Neurochemical Research, VoL 16, No. 6, 1991, pp. 687-692
Reduction of Cellular Damage Induced by Cerebral Ischemia in Rats* Albert W. N. Leung 1, Zhixian Mo 2, and Youshan Zheng z (AcceptedJanuary 29, 1991)
A model of incomplete cerebral ischemia involving bilateral ligation of the common carotid arteries in rats, was used to examine the potential of a Chinese herbal preparation and of nifedipine to reduce cell damage following cerebral ischemia. The herbal preparation contained ginsengosides and extracts of Panax notoginseng, Ligusticum chuanxiong Hort., Carthamus tinctorius L. and Salvia militorrhiza Bge. Histological evidence of cell damage and the formation of peroxidation products were both reduced in rats pretreated with the herbal preparation or with nifedipine. It has been suggested that the free radical reaction is involved in tissue damage, particularly in the pathological neurocyte injury of cerebral ishemia. The results show that in this model of incomplete cerebral ischemia, the degree of lipid peroxidation can be lowered by the pretreatment with Chinese herbs containing ginsengosides or with nifedipine. These drugs maybe beneficial in the treatment of cerebral ischemia in humans. KEY WORDS: Cerebral ischemia; malondialdehyde; nifedipine; Chinese herbal ginsengosides.
Nifedipine is a potent vasodilator with effects on the coronary and other peripheral blood vessels. In recent years, the actions of calcium antagonists on the cerebrovasculature have attracted more and more attention. There are now many reports demonstrating that nifedipine relieves cerebral arterial spasm (2,3) and has a brain protective effect (4). According to the Chinese pharmaceutical tradition, the herbal preparation, composed of ginsengosides and extracts of Panax notoginseng, Ligusticum chuanxiong Hort., Carthamus tinctorius L. and Salvia militorrhiza Bge, has actions of increasing blood circulation to dissipate blood stasis, thus benefiting vital energy and invigorating the brain. It has been used in China for hundreds of years to treat cardiovascular and cerebrovascular diseases, such as coronary heart disease, cerebral arteriosclerosis and cerebral function failure. Animal experiments have shown that this herbal preparation dilates small blood vessels and improves microcirculation in rats (5). The aim of present investigation is to compare the actions of nifedipine and the herbal preparation in reducing cellular damage caused by cerebral ischemia.
INTRODUCTION Stroke, one of the commonest causes of death in Western countries, is caused by ischemic cerebral lesions resulting from a reduction in the peffusion of brain tissue by blood. About a fifth of the cardiac output normally passes through the carotid and vertebral arteries to supply the brain and a reduction in total cerebral blood flow to less than half of normal will repair cerebral function (1). The present paper reports attempts to reduce the cellular damage due to cerebral ischemia in a rat model by pretreatment with the calcium channel blocker nifedipine or a Chinese herbal preparation containing ginsengosides. Nifedipine is used in the clinical treatment of various cardiovascular disorders while these Chinese herbs have a long history of use in promoting blood circulation. 1 Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N. T., Hong Kong. Department of Chinese Medicine, First Military Medical College, Guangzhou, China. * Special issue dedicated to Dr. Lawrence Austin.
687
0364-3190/91/0600-0687506.50/09 1991PlenumPublishingCorporation
688 EXERIMENTAL
Leung, Mo, and Zheng PROCEDURE
Nifedipine was kindly donated by Bayer (Germany), 1,1,3,3tetraethoxypropane (TEP) was purchased from Sigma and 2-thiobarbituric acid (TBA) from FIuka. The Chinese herbal preparation was supplied by Hong Xing PharmaceuticalCompany of Guangdong, China. Sprague-Dawley rats of both sexes weighing 200-250 g were used in this study. The animals were divided randomly into the following 4 groups; (1) sham-operation saline control, (2) ischemia saline con-
trol, (3) ischemia pretreated with nifedipine (2.5 rag/100 g -body weight p.o.), and (4) ischemia pretreated with the Chinese herbal preparation at either a low dose (0.2 g/100 g p.o.) or a high dose (0.4 g/100 g p.o.) The p.o. injections were given daily in the morning each day and the same volume of normat saline was given to the control groups. Incomplete cerebral ischemia was established under pentobarbital anesthesia by ligating bilaterally the common carotid arteries, which produces about a 70% reduction in cerebral blood flow. After 15 hours of ischemia the rats were killed by decapitation, the brain was excised,
Fig. 1. Electron micrograph of neuronal cells of ischaemic rat in the control group ( • 13000)
Cerebral Ischemia in Rats
689
Fig. 2. Electron micrograph of neuronal cells of ischaemic rat which had been pretreated with low dose of Chinese herbal composite ( x 11000)
and a small sample of right forebrain was taken for microscopy. The left hemisphere was homogenized as soon after death as possible in a ratio of 1 g of wet tissue to 9 ml of ice-cold 0.9% NaC1 by using a glass homogenizer. The measurement of peroxidation products (primarily malondialdehyde) was carried out with a modification of the thiobarbituric acid method described by Yagi [6]. A more sensitive method using HPLC (7) also gave similar results. The reaction mixture contained 0.05 rnl of homogenate and 0.2 ml of 8.1% aqueous solution of thiobarbituric acid. The
mixture was made up of a ,column of 4.0 ml with distilled water and heated at 95~ for 60 rain. After cooling with tap water, 1.0 ml of distilled water and 5.0 ml of a mixture of n-butanol and pyridine (15:1 v/v) were added and the tube was shaken vigorously. After centrifugation at 4000 rpm for 10 rain, the absorbance of the organic layer (upper layer) was measured in a MPF-4 Spectrofluorometer(Japan), Ex:515 nm, Em:553nm. Standards were prepared by using 0.1 ml of TEP (5 nmol/ml) in place of homogenate. The statistical significance of group differences was determined with student's t test.
690
Leung, Mo, and Zheng
Fig. 3. Electronmicrographof neuronalcells of isehaemierat which had been pretreatedwith high dose of Chineseherbal composite( x 8000)
Results
Nifedipine. Brain slices from right forebrain were observed in 10 vision fields under high power magnification. The number of normal, degenerating and necrotic cells was counted. The percentage of normal cells in nifedipine pretreated group was significantly higher than
that of the saline ischemia group. Similarly, the number of degenerating cells was 10% and necrotic cells 12% lower than the ischemia control (Table I). The content of thiobarbiturate reactive products (TBAR) in the ischemia saline control group was elevated as compared with that of the sham-operated saline control group. Nifedipine pretreatment reduced the increase in TBAR lev-
Cerebral Ischemia in Rats
691
Table I. Effect of nifedipine and Chinese Herbal Preparation on Neurocytes of Rats
% of all cells in 10 vision-fields Group
normal cells
Saline-ischemia control Nifedipine Herbal preparation (Low dose) Herbal preparation (high dose)
16.39 -- 2.96
degenerative ceils
Mar membranes were intact, rough endoplasmic reticulum appeared in an orderly arrangement, other cellular organelles were not altered and chromatin showed a welldistributed pattern (Figure 3).
necrotic cells
29.31 --_ 3.37 54.58 _+ 4.78
DISCUSSION 25.61 • 3.66* 26.48 • 5.14 47.93 • 4.78* 23.88 --- 3.64* 27.66 - 2.03 47.44 • 5.17" 25.81 4- 2.81' 27.39 • 4.46 46.79 • 4.83*
n = 120, X • SD. *P < 0.05 vs. saline-ischemia control.
Table II. Effect of Nifedipine and Chinese Herbal Preparation on the Content of TBAR of Rats
Group Saline Sham-operation control Saline, ischemia control Nifedipine Herbal preparation (low dose) Herbal preparation (high dose)
Content of TBAR (nmol/mg tissue) 0.765 1.24 0.783 0.817 0.688
--- 0.089** _-_ 0.177 - 0.091"* _-2 0.084** • 0.065**
n = 10, X--- SD. **P
Reduction of Cellular Damage Induced by Cerebral Ischemia in Rats* Albert W. N. Leung 1, Zhixian Mo 2, and Youshan Zheng z (AcceptedJanuary 29, 1991)
A model of incomplete cerebral ischemia involving bilateral ligation of the common carotid arteries in rats, was used to examine the potential of a Chinese herbal preparation and of nifedipine to reduce cell damage following cerebral ischemia. The herbal preparation contained ginsengosides and extracts of Panax notoginseng, Ligusticum chuanxiong Hort., Carthamus tinctorius L. and Salvia militorrhiza Bge. Histological evidence of cell damage and the formation of peroxidation products were both reduced in rats pretreated with the herbal preparation or with nifedipine. It has been suggested that the free radical reaction is involved in tissue damage, particularly in the pathological neurocyte injury of cerebral ishemia. The results show that in this model of incomplete cerebral ischemia, the degree of lipid peroxidation can be lowered by the pretreatment with Chinese herbs containing ginsengosides or with nifedipine. These drugs maybe beneficial in the treatment of cerebral ischemia in humans. KEY WORDS: Cerebral ischemia; malondialdehyde; nifedipine; Chinese herbal ginsengosides.
Nifedipine is a potent vasodilator with effects on the coronary and other peripheral blood vessels. In recent years, the actions of calcium antagonists on the cerebrovasculature have attracted more and more attention. There are now many reports demonstrating that nifedipine relieves cerebral arterial spasm (2,3) and has a brain protective effect (4). According to the Chinese pharmaceutical tradition, the herbal preparation, composed of ginsengosides and extracts of Panax notoginseng, Ligusticum chuanxiong Hort., Carthamus tinctorius L. and Salvia militorrhiza Bge, has actions of increasing blood circulation to dissipate blood stasis, thus benefiting vital energy and invigorating the brain. It has been used in China for hundreds of years to treat cardiovascular and cerebrovascular diseases, such as coronary heart disease, cerebral arteriosclerosis and cerebral function failure. Animal experiments have shown that this herbal preparation dilates small blood vessels and improves microcirculation in rats (5). The aim of present investigation is to compare the actions of nifedipine and the herbal preparation in reducing cellular damage caused by cerebral ischemia.
INTRODUCTION Stroke, one of the commonest causes of death in Western countries, is caused by ischemic cerebral lesions resulting from a reduction in the peffusion of brain tissue by blood. About a fifth of the cardiac output normally passes through the carotid and vertebral arteries to supply the brain and a reduction in total cerebral blood flow to less than half of normal will repair cerebral function (1). The present paper reports attempts to reduce the cellular damage due to cerebral ischemia in a rat model by pretreatment with the calcium channel blocker nifedipine or a Chinese herbal preparation containing ginsengosides. Nifedipine is used in the clinical treatment of various cardiovascular disorders while these Chinese herbs have a long history of use in promoting blood circulation. 1 Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N. T., Hong Kong. Department of Chinese Medicine, First Military Medical College, Guangzhou, China. * Special issue dedicated to Dr. Lawrence Austin.
687
0364-3190/91/0600-0687506.50/09 1991PlenumPublishingCorporation
688 EXERIMENTAL
Leung, Mo, and Zheng PROCEDURE
Nifedipine was kindly donated by Bayer (Germany), 1,1,3,3tetraethoxypropane (TEP) was purchased from Sigma and 2-thiobarbituric acid (TBA) from FIuka. The Chinese herbal preparation was supplied by Hong Xing PharmaceuticalCompany of Guangdong, China. Sprague-Dawley rats of both sexes weighing 200-250 g were used in this study. The animals were divided randomly into the following 4 groups; (1) sham-operation saline control, (2) ischemia saline con-
trol, (3) ischemia pretreated with nifedipine (2.5 rag/100 g -body weight p.o.), and (4) ischemia pretreated with the Chinese herbal preparation at either a low dose (0.2 g/100 g p.o.) or a high dose (0.4 g/100 g p.o.) The p.o. injections were given daily in the morning each day and the same volume of normat saline was given to the control groups. Incomplete cerebral ischemia was established under pentobarbital anesthesia by ligating bilaterally the common carotid arteries, which produces about a 70% reduction in cerebral blood flow. After 15 hours of ischemia the rats were killed by decapitation, the brain was excised,
Fig. 1. Electron micrograph of neuronal cells of ischaemic rat in the control group ( • 13000)
Cerebral Ischemia in Rats
689
Fig. 2. Electron micrograph of neuronal cells of ischaemic rat which had been pretreated with low dose of Chinese herbal composite ( x 11000)
and a small sample of right forebrain was taken for microscopy. The left hemisphere was homogenized as soon after death as possible in a ratio of 1 g of wet tissue to 9 ml of ice-cold 0.9% NaC1 by using a glass homogenizer. The measurement of peroxidation products (primarily malondialdehyde) was carried out with a modification of the thiobarbituric acid method described by Yagi [6]. A more sensitive method using HPLC (7) also gave similar results. The reaction mixture contained 0.05 rnl of homogenate and 0.2 ml of 8.1% aqueous solution of thiobarbituric acid. The
mixture was made up of a ,column of 4.0 ml with distilled water and heated at 95~ for 60 rain. After cooling with tap water, 1.0 ml of distilled water and 5.0 ml of a mixture of n-butanol and pyridine (15:1 v/v) were added and the tube was shaken vigorously. After centrifugation at 4000 rpm for 10 rain, the absorbance of the organic layer (upper layer) was measured in a MPF-4 Spectrofluorometer(Japan), Ex:515 nm, Em:553nm. Standards were prepared by using 0.1 ml of TEP (5 nmol/ml) in place of homogenate. The statistical significance of group differences was determined with student's t test.
690
Leung, Mo, and Zheng
Fig. 3. Electronmicrographof neuronalcells of isehaemierat which had been pretreatedwith high dose of Chineseherbal composite( x 8000)
Results
Nifedipine. Brain slices from right forebrain were observed in 10 vision fields under high power magnification. The number of normal, degenerating and necrotic cells was counted. The percentage of normal cells in nifedipine pretreated group was significantly higher than
that of the saline ischemia group. Similarly, the number of degenerating cells was 10% and necrotic cells 12% lower than the ischemia control (Table I). The content of thiobarbiturate reactive products (TBAR) in the ischemia saline control group was elevated as compared with that of the sham-operated saline control group. Nifedipine pretreatment reduced the increase in TBAR lev-
Cerebral Ischemia in Rats
691
Table I. Effect of nifedipine and Chinese Herbal Preparation on Neurocytes of Rats
% of all cells in 10 vision-fields Group
normal cells
Saline-ischemia control Nifedipine Herbal preparation (Low dose) Herbal preparation (high dose)
16.39 -- 2.96
degenerative ceils
Mar membranes were intact, rough endoplasmic reticulum appeared in an orderly arrangement, other cellular organelles were not altered and chromatin showed a welldistributed pattern (Figure 3).
necrotic cells
29.31 --_ 3.37 54.58 _+ 4.78
DISCUSSION 25.61 • 3.66* 26.48 • 5.14 47.93 • 4.78* 23.88 --- 3.64* 27.66 - 2.03 47.44 • 5.17" 25.81 4- 2.81' 27.39 • 4.46 46.79 • 4.83*
n = 120, X • SD. *P < 0.05 vs. saline-ischemia control.
Table II. Effect of Nifedipine and Chinese Herbal Preparation on the Content of TBAR of Rats
Group Saline Sham-operation control Saline, ischemia control Nifedipine Herbal preparation (low dose) Herbal preparation (high dose)
Content of TBAR (nmol/mg tissue) 0.765 1.24 0.783 0.817 0.688
--- 0.089** _-_ 0.177 - 0.091"* _-2 0.084** • 0.065**
n = 10, X--- SD. **P
