Effect of Salvia Miltiorrhiza Bunge on cerebral infarct in ischemia-reperfusion injured rats

Effect of Salvia Miltiorrhiza Bunge on cerebral infarct in ischemia-reperfusion injured rats

Chih-Jui Lao

Abstract: According to the theory of traditional Chinese medicine, cerebral infarction results from blood stasis, and the method of quickening the blood and dispelling stasis is used to treat cerebral infarct. Salvia Miltorrhiza Bunge (SM) is a Chinese herb, which is considered to have an action of quickening the blood and dispelling stasis, and is frequently used to treat related disorders of blood stasis such as cerebrovascular accident and ischemic heart disease. The aim of the present study was to investigate the effect of SM on cerebral infarct in ischemia-reperfusion injured rats. A total of 30 Sprague-Dawley (SD) rats were studied. A model of focal cerebral infarct was developed by occluding both common carotid arteries and the right middle cerebral artery for 90 minutes. After 24 hours reperfusion, the rats were killed and the brain tissue was stained with 2, 3, 5-triphenyl-tetrazolium chloride (TTC). The areas of cerebral infarct were calculated, and lumino-chemiluminesence (CL) counts and lucigenin-CL counts of peripheral blood taken at this time were measured. The changes in the area of cerebral infarct were used as an index to evaluate the effect of SM on cerebral infarct. The results indicated that pretreatment with intraperitoneal injection of 30 mg/kg and 15 mg/kg SM reduced the area of cerebral infarct and also reduced the luminol-CL counts of peripheral blood in ischemia-reperfusion injured rats. This study has demonstrated that SM can reduce the area of cerebral infarct in ischemia-reperfusion injured rats, suggesting it may be useful in the treatment of cerebral infarct in humans. The therapeutic effect of SM may be partly due to its free radical scavenging activities.

Keywords: Salvia Miltorrhiza Bunge; Cerebral Infarct; Ischemia-reperfusion; Free Radicals.

Introduction

In traditional Chinese medicine, the method of quickening the blood and dispelling stasis is used to treat cerebrovascular accident and ischemic heart disease because the main etiology of these diseases are considered closely related to blood stasis (Sun, 1992). Salvia miltiorrhiza bunge (SM) is a Chinese herb, which is considered to have the action of quickening the blood and dispelling stasis. Therefore, it is frequently used to treat cerebrovascular accident or ischemic heart disease (Sun, 1992). Study of Chen (1996) has shown that SM may improve microcirculation, blood viscosity and the flexibility of erythrocytes in rabbits or in rats with blood stasis.

The occlusion of the middle cerebral artery from its origin to its junction with the inferior cerebral vein, or from 2 mm proximal to the olfactory tract to the inferior cerebral vein has been previously used to establish a model of focal cerebral infarct in Sprague-Dawley (SD) rats (Bederson et al., 1986; Ginsberg and Busto, 1989). In addition, the cerebral infarct size may be calculated with 2, 3, 5-triphenyl-tetrazolium chloride (TTC) staining (Schaarschmidt et al., 1997; Yang et al., 1998). Several studies have concluded that ischemic brain damage mainly results from the production of superoxide anion radicals in the reperfusion period (McCord, 1985; Hallenbeck and Dutka, 1990; Nelson et al., 1992; Chan, 1996). In addition, free radical generation during ischemia plays a critical role in triggering ischemic neuronal damage, which causes neuronal death, and this damage occurs selectively in vulnerable regions of the brain (Kitagawa et al., 1990). The superoxide dismutase (SOD) activity in serum is reduced in patients with acute cerebral ischemic injury (Spranger et al., 1997), and in rats with middle cerebral artery occlusion (Michowiz et al., 1990). In addition, antioxidants such as alpha-tocopherol and pyridoindole stobadine may be effective in preventing hippocampus reoxygenation injury (Horakova et al., 1997).

The aim of the present study was to determine the effect of SM on focal cerebral infarct and to investigate its mechanisms of action. We established a model of focal cerebral infarct in ischemia-reperfusion injured rats by occluding the common carotid arteries bilaterally and the right middle cerebral artery for 90 minutes. After 24 hours of reperfusion, the rats were sacrificed, the brain tissue was stained with TTC, and the infarct size was calculated using an image-analysis system. The luminol-chemiluminesence (CL) and lucigenin-CL counts of peripheral blood were also measured simultaneously.

Materials and Methods

Extraction of SM

The SM used in this study originated in the Sichuan province of China. SM was collected in crude form in Taiwan, and authenticated by the Institute of Chinese Pharmacy, China Medical College, and by the high performance liquid chromatography (HPLC) system (Hitachi Instruments Service Co., Ltd., Interface D-700, Pump L-7100, UV-Vis Detector L-7420, Ibaraki-Ken, Japan) using Tanshinone-IIA (Koda Pharmaceutical Com. Ltd., Taoyuan, Taiwan) as a standard in the Koda Pharmaceutical Com. Ltd. (Taoyuan, Taiwan). Three kilograms of crude SM was extracted twice with 12 liters of water. The extracts were filtered, freeze-dried, and then stored in a drier box. The total yield was 639.64 g (21.32%) of SM.

Animal

Adult male SD rats, weighing 350-400 g, were housed in iron cages and maintained on a 12-hour light-dark cycle at 25[degrees]C. All animal experiments were undertaken in accordance with the Guidelines Principles for the Care and Use of Laboratory Animals.

Establishment of an Animal Model

A total of 30 SD rats were used in the present study. The rats were anesthetized with an intraperitoneal injection (i.p.) of chloral hydrate (400 mg/kg). The rat’s blood pressure and heart rate were monitored by a heart rate-blood pressure measuring apparatus (LE 5001 pressure meter, Panlab. S. L. L., Barcelona, Spain), and body temperature was maintained at 37 [+ or -] 0.5[degrees]C with a heated pad throughout the experimental procedure. The experimental procedure was divided into two steps. First, the rats were placed in a supine position and the common carotid arteries were exposed through a midline incision in the neck. Then both arteries were wrapped by a loop of plastic line (0.1 mm in diameter) and a PE-50 tube (0.2 mm in diameter), respectively. Second, the head of each rat was fixed in a stereotactic apparatus in a prone position. The scalp was incised to create a wound (1.5 cm in length) from the midpoint of the binaural line, then a bone window 3.5 mm in diameter was made after the temporal muscles were separated and the temporal bone was exposed. The olfactory tract and right middle artery were thus clearly visible. Using a nylon line (8-0) placed through a surgical needle, a loose tie was made that was then placed on the right middle cerebral artery just at the upper margin of the olfactory tract. The markers for laser Doppler perfusion were monitored (DRT4, Moor Instruments Inc. Wilmington, USA) down from 900 to 200 when the blood flow of the common carotid arteries was blocked by drawing the loops of plastic line. Then the markers of laser Doppler perfusion were monitored down from 200 to 50 when the blood flow of the right middle cerebral artery was blocked by drawing the loose tie of nylon line. After the blood flow of the common carotid arteries and right middle cerebral artery was blocked for 90 minutes, the blood flow was re-established.

The rats were randomly divided into five groups of six rats as follows: (A) sham group: the common carotid arteries and the right middle cerebral artery were exposed, but the blood flow was not blocked; (B) control group: the blood flow of the common carotid arteries and the right middle cerebral artery was blocked for 90 minutes followed by reperfusion for 24 hours; (C) SM30 group: the methods of blood flow blocking were identical to the control group, but 30 mg/kg SM i.p. (In 1 ml PBS solution) was administrated 30 minutes prior to blocking the blood flow; (D) SM15 group: the methods were identical to the SM30 group, but using 15 mg/kg SM (1n 1 ml PBS solution); and (E) contrast group: the methods were identical to the SM30 group, but 1 ml PBS solution was administrated 30 minutes prior to blocking the blood flow.

Measurement of Infarct Size

After 24 hours reperfusion, whole blood samples (2 ml) were obtained by transcardiac puncture with heparinzed plastic syringes under anesthesia with chloral hydrate (400 mg/kg) i.p., then the brains of the rats were removed after transcardiac perfusion of 0.9% NaCl and 4% Paraformaldehyde. The brain of each rat was sectioned in the coronals plane into 2 mm thickness pieces using a plastic model of the rat brain. The samples were then placed in 2% TTC solution in a 37[degrees]C room for 15 minutes allowing the white cerebral infarct area and the red-purple normal brain tissue to be differentiated clearly. Finally the samples were fixed by 10% formalin solution.

The cerebral infarct areas of the first six pieces from the frontal lobe were measured using an image-analysis system (Image-Pro Lito Version 3.0, Media Cybernetics, USA). The ratio of infarct area to total brain area in each piece of rat brain was calculated, and the data were represented as a percentage (%).

Measurement of Free Radicals

The blood samples were immediately wrapped with aluminum foil to prevent light exposure and kept in an icebox until testing for CL, which was measured within 2 hours of collection. A tube of 1 ml blood with EDTA was used for counting white blood cells (WBC). The method for measuring luminol-CL was similar to that previously described (Sun et al., 1996; Hsieh et al., 1999a and 2000). Briefly, 0.2 ml of whole blood was mixed with 0.1 ml of PBS in a special chamber unit (Model CLD-110, Tohoku Electronic Industrial Co.). The CL was then measured in an absolutely dark chamber of the Chemiluminesence Analyzing System (Tohoku Electronic Industrial Co., Sendai, Japan). This system includes a photon dector (Model CLD-110), a CL counter (Model CLD-110), a water circulator (Model CH-201), and a 32-bit IBM personnel computer. After 200 seconds, 1.0 ml of 25 mM luminol (Sigma Co., USA) in PBS was injected into the stainless steel cell and the CL of the blood sample was measured continuously. After 600 seconds, 0.2 ml of Zymosan-A (Sigma Co., USA) was added, and the CL in the blood sample was measured continuously for a total of 1020 seconds. Integrating the area under the curve and subtracting it from the background level gave the total CL counts. The production of CL per WBC was calculated by dividing the blood CL levels by the WBC count, and expressed as CL/1000 WBC.

The method for measuring lucigenin-CL was similar to that described previously (Sun et al., 1996; Chen et al., 1997; Hsieh et al., 1999a and 2000). Briefly, 0.1 ml PBS (pH 7.40) was added to 0.2 ml of blood. The CL counts were then measured in an absolutely dark chamber of the CL Analyzing System as described above. After 200 seconds, 1.0 ml of 0.01 mM lucigenin (Sigma Co., USA) in PBS was injected into the stainless steel cell and the CL of the blood sample was measured continuously. After 600 seconds, 0.2 ml Zymosan-A was added by the same method, and the CL of the blood sample was measured continuously for a total of 1020 seconds. The total CL and production of CL per WBC were calculated as described above.

Statistical Analysis

The data are represented as mean [+ or -] SD. One-way analysis of variance (ANOVA) followed by Scheffe’s test was used for comparisons among groups, p < 0.05 was considered statistically significant.

Results

The blood flow of both the common carotid arteries and the right middle cerebral artery was blocked by constricting the arteries for 90 minutes in 24 SD rats. We found that all of the rats developed cerebral infarct after 24 hours reperfusion. After TTC staining, the infarct area of the rat brain was visibly white in color, whereas the non-infarct area was a red-purple color (Fig. 1). In the control and contrast groups, neurological deficits of the animal were noted on the side contralateral to the infarct, and included left forelimb flexion, decrease of resistance to lateral push toward the left paretic side, and ambulation in a circle toward the left paretic side.

[FIGURE 1 OMITTED]

Effect of SM on Cerebral Infarct Size in Ischemia-reperfusion Injured Rats

The cerebral infarct area in the SM30 (0.63 [+ or -] 1.03%) and the SM15 (1.63 [+ or -] 2.19%) groups had a lower ratio than that of the control (9.92 [+ or -] 2.56%) and PBS (6.86 [+ or -] 1.45%) groups (All p 0.05, Figs. 1 and 2).

[FIGURE 2 OMITTED]

Effect of SM on Luminol-CL and Lucigenin-CL Counts in Ischemia-reperfusion Injured Rats

In ischemia-reperfusion injured rats, the luminol-CL counts in peripheral blood were lower in the SM30 group than in the control or PBS groups (p < 0.01, p 0.05, Fig. 3).

[FIGURE 3 OMITTED]

In ischemia-reperfusion injured rats, lucigenin-CL counts in peripheral blood showed no significant differences among the sham, control, SM30, SM15 and PBS groups (all p > 0.05, Fig. 3).

Discussion

In the present study, blocking the blood flow of both common carotid arteries and the right middle cerebral artery for 90 minutes developed a rat model of cerebral infarct. Twenty-four hours after reperfusion, this animal model has been shown to be similar to cerebral infarct in humans, and is similar to several previously studied models (Bederson et al., 1986; Ginsberg and Busto, 1989). In this study, neurological deficits similar to those previously reported with this model were found; including left forelimb flexion, decrease of resistance when the rat was pushed laterally toward the left paretic side, and ambulation in a circle toward the left paretic side (Bederson et al., 1986).

Our previous results (Hsieh et al., 2001) and reports of Bederson et al. (1986) have shown that neurological deficit correlated to size of cerebral infarct. The results in the present study indicated that SM30 mg/kg and 15 mg/kg SM i.p. 30 minutes prior to blocking the blood flow of both common carotid arteries and the right middle cerebral artery reduced the area of the cerebral infarct, suggesting that SM may be useful in the treatment of cerebral infarct in humans. Many studies have demonstrated that oxygen-derived free radicals play a critical role in cell death in ischemia-reperfusion tissue injury conditions including ischemic heart disease, cerebral ischemia, and liver and lung diseases (Turrens et al., 1982; McCord, 1985; Werns and Lucchesi, 1990; Henry et al., 1990; Caraceni et al., 1994; Maxwell, 1995). Previous studies have shown that oxygen free radicals including superoxide anion, hydrogen peroxide, hydroxyl radical and nitric oxide play a critical role in ischemia-induced brain damage (Nelson et al., 1992; Chan, 1996). Free radicals develop during the brief period of ischemia, and play an important role in triggering the ischemic neuronal damages causing delayed neuronal death (Kitagawa et al., 1990). In addition, SOD is a major superoxide anion scavenging system, which decreases superoxide activity in patients with acute cerebral infarct (Spranger et al., 1997) and in rats with middle cerebral artery occlusion (Michowiz et al., 1990). Our results indicated that lucigenin-CL counts were not significantly different among the sham group without blocking blood flow, the control group, the SM-treated group and the PBS-treated group. In addition, the luminol-CL counts were lower in the SM30 group than in the control and PBS groups. These results seem to be partly in contradiction because lucigenin-CL counts are primarily sensitive to superoxide anion (Caraceni et al., 1994; Dirnagel et al., 1995; Hsieh et al., 1999a and 2000), whereas the luminol-CL level is an indicator of the production of reactive oxygen species, including superoxide anion, hydroxyl radicals, hydrogen peroxide and hypochlorous acid (Kaever et al., 1992; Sun et al., 1996; Hsieh et al., 1999a and 2000), which can be generated by activated neutrophils (Takahashi et al., 1991).

Our previous study has shown that a stable model of focal cerebral infarct might be established by blocking the blood flow of both common carotid arteries and the right middle cerebral artery for 90 minutes, then re-establishing blood flow for 24 hours (Hsieh et al., 2001). Similar results were also reported by Nakashima et al. (1999). In addition, we found that the time course of wet dog shakes correlated positively with luminol-CL and lucigenin-CL counts in the whole blood in kainic acid-treated rats (Hsieh et al., 1999b), and intracerebral microinjection of kainic acid may induce histopathological and molecular changes similar to those occurring in brain infarct (Jorgensen et al., 1991; Liu et al., 1996). Therefore, we measured luminol-CL and lucigenin counts after 24 hours of blocking the blood flow of both common carotid arteries and the right middle cerebral artery. Mastuo et al. (1995) found that neutrophils are a major source of oxygen radicals during reperfusion after cerebral ischemia. In addition, SM has been reported to inhibit circulating neutrophil function in rabbits with myocardial infarction (Li and Tang, 1991). Propanoic acid is an ingredient of SM and is considered a strong superoxide anion scavenger, with a protective action on lipid peroxidation by oxygen stress in ischemia-reperfusion pig cardiac muscle. It may also improve microcirculation and reduce platelet aggregation (Chi, 1999).

In conclusion, the results of this study demonstrate that SM can reduce the area of cerebral infarct in ischemia-reperfusion injured rats, suggesting it may be useful in the treatment of cerebral infarct in humans. The free radical scavenging activities of SM may partly contribute to its therapeutic effect.

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Chih-Jui Lao, *, ([double dagger]) Jaung-Geng Lin, * Jon-Son Kuo, ([dagger]) Sun-Yin Chiang, * Shu-Chao Chen, ([double dagger]) En-Tzu Liao * and Ching-Liang Hsieh ([section])

* Institute of Chinese Medical Science, China Medical College, Taichung, Taiwan, R.O.C.

([dagger]) Institute of Medical Science, College of Medicine Tzu Chi University, Hualien, Taiwan, R.O.C.

([double dagger]) Lao Chih-Jui Clinics, Taichung, Taiwan, R.O.C.

([section] Internal Medicine Section of Chinese Medicine Department China Medical College Hospital, Taichung, Taiwan, R.O.C. Institute of Integration Chinese and Western Medicine China Medical College, Taichung, Taiwan, R.O.C.

Correspondence to: Dr. Ching-Liang Hsieh, Internal Medicine Section of Chinese Medicine Department, China Medical College Hospital, 2, Yuh-Der Road, Taichung, Taiwan, R.O.C. Tel: (+886) 4-2206-2121 (ext. 5062), Fax: (+886) 4-2206-2121 (ext. 5064), E-mail: clhsieh4321@yahoo.com.tw

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