significant difference in the neurological deficit score in rats treated with 1 mg/mL and 1.5 mg/mL EA (P b 0.05). Fig. 1B displayed that the neurological deficit score of rats was significantly increased 3 h after the injection of 1.5 mg/mL EA, reached a maximum at 6 h and sustained for 24 h (P b 0.05).
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3.2. Brain weight to body weight ratio of rats with focal cerebral ischemia induced by EA
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Fig. 2A exhibited that the brain weight to body weight ratio was dose-dependently raised after rats were treated with EA for 24 h as compared to the sham group (P b 0.05). Fig. 2B indicated that the brain weight to body weight ratio of rats was raised 3 h after the injection of 1.5 mg/mL EA, and sustained for more than 24 h (P b 0.05).
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3.3. Cerebral infarction ratio of rats with focal cerebral ischemia induced by EA
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As shown in Fig. 3, the cerebral infarction ratio was enlarged in a dose-dependent manner 24 h after injecting EA to rats (P b 0.05). At 0.5 mg/mL EA, infarction foci were sporadic and spotty. At 1.0 mg/mL EA, the cerebral hemisphere showed patchy necrosis and the cerebral infarction ratio reached 18%. At 1.5 mg/mL EA, most of the cerebral hemisphere exhibited severe infarction and the cerebral infarction ratio reached 29.9 ± 1.9%. Likewise, EA time-dependently enlarged the cerebral infarction ratio of rats (P b 0.05). The cerebral infarction ratio was obviously raised 3 h after injecting 1.5 mg/mL EA (P b 0.05), and slowly increased after 12 h (P b 0.05) (Fig. 4).
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As seen from Figs. 6–8, all the concentrations of EA increased 234 LDH activity and decreased SOD activity in serum (P b 0.05), whereas 235 middle and high concentrations of EA increased MDA level (P b 0.05). 236
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The histopathological analysis showed that MCA injection of EA to rats caused the morphological abnormalities, including degeneration
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In the clinical condition, coagulation factor XII is activated by collagen in the vessel wall once endothelium is injured by the pathological factors, such as hypertension and ox-LDL. The activated coagulation factor XII is able to trigger the intrinsic coagulation system to form fibrin. Meanwhile, platelets are also activated by thrombin from the activated coagulation system. Finally, combination of fibrin and activated platelets participates in formation of the mixed thrombosis. Many studies demonstrate that EA is an effective activator of coagulation factor XII. Therefore, we attempted to use EA to induce thrombosis in MCA and to develop a new model of focal cerebral ischemia in rats. The present study showed that injection of EA into MCA was able to produce focal cerebral infarction in rats, increased the neurological deficit score and the brain weight to body weight ratio in dose- and time-dependent manners. EA also raised LDH activity and MDA level, but decreased SOD activity in serum in a dose-related fashion. The cerebral infarction size has widely been accepted as the most precise and objective indicator in study of the animal ischemic stroke (Durukan & Tatlisumak, 2007). In this study, treatment of rats with injection of EA via MCA was able to generate focal cerebral infarction in both dose-dependent manner and time-dependent way. The cerebral infarction ratio reached 29.9% after the injection of 1.5 mg/mL EA for 24 h, which is comparable to the infarction size from MCAO model by intraluminal suture (Xiao, Liu, Hu, Li, & Zhang, 2007). The results from the histopathological examination provided further evidence in support of the cerebral damage in focal cerebral ischemia induced by EA. In terms of the infarction position and pattern, the cerebral ischemia exists in the territory supplied by MCA, suggesting that EA is likely to occlude large branch of MCA, and spontaneous reperfusion and collateral perfusion do not exist possibly at 24 h after injection of EA. Cerebral ischemia may cause neurological abnormalities, the severity of which is correlated with the cerebral infarction size. Therefore, the neurological deficit was used for reflecting formation and severity of focal cerebral infarction. The present results showed that the injection of EA to rats significantly increased the neurological deficit score.
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3.4. Morphological changes of brain in rats with focal cerebral ischemia induced by EA
4. Discussion
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3.5. Changes of LDH, SOD and MDA in serum of rats with focal cerebral 232 ischemia induced by EA 233
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Data were expressed as mean ± S.D. and statistical tests were performed with one-way ANOVOA followed by Tukey's test to determine differences among groups. P b 0.05 was considered to be significant.
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and necrosis of neurons, focal cerebral edema, infiltration of inflamma- 229 tory cells as well as dilated capillaries in comparison with the sham 230 group (Fig. 5). 231
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Fig. 1. Neurological deficit of rats with focal cerebral ischemia induced by EA. The neurological deficit score was evaluated 6 h after injecting the different doses of EA to rats (A) or at the indicated time after injecting 1.5 mg/mL EA to rats (B) according to a modified Bederson's method with a scale of 0–10. Data was expressed as mean ± S.D. (n = 8–10). *P b 0.05, compared with sham group.
Please cite this article as: Pang, X., et al., Ellagic acid-induced thrombotic focal cerebral ischemic model in rats, Journal of Pharmacological and Toxicological Methods (2014), http://dx.doi.org/10.1016/j.vascn.2014.01.001
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ischemia induced by EA, implying the involvement of lipid peroxidation in EA-induced focal cerebral ischemia. At present, the majority of studies on stroke are conducted in small animals due to lower cost and greater acceptability from ethical viewpoints. The rat is an ideal subject in study of stroke because of its similarity to human in cerebrovascular anatomy and physiology (Mhairi Macrae, 1992; Tamura, Graham, McCulloch, & Teasdale, 1981). In order to avoid the variability caused by female hormone cycling and weight differences, we chose male SD rats weighing 280 to 320 g as study subjects in the present study. Body temperature and blood glucose are two important factors influencing focal cerebral ischemia. It is documented that body temperature is able to affect stroke severity, infarct size, mortality, and outcome (Castillo, Davalos, Marrugat, & Noya, 1998; MacLellan, Davies, Fingas, & Colbourne, 2006; Reith et al., 1996). During the process of the animal surgery, the rat rectal temperature was maintained with a heat lamp at 37.0 °C. The majority of studies had shown that hyperglycemia worsens the ischemic damage (Huang, Wei, & Quast, 1996; Nedergaard, 1987). Therefore, rats were fasted for 24 h before the surgery in order to minimize variability resulted from blood glucose levels in this study. Many animal models of focal cerebral ischemia have been developed, and current translational models do not adequately simulate the pathogenesis of human cerebral ischemia. At present, focal cerebral ischemia model most commonly used is the MCAO model using an intraluminal suture. However, this is a mechanical occlusion model, which may differ from the human cerebral ischemia in its pathogenesis.
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Moreover, all rats with the cerebral infarction ratio greater than 20% revealed a significant neurological deficit, which is consistent with Bederson's report (Bederson et al., 1986). Blood brain barrier (BBB) is a separation of circulating blood from brain and cerebrospinal fluid in the central nervous system. Focal cerebral ischemia disrupts BBB, which results in cerebral edema. In the study, the brain weight to body weight ratio was assessed to represent the degree of BBB damage and cerebral edema in focal cerebral ischemia induced by EA. The results indicated that the brain weight to body weight ratio was increased 3 h after the injection of EA and sustained for 24 h, suggesting the existence of BBB damage and cerebral edema in focal cerebral ischemia induced by EA. LDH is one of intracellular enzymes. When neurons are injured, LDH is released into blood. Therefore, serum LDH activity is often used as a marker of the cell injury. The present experiment revealed that serum LDH activity was significantly increased in a dose-related fashion after injection of EA into MCA, which was due to the neuron injury secondary to focal cerebral ischemia caused by EA. It is well known that serum SOD activity decreases and MDA production increases in patient with acute ischemic stroke. It is confirmed that lipid peroxides produced in focal cerebral ischemia cause the cerebral damage and play a crucial role in pathogenesis of focal cerebral ischemia (Aygul, Kotan, Demirbas, Ulvi, & Deniz, 2006; Chen et al., 2011; Hall & Braughler, 1989; Tomizawa et al., 2005). Therefore, MDA level and SOD activity are used as the biomarkers of oxidative stress under the status of focal cerebral ischemia. Our study illustrated that SOD activity decreased and MDA level increased in serum of rats with focal cerebral
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Fig. 2. Brain weight to body weight ratio of rats with focal cerebral ischemia induced by EA. After injecting the different doses of EA to rats for 24 h (A) or at the indicated time after injecting 1.5 mg/mL EA to rats (B), brains were removed and weighed immediately. The brain weight to body weight ratio was calculated according to the formula [brain weight to body weight ratio (%) = (brain weight/body weight) × 100]. Data was expressed as mean ± S.D. (n = 8–10). *P b 0.05, compared with sham group.
Fig. 3. Dose–effect relationship of the cerebral infarction size of focal cerebral ischemia induced by EA. After injecting the different doses of EA to rats for 24 h, brains were removed and sliced. Then, the slices were stained with TTC. The unstained area (pale) was the infarcted area, while the stained area (red) was the normal area. The cerebral infarction size was presented as the infarction ratio, which was calculated according to the formula [infarction ratio (%) = (infarction volume/whole brain volume) × 100]. (A) the representative brain slices, (B) the statistical results. Data was expressed as mean ± S.D. (n = 8–10). *P b 0.05, compared with sham group.
Please cite this article as: Pang, X., et al., Ellagic acid-induced thrombotic focal cerebral ischemic model in rats, Journal of Pharmacological and Toxicological Methods (2014), http://dx.doi.org/10.1016/j.vascn.2014.01.001
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Fig. 4. Time-effect relationship of the cerebral infarction size of focal cerebral ischemia induced by EA. After injecting 1.5 mg/mL EA to rats for the indicated time, brains were removed and sliced. Then, the slices were stained with TTC. The cerebral infarction size was presented as the infarction ratio, which was calculated according to the formula [infarction ratio (%) = (infarction volume/whole brain volume) × 100]. Data was expressed as mean ± S.D. (n = 8–10). *P b 0.05, compared with sham group.
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present experiment. Zhang et al. injected thrombin plus blood clot into MCA to induce focal cerebral ischemia (Zhang et al., 1997). They use the expensive and complicated methods in the model, e.g. MRI, laser-Doppler flowmetry etc. In fact, this model is a merged model of thrombosis and embolism. Moreover, performance for focal cerebral ischemia is more complicated than our model. Therefore, the model is not suitable for the experimental study of the cerebral ischemia in animals. Since the model is induced by thrombin plus blood clot, it is more suitable for scanning of thrombin inhibitor, and not suitable for evaluation of inhibitor of other coagulation factors over thrombin in coagulation process, such as coagulation factor X inhibitor. In the present model, we injected EA into MCA to induce focal cerebral ischemia in rats. This model is similar to human cerebral ischemia in pathogenesis. It may be used both for pathological study of ischemic stroke and for pharmacological evaluation of drugs against the thromboembolic cerebrovascular diseases, including anti-thrombotic drug and thrombolytic drug. Moreover, this model exhibits good reproducibility and relatively low death rate of rats. But, the model is not suitable for study of the cerebral ischemia and reperfusion in comparison with MCAO model. Collectively, it is recommended that 1.5 mg/mL EA is injected into MCA of rats and all the parameters were examined 24 h after the injection. In addition, adult male SD rats were fasted for 24 h before the
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This model is not also suitable for the study of thrombolytic and antithrombotic drugs. Hara et al. previously injected EA into the common carotid artery of rats to induce the cerebral ischemia for observing effect of thrombin inhibitor (Hara et al., 1994). They do not systematically evaluate the model. Moreover, this is a global ischemic model, which is different from the focal cerebral ischemic model established in the
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Fig. 6. Changes of serum LDH activity in rats with focal cerebral ischemia induced by EA. After injecting the different doses of EA to rats for 24 h, blood were collected for preparation of serum. Then, LDH activity was determined at 450 nm by spectrophotometry. Data was expressed as mean ± S.D. (n = 8–10). *P b 0.05, compared with sham group.
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Fig. 5. Morphological changes of brain in rats with focal cerebral ischemia induced by EA. After injecting 1.5 mg/mL EA to rats for 24 h, brains were removed and fixed in 10% formalin. Then, the brain tissue was embedded in paraffin and sectioned at 4 μm thickness in the coronal plane. The sections were stained with haematoxylin and eosin, and examined under light microscopy (×400).
Fig. 7. Changes of serum SOD activity in rats with focal cerebral ischemia induced by EA. After injecting the different doses of EA to rats for 24 h, blood were collected for preparation of serum. Then, SOD activity was determined at 550 nm by spectrophotometry. Data was expressed as mean ± S.D. (n = 8–10). *P b 0.05, compared with sham group.
Please cite this article as: Pang, X., et al., Ellagic acid-induced thrombotic focal cerebral ischemic model in rats, Journal of Pharmacological and Toxicological Methods (2014), http://dx.doi.org/10.1016/j.vascn.2014.01.001
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experiment and body temperature of rats was maintained at 37.0 °C during the surgery.
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Fig. 8. Changes of serum MDA concentration in rats with focal cerebral ischemia induced by EA. After injecting the different doses of EA to rats for 24 h, blood were collected for preparation of serum. Then, MDA concentration was determined at 532 nm by spectrophotometry. Data was expressed as mean ± S.D. (n = 8–10). *P b 0.05, compared with sham group.
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Contents lists available at ScienceDirect
Journal of Pharmacological and Toxicological Methods journal homepage: www.elsevier.com/locate/jpharmtox
Original article
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Ellagic acid-induced thrombotic focal cerebral ischemic model in rats
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Xiaoming Pang 1, Tianxia Li 1, Liuxin Feng, Jingjing Zhao, Xiaolu Zhang, Juntian Liu ⁎
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Department of Pharmacology, Xi'an Jiaotong University School of Medicine, Xi'an, China
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Introduction: Ischemic stroke is a common cause of human disability and death. Animal models of focal cerebral ischemia are widely utilized to mimic human ischemic stroke. Although models of focal cerebral ischemia have been well established, very few evidence is based on triggering the intrinsic coagulation system to induce focal cerebral ischemia. Ellagic acid (EA) has been identified to trigger the intrinsic coagulation system via activating coagulation factor XII. However, it remains unknown whether EA can serve as a novel pharmacological approach to induce a new model of focal cerebral ischemia in rats. Methods: EA was used for inducing focal cerebral ischemia in adult rats. The dose- and time-dependent effects of EA were characterized. The cerebral infarction ratio was determined with triphenyltetrazolium chloride staining, and the histopathological analysis of the brain tissue was performed under light microscopy. The neurological deficit score was evaluated by a modified method of Bederson. Malondialdehyde (MDA) level and lactate dehydrogenase (LDH) and superoxide dismutase (SOD) activities in serum were determined by spectrophotometry. Results: Injection of EA into the middle cerebral artery of rats was able to generate focal cerebral infarction and increased the neurological deficit score and the brain weight to body weight ratio in dose- and time-dependent manners. Furthermore, EA raised serum LDH activity and MDA level and decreased serum SOD activity in a dose-related fashion. Discussion: This is the first evidence to show that EA induces focal cerebral ischemia in rats, which is similar to human ischemia stroke in pathogenesis. This model holds promise for pathological, pharmacological and clinical studies of ischemic stroke. © 2014 Published by Elsevier Inc.
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Article history: Received 18 August 2013 Accepted 2 January 2014 Available online xxxx
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Keywords: Animal model Ellagic acid Focal cerebral ischemia Stroke
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1. Introduction
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Stroke is a heterogeneous disease with a complex pathophysiology and causes death and disability of patients as well as a significantly economic burden. With the fact that life expectancy increases, stroke is projected to be a more challenging disease in the future (Mukherjee & Patil, 2011). Ischemic stroke which is caused by an embolic or thrombotic occlusion of an artery supplying a specific territory of brain accounts for 80% of all strokes. Animal models of focal cerebral ischemia have been developed to mimic human stroke and play a major role in the experimental study of stroke (Durukan & Tatlisumak, 2007). Over the past years, animal models have largely advanced our knowledge on the pathophysiology and etiology of ischemic stroke (Mergenthaler, Dirnagl, & Meisel, 2004). However, due to the heterogeneity of human stroke, most translational stroke trials that aim to introduce basic experimental findings into clinical therapeutic strategies have failed, which is mainly attributed to ischemic stroke models not simulating pathogenesis of human stroke.
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Abbreviations: MCAO, middle cerebral artery occlusion; EA, ellagic acid; TTC, 2,3,5triphenyltetrazolium chloride; MDA, malondialdehyde; LDH, lactate dehydrogenase; SOD, superoxide dismutase; MCA, middle cerebral artery; BBB, blood brain barrier. ⁎ Corresponding author at: Postbox 58, Xi'an Jiaotong University School of Medicine, 76 West Yanta Road, Xi'an 710061, China. Tel./fax: +86 29 82655188. E-mail address: [email protected] (J. Liu). 1 Co-first author.
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The focal cerebral ischemia model commonly used in the animal experiment is the middle cerebral artery occlusion (MCAO) model via intraluminal suture, which is developed in rats by Koizumi et al. and, is subsequently modified (Belayev, Alonso, Busto, Zhao, & Ginsberg, 1996; Longa, Weinstein, Carlson, & Cummins, 1989). By controlling the suture, both transient ischemia and permanent ischemia are performed using this model. However, the intraluminal suture in MCAO model varies largely from most human focal cerebral ischemia because it is actually a mechanical occlusion model. Thromboembolism is one of the most frequent causes of ischemic stroke in humans. Therefore, development of thromboembolic focal cerebral ischemia models will serve as a unique approach to mimic human stroke, which exhibits an advantage over other stroke models. Although thromboembolic models have previously been reported (Kudo, Aoyama, Ichimori, & Fukunaga, 1982; Zhang et al., 1997), few models are developed based on triggering the intrinsic coagulation system to induce thrombosis in vivo. Ellagic acid (EA), 2,3,7,8-tetrahydroxy-chromeno [5,4,3-cde] chromene-5, 10-dione, is a plant phenol found in various fruits. It has been confirmed that EA triggers the intrinsic coagulation via activating coagulation factor XII, and promotes platelet aggregation by thrombin (Damas, Remacle-Volon, & Adam, 1989; Ratnoff & Crum, 1964), which have been applied in inducing the hypercoagulable state (Botti, 1966; Girolami, Cella, Burul, & Zucchetto, 1976; Iomhair & Lavelle, 1996; Ratnoff & Saito, 1982; Shiozaki et al., 1994). Hara et al. first established the global cerebral ischemia by injection of EA suspension into the common carotid artery of rats, and conducted a primary evaluation
1056-8719/$ – see front matter © 2014 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.vascn.2014.01.001
Please cite this article as: Pang, X., et al., Ellagic acid-induced thrombotic focal cerebral ischemic model in rats, Journal of Pharmacological and Toxicological Methods (2014), http://dx.doi.org/10.1016/j.vascn.2014.01.001
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2.1. Chemical reagents
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EA was purchased from Sigma (St. Louis, MO, USA), and dissolved in 1 M NaOH, and then pH in the solution was adjusted to 7.4 with 1 M HCl. 2,3,5-Triphenyltetrazolium chloride (TTC) was obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Assay kits for detecting malondialdehyde (MDA), lactate dehydrogenase (LDH) and superoxide dismutase (SOD) were provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
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2.2. Animals
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Male Sprague–Dawley rats weighing 280–320 g were purchased from the Experimental Animal Center of Xi'an Jiaotong University School of Medicine, Xi'an, China. Animals were housed at 20–25 °C with a relative humidity of 50%–60% and a 12 h light/dark cycle. Food and water were provided ad libitum. Rats were fasted for 24 h before the experiment. All the experimental procedures carried out in this study were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Xi'an Jiaotong University, and approved by the Institutional Animal Care Committee of Xi'an Jiaotong University.
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2.3. Establishment of the focal cerebral ischemia model in rats
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2.4. Experimental protocol
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In order to present a comprehensive evaluation on the model, we examined dose- and time-dependent characteristics of EA-induced focal cerebral ischemia. In the dose–effect experiment, rats were randomly divided into the sham group and three EA-treated groups (0.5, 1.0, 1.5 mg/mL; n = 8–10). The neurological deficit score, the cerebral infarction size, the brain weight to body weight ratio, LDH, SOD, MDA in serum and morphological changes were evaluated at 24 h after the injection of EA or saline. In the time-effect experiment, rats were randomly divided into the sham group and EA-treated group
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The surgical process of the focal cerebral ischemia model induced by EA was modified according to Zhang's thrombotic focal cerebral ischemia model (Zhang et al., 1997) and Longa's MCAO model (Longa et al., 1989). Rats were anesthetized with intraperitoneal injection of chloral hydrate (300 mg/kg). The left common carotid artery was exposed at the level of the external and internal carotid artery bifurcation under the sterile condition. Catheter (0.3 mm, O.D.) filled with EA or saline was inserted into the left common carotid artery and gently advanced into the internal carotid artery for a length of about 20–21 mm until a slight resistance was felt. Such resistance indicated that the catheter just passed over the proximal segment of the anterior cerebral artery. Then, the catheter was pulled back 2 mm and at this point, the intraluminal catheter was about 2 mm to the origin of the middle cerebral artery (MCA). Next, 0.1 mL EA was slowly injected into MCA of rats through the catheter in the model group (EA-treated group), while 0.1 mL normal saline was given to rats in the sham group in an identical fashion to the EA-treated group instead. Finally, the catheter was pulled out 5 min after the injection. During the whole process of animal surgery, the rat rectal temperature was maintained at 37.0 °C with a heat lamp.
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In the end of each experiment, rats were sacrificed by decapitation under the deep anesthesia with chloral hydrate. Brains were removed, and weighed immediately. The brain weight to body weight ratio was calculated according to the following formula:
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The neurological deficit score was recorded 6 h after the injection of EA in the dose-effect experiment, and at the indicated time after the injection of EA in the time-effect experiment. The neurological status of each rat was carefully evaluated by an observer who was blinded to the experimental procedure following a modified method of Bederson (Bederson et al., 1986). ① Rats were gently held by the tail, suspended one meter above the floor, and observed for forelimb flexion. Normal rats extended both forelimbs toward the floor. Rats that consistently flexed the forelimb contralateral to the injured hemisphere were scored as 4, otherwise scored as 0. ② Rats were pushed in the contralateral direction and scored as: 0 (resistance to lateral push), 1 (initially reduced but progressive resistance), 2 (reduced resistance), or 3 (lateral down fall). ③ Rats were then allowed to move freely to observe circling behavior. The movement was scored as: 0 (straight movement), 1 (movement to the right), 2 (circling movement), or 3 (no movement). The scores of three tests were summed and represented the neurological deficit score (0 to 10).
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Brain weight to body weight ratio ð%Þ ¼ ðbrain weight=body weightÞ 100
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(1.5 mg/mL; n = 8–10). The above-described measurements were 140 analyzed 3, 6, 12, 24 h after the injection of EA or saline. 141
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(Hara, Iwamoto, Ishihara, & Tomikawa, 1994). However, there is no study to identify whether EA induces focal cerebral ischemia and the comprehensive evaluation. The aim of the present study was to develop a stable EA-induced focal cerebral ischemia model similar to human thrombotic ischemia stroke in rats.
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2.7. Detection of cerebral infarction size
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Brains were removed and sliced into six 2-mm-thick coronal slices (first to sixth from rostral to caudal). The brain slices were incubated in 2% phosphate buffer solution of TTC for 30 min at 37 ºC, and immersed in 10% formalin overnight. The unstained area (pale) was the infarcted area, while the stained area (red) was the normal area. The slices were photographed with a digital camera and the infarction volume was calculated by summing the infarction volume of sequential sections with a commercial image processing software program (Photoshop, Adobe Systems; Mountain View, CA, USA). The investigator who analyzed the image was blinded to the experimental groups. The cerebral infarction size was presented as the infarction ratio, which was calculated according to the following formula:
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Infarction ratio ð%Þ ¼ ðinfarction volume=whole brain volumeÞ 100 181 180
2.8. Determination of MDA, LDH and SOD
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Blood was collected via the abdominal aorta in the end of the experiment. Then, the separated serum was used to spectrophotometrically determine LDH activity at 450 nm, SOD activity at 550 nm and MDA concentration at 532 nm following the manufacturer's instructions.
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Brains were removed and fixed in 10% formalin in the end of the experiment. Then, the brain tissue was embedded in paraffin and sectioned at 4 μm thickness in the coronal plane. The sections were stained with haematoxylin and eosin, and examined under light microscopy.
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3.1. Neurological deficit of rats with focal cerebral ischemia induced by EA
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The result in Fig. 1A showed that the neurological deficit score of rats was increased in a dose-dependent manner 24 h after the injection of EA. Compared with the sham group, there is a significant difference in the neurological deficit score in rats treated with 1 mg/mL and 1.5 mg/mL EA (P b 0.05). Fig. 1B displayed that the neurological deficit score of rats was significantly increased 3 h after the injection of 1.5 mg/mL EA, reached a maximum at 6 h and sustained for 24 h (P b 0.05).
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3.2. Brain weight to body weight ratio of rats with focal cerebral ischemia induced by EA
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Fig. 2A exhibited that the brain weight to body weight ratio was dose-dependently raised after rats were treated with EA for 24 h as compared to the sham group (P b 0.05). Fig. 2B indicated that the brain weight to body weight ratio of rats was raised 3 h after the injection of 1.5 mg/mL EA, and sustained for more than 24 h (P b 0.05).
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3.3. Cerebral infarction ratio of rats with focal cerebral ischemia induced by EA
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As shown in Fig. 3, the cerebral infarction ratio was enlarged in a dose-dependent manner 24 h after injecting EA to rats (P b 0.05). At 0.5 mg/mL EA, infarction foci were sporadic and spotty. At 1.0 mg/mL EA, the cerebral hemisphere showed patchy necrosis and the cerebral infarction ratio reached 18%. At 1.5 mg/mL EA, most of the cerebral hemisphere exhibited severe infarction and the cerebral infarction ratio reached 29.9 ± 1.9%. Likewise, EA time-dependently enlarged the cerebral infarction ratio of rats (P b 0.05). The cerebral infarction ratio was obviously raised 3 h after injecting 1.5 mg/mL EA (P b 0.05), and slowly increased after 12 h (P b 0.05) (Fig. 4).
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As seen from Figs. 6–8, all the concentrations of EA increased 234 LDH activity and decreased SOD activity in serum (P b 0.05), whereas 235 middle and high concentrations of EA increased MDA level (P b 0.05). 236
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The histopathological analysis showed that MCA injection of EA to rats caused the morphological abnormalities, including degeneration
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In the clinical condition, coagulation factor XII is activated by collagen in the vessel wall once endothelium is injured by the pathological factors, such as hypertension and ox-LDL. The activated coagulation factor XII is able to trigger the intrinsic coagulation system to form fibrin. Meanwhile, platelets are also activated by thrombin from the activated coagulation system. Finally, combination of fibrin and activated platelets participates in formation of the mixed thrombosis. Many studies demonstrate that EA is an effective activator of coagulation factor XII. Therefore, we attempted to use EA to induce thrombosis in MCA and to develop a new model of focal cerebral ischemia in rats. The present study showed that injection of EA into MCA was able to produce focal cerebral infarction in rats, increased the neurological deficit score and the brain weight to body weight ratio in dose- and time-dependent manners. EA also raised LDH activity and MDA level, but decreased SOD activity in serum in a dose-related fashion. The cerebral infarction size has widely been accepted as the most precise and objective indicator in study of the animal ischemic stroke (Durukan & Tatlisumak, 2007). In this study, treatment of rats with injection of EA via MCA was able to generate focal cerebral infarction in both dose-dependent manner and time-dependent way. The cerebral infarction ratio reached 29.9% after the injection of 1.5 mg/mL EA for 24 h, which is comparable to the infarction size from MCAO model by intraluminal suture (Xiao, Liu, Hu, Li, & Zhang, 2007). The results from the histopathological examination provided further evidence in support of the cerebral damage in focal cerebral ischemia induced by EA. In terms of the infarction position and pattern, the cerebral ischemia exists in the territory supplied by MCA, suggesting that EA is likely to occlude large branch of MCA, and spontaneous reperfusion and collateral perfusion do not exist possibly at 24 h after injection of EA. Cerebral ischemia may cause neurological abnormalities, the severity of which is correlated with the cerebral infarction size. Therefore, the neurological deficit was used for reflecting formation and severity of focal cerebral infarction. The present results showed that the injection of EA to rats significantly increased the neurological deficit score.
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3.4. Morphological changes of brain in rats with focal cerebral ischemia induced by EA
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and necrosis of neurons, focal cerebral edema, infiltration of inflamma- 229 tory cells as well as dilated capillaries in comparison with the sham 230 group (Fig. 5). 231
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Fig. 1. Neurological deficit of rats with focal cerebral ischemia induced by EA. The neurological deficit score was evaluated 6 h after injecting the different doses of EA to rats (A) or at the indicated time after injecting 1.5 mg/mL EA to rats (B) according to a modified Bederson's method with a scale of 0–10. Data was expressed as mean ± S.D. (n = 8–10). *P b 0.05, compared with sham group.
Please cite this article as: Pang, X., et al., Ellagic acid-induced thrombotic focal cerebral ischemic model in rats, Journal of Pharmacological and Toxicological Methods (2014), http://dx.doi.org/10.1016/j.vascn.2014.01.001
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ischemia induced by EA, implying the involvement of lipid peroxidation in EA-induced focal cerebral ischemia. At present, the majority of studies on stroke are conducted in small animals due to lower cost and greater acceptability from ethical viewpoints. The rat is an ideal subject in study of stroke because of its similarity to human in cerebrovascular anatomy and physiology (Mhairi Macrae, 1992; Tamura, Graham, McCulloch, & Teasdale, 1981). In order to avoid the variability caused by female hormone cycling and weight differences, we chose male SD rats weighing 280 to 320 g as study subjects in the present study. Body temperature and blood glucose are two important factors influencing focal cerebral ischemia. It is documented that body temperature is able to affect stroke severity, infarct size, mortality, and outcome (Castillo, Davalos, Marrugat, & Noya, 1998; MacLellan, Davies, Fingas, & Colbourne, 2006; Reith et al., 1996). During the process of the animal surgery, the rat rectal temperature was maintained with a heat lamp at 37.0 °C. The majority of studies had shown that hyperglycemia worsens the ischemic damage (Huang, Wei, & Quast, 1996; Nedergaard, 1987). Therefore, rats were fasted for 24 h before the surgery in order to minimize variability resulted from blood glucose levels in this study. Many animal models of focal cerebral ischemia have been developed, and current translational models do not adequately simulate the pathogenesis of human cerebral ischemia. At present, focal cerebral ischemia model most commonly used is the MCAO model using an intraluminal suture. However, this is a mechanical occlusion model, which may differ from the human cerebral ischemia in its pathogenesis.
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Moreover, all rats with the cerebral infarction ratio greater than 20% revealed a significant neurological deficit, which is consistent with Bederson's report (Bederson et al., 1986). Blood brain barrier (BBB) is a separation of circulating blood from brain and cerebrospinal fluid in the central nervous system. Focal cerebral ischemia disrupts BBB, which results in cerebral edema. In the study, the brain weight to body weight ratio was assessed to represent the degree of BBB damage and cerebral edema in focal cerebral ischemia induced by EA. The results indicated that the brain weight to body weight ratio was increased 3 h after the injection of EA and sustained for 24 h, suggesting the existence of BBB damage and cerebral edema in focal cerebral ischemia induced by EA. LDH is one of intracellular enzymes. When neurons are injured, LDH is released into blood. Therefore, serum LDH activity is often used as a marker of the cell injury. The present experiment revealed that serum LDH activity was significantly increased in a dose-related fashion after injection of EA into MCA, which was due to the neuron injury secondary to focal cerebral ischemia caused by EA. It is well known that serum SOD activity decreases and MDA production increases in patient with acute ischemic stroke. It is confirmed that lipid peroxides produced in focal cerebral ischemia cause the cerebral damage and play a crucial role in pathogenesis of focal cerebral ischemia (Aygul, Kotan, Demirbas, Ulvi, & Deniz, 2006; Chen et al., 2011; Hall & Braughler, 1989; Tomizawa et al., 2005). Therefore, MDA level and SOD activity are used as the biomarkers of oxidative stress under the status of focal cerebral ischemia. Our study illustrated that SOD activity decreased and MDA level increased in serum of rats with focal cerebral
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Fig. 2. Brain weight to body weight ratio of rats with focal cerebral ischemia induced by EA. After injecting the different doses of EA to rats for 24 h (A) or at the indicated time after injecting 1.5 mg/mL EA to rats (B), brains were removed and weighed immediately. The brain weight to body weight ratio was calculated according to the formula [brain weight to body weight ratio (%) = (brain weight/body weight) × 100]. Data was expressed as mean ± S.D. (n = 8–10). *P b 0.05, compared with sham group.
Fig. 3. Dose–effect relationship of the cerebral infarction size of focal cerebral ischemia induced by EA. After injecting the different doses of EA to rats for 24 h, brains were removed and sliced. Then, the slices were stained with TTC. The unstained area (pale) was the infarcted area, while the stained area (red) was the normal area. The cerebral infarction size was presented as the infarction ratio, which was calculated according to the formula [infarction ratio (%) = (infarction volume/whole brain volume) × 100]. (A) the representative brain slices, (B) the statistical results. Data was expressed as mean ± S.D. (n = 8–10). *P b 0.05, compared with sham group.
Please cite this article as: Pang, X., et al., Ellagic acid-induced thrombotic focal cerebral ischemic model in rats, Journal of Pharmacological and Toxicological Methods (2014), http://dx.doi.org/10.1016/j.vascn.2014.01.001
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Fig. 4. Time-effect relationship of the cerebral infarction size of focal cerebral ischemia induced by EA. After injecting 1.5 mg/mL EA to rats for the indicated time, brains were removed and sliced. Then, the slices were stained with TTC. The cerebral infarction size was presented as the infarction ratio, which was calculated according to the formula [infarction ratio (%) = (infarction volume/whole brain volume) × 100]. Data was expressed as mean ± S.D. (n = 8–10). *P b 0.05, compared with sham group.
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present experiment. Zhang et al. injected thrombin plus blood clot into MCA to induce focal cerebral ischemia (Zhang et al., 1997). They use the expensive and complicated methods in the model, e.g. MRI, laser-Doppler flowmetry etc. In fact, this model is a merged model of thrombosis and embolism. Moreover, performance for focal cerebral ischemia is more complicated than our model. Therefore, the model is not suitable for the experimental study of the cerebral ischemia in animals. Since the model is induced by thrombin plus blood clot, it is more suitable for scanning of thrombin inhibitor, and not suitable for evaluation of inhibitor of other coagulation factors over thrombin in coagulation process, such as coagulation factor X inhibitor. In the present model, we injected EA into MCA to induce focal cerebral ischemia in rats. This model is similar to human cerebral ischemia in pathogenesis. It may be used both for pathological study of ischemic stroke and for pharmacological evaluation of drugs against the thromboembolic cerebrovascular diseases, including anti-thrombotic drug and thrombolytic drug. Moreover, this model exhibits good reproducibility and relatively low death rate of rats. But, the model is not suitable for study of the cerebral ischemia and reperfusion in comparison with MCAO model. Collectively, it is recommended that 1.5 mg/mL EA is injected into MCA of rats and all the parameters were examined 24 h after the injection. In addition, adult male SD rats were fasted for 24 h before the
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This model is not also suitable for the study of thrombolytic and antithrombotic drugs. Hara et al. previously injected EA into the common carotid artery of rats to induce the cerebral ischemia for observing effect of thrombin inhibitor (Hara et al., 1994). They do not systematically evaluate the model. Moreover, this is a global ischemic model, which is different from the focal cerebral ischemic model established in the
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Fig. 6. Changes of serum LDH activity in rats with focal cerebral ischemia induced by EA. After injecting the different doses of EA to rats for 24 h, blood were collected for preparation of serum. Then, LDH activity was determined at 450 nm by spectrophotometry. Data was expressed as mean ± S.D. (n = 8–10). *P b 0.05, compared with sham group.
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Fig. 5. Morphological changes of brain in rats with focal cerebral ischemia induced by EA. After injecting 1.5 mg/mL EA to rats for 24 h, brains were removed and fixed in 10% formalin. Then, the brain tissue was embedded in paraffin and sectioned at 4 μm thickness in the coronal plane. The sections were stained with haematoxylin and eosin, and examined under light microscopy (×400).
Fig. 7. Changes of serum SOD activity in rats with focal cerebral ischemia induced by EA. After injecting the different doses of EA to rats for 24 h, blood were collected for preparation of serum. Then, SOD activity was determined at 550 nm by spectrophotometry. Data was expressed as mean ± S.D. (n = 8–10). *P b 0.05, compared with sham group.
Please cite this article as: Pang, X., et al., Ellagic acid-induced thrombotic focal cerebral ischemic model in rats, Journal of Pharmacological and Toxicological Methods (2014), http://dx.doi.org/10.1016/j.vascn.2014.01.001
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experiment and body temperature of rats was maintained at 37.0 °C during the surgery.
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Fig. 8. Changes of serum MDA concentration in rats with focal cerebral ischemia induced by EA. After injecting the different doses of EA to rats for 24 h, blood were collected for preparation of serum. Then, MDA concentration was determined at 532 nm by spectrophotometry. Data was expressed as mean ± S.D. (n = 8–10). *P b 0.05, compared with sham group.
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Please cite this article as: Pang, X., et al., Ellagic acid-induced thrombotic focal cerebral ischemic model in rats, Journal of Pharmacological and Toxicological Methods (2014), http://dx.doi.org/10.1016/j.vascn.2014.01.001
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