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Effects Of Acupuncture On SOD (Superoxide
Dismutase) And LPO (Lipid Peroxides) In Rabbit
Models Of Acute Cerebral Hemorrhage
Luo Song, MD
Professor Liao Fang Zheng
ABSTRACT
Background Research indicates that the mortality rate of cerebral hemorrhage is generally 30%-40%, and the mortality rate occurring at some anatomic regions reaches 60%. A total of 15%-40% of mortality due to cerebral hemorrhage happens at the acute phase.
Objective To research the mechanism of acupuncture treatment on acute cerebral hemorrhage (ACH).
Design, Setting, and Subjects On the basis of an atherosclerosis cerebral hemorrhage rabbit model built by feeding ketogenic forage and injecting blood into the brain – and according to the therapeutic principle of "Tonifying Kidney and Promoting Blood Circulation, Reviving Brain and Inducing Resuscitation" – we used acupuncture points Renzhong (GV 26), Fengfu (GV 16), Quze (PC 3), Neiguan (PC 6), Sanying Jiao (SP 6), Xuehai (SP 10), and Taixi (KI 3) to observe the effect of acupuncture on several indexes of ACH cases in groups.
Results In the 3 segments, compared with group 1, the superoxide dismutase (SOD) activity decreased in groups 2 and 3, and significantly in group 2. Twenty-four and 72 hours after cerebral hemorrhage, the SOD activity in group 3 was higher than that in group 2. Compared with SOD activity 24 hours after hemorrhage, it was further decreased at 72 hours in group 2, while it was elevated in group 3. Compared with SOD activity at 72 hours after hemorrhage, it was elevated at 1 week in group 2, while less than that in group 3. Superoxide dismutase activity in group 3 was higher than that at 24 hours. In the 3 segments, compared with group 1, the lipid peroxidase (LPO) level increased significantly in group 2. Twenty-four and 72 hours after hemorrhage, the LPO in group 3 was lower than that in group 2. Compared with LPO at 24 hours, it was further increased at 72 hours in groups 2 and 3. Compared with LPO at 72 hours after cerebral hemorrhage, it was decreased at 1 week in groups 2 and 3; the LPO in group 3 was significantly less than that in group 2; the LPO in groups 2 and 3 was lower than that at 24 hours.
Conclusion Compared with the model group, acupuncture therapy used for early ACH may significantly improve the activity of SOD and reduce the content of LPO in the brain, which could protect the function of the brain.
KEY WORDS
Acupuncture, Cerebral Hemorrhage, Acute Cerebral Hemorrhage, Free Radicals, Superoxide Dismutase, Lipid Peroxides Rabbits
INTRODUCTION
Cerebrovascular disease (CVD), cardiac disease, and malignant tumors have the greatest human incidence, mortality, and disability rates. According to the MONICA1-3 program, an epidemiological investigation sponsored by the World Health Organization involving 4.5 million people from 11 countries, the average incidence of CVD is 101-285/100,000 men and 47-198/100,000 women. Along with an accelerating aging population, there is a more prominent threat of CVD. Investigation indicates that the mortality rate of cerebral hemorrhage is generally 30%-40%, and the mortality rate occurring at some anatomic regions reaches 60%. A total of 15%-40% of mortality due to cerebral hemorrhage happens at the acute phase.4-7 Therefore, to administer effective treatment for acute cerebral hemorrhage (ACH), it is essential to reduce mortality due to CVD. Our goal was to provide objective evidence for determining at what point acupuncture intervention should occur to treat patients at the early stage of cerebral hemorrhage, through the investigation into effects of acupuncture on superoxide dismutase (SOD) and lipid peroxides (LPO) in a rabbit model of ACH.
METHODS
Animals and Experimental Grouping
Fifty-four rabbits, an equal number male and female, weighing 2.0-2.5 kg, were provided by the Animal Center of Chengdu Institute for Blood Transfusion, China Academy of Medical Science. (All rabbits were in accordance with class III laboratory animal standards and were treated humanely, and our research conformed to standards of ethical animal research practices.) From the group, 18 rabbits were chosen at random as a control group and were fed common forage. The remaining rabbits were divided at random into 2 groups, the model group (n=18) and the acupuncture group (n=18).
Generation of ACH Rabbit Models
According to the methods previously introduced,8 atherosclerosis rabbit models were successfully created after the animals were fed high-fat forage for 6 weeks. Based on the above-mentioned atherosclerosis rabbit models, cerebral hemorrhage models were generated by means of slight modification of the method reported by Suiyu et al.9 The hairs on the rabbit's vertex were cut prior to limbus auricular vein anesthesia with 3% pentobarbital sodium solution in 1 mL/kg body weight. An eyelet was holed at the site 0.3 cm on the left lateral to and 0.5 cm anterior to the middle line of lambdoid suture of the skull of each atherosclerosis model. Then, 0.5 mL of blood taken from the rabbit's heart was injected into the brain using a No. 7 needle that punctured the eyelet with a depth of 1.4 cm, for the purpose of forming a hematoma in the mesencephalon. Afterward, the surgical wound was sutured following sterilization.
Method of Acupuncture
Rabbit models of cerebral hemorrhage were created in the acupuncture group and the model group after 6 weeks of administering high-fat forage. After generation of the models, a total of 12 acupoints were used in the acupuncture group: Suigo (GV 26), Fengfu (GV 16), bilateral Quze (PC 3), Neiguan (PC 6), Sanying Jiao (SP 6), bilateral Xuehai (SP 10), and Taixi (KI 3).10 One-cun (25 mm in length and 0.3 mm in diameter) filiform needles (Suzhou, China) were applied. Neiguan was punctured perpendicularly 1 cm; Shuigou (GV 26) was punctured obliquely upward 0.5 cm; Fengfu was punctured obliquely downward 0.5 cm; Quze was punctured perpendicularly 1 cm, and a reducing method was applied by twirling and rotating the needles in these acupuncture points. Xuehai, Sanying Jiao, and Taixi were punctured perpendicularly 0.5 cm by an even reinforcing and reducing method. All the points received 1-minute manipulation and 30-minute needle retaining, twice a day. We twirled the needles both clockwise and counterclockwise less than 360° each in reinforcing and reducing methods; we stressed the clockwise rotating in reinforcing, and the contrary in the reducing method. During the treatment, we manipulated the needles for 1 minute at the beginning, 15 times per minute for reinforcing, and 30 times per minute for reducing. All treatments were performed by a professional physician certified by the Sichuan Health Bureau.
The rabbits were separately sacrificed in groups of 6 at 24 hours, 72 hours, and 1 week after generation of the models, respectively. At that time, brain tissue was harvested by prompt decapitation.
Detection of SOD and LPO in Brain Tissue
After sacrificing the rabbits, we collected 2 peripheral brain tissues of the hematoma, each weighing 0.5 g, and promptly stored them on ice. The brain tissues were stored in -196°F liquid nitrogen for detection with UV-260, UV-visible spectrophotometer (Japan Shimadzu).
Activity of SOD was detected by means of pyrogallic acid auto-oxidation, taking u/g-wet tissue as the basic unit. Content of LPO was detected by means of TBA chromatometry,11 taking nmol/mL tissue homogenate as the basic unit.
Statistical Analysis
All experimental data were analyzed by PEMS Version 2.1 (packaged for Encyclopedia Medical Statistics) and developed by the Teaching and Research Section of West China University of Medical Science. The results were expressed as mean 6 SD. When making a comparison between 2 groups, the t test was adopted if variance was homogeneous.
RESULTS
In the 3 phases, in comparison with the control group, activity of SOD was decreased significantly in the model group and the acupuncture group (P,.01), especially in the model group. Twenty-four and 72 hours after cerebral hemorrhage, activity of SOD in the acupuncture group was markedly higher than that in the model group (P,.01) (Table 1). Compared with activity of SOD at 24 hours, it was further decreased at 72 hours after hemorrhage in the model group with markedly significant difference (P,.01); conversely, activity of SOD in the acupuncture group was increased (P,.05). One week after cerebral hemorrhage, compared with activity of SOD at 72 hours, it was elevated to a certain degree in the model group, but its level was still significantly lower than that in the acupuncture group (P,.01); activity of SOD in the acupuncture group was higher than that at 24 hours after cerebral hemorrhage (P,.05).
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Table 1. Cerebral Tissue Superoxide Dismutase, u/g-wet tissue
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Group
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n
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24 h
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72 h
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1 wk
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Control
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18
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38.50±2.80
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39.10±2.23
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39.80±2.13
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Model
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18
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23.94±2.70*
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14.49±2.84*
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§22.83±1.26*¶
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Acupuncture
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18
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34.08±1.04*†
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35.90±1.21*†‡
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36.01±1.50*†‡
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Compared with control: *P<.01; compared with model: †P<.01; compared with that after 24 hours: ‡P<.05, §P<.01; compared with that after 72 hours: ¶P<.01.
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Table 2. Cerebral Tissue Lipid Peroxidation, nmol/mL
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Group
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n
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24 h
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72 h
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1 wk
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Control
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18
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18.67±4.51
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18.44±4.72
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18.67±4.53
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Model
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18
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43.30±2.15*
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74.40±8.5†§
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35.25±2.69*‡¶
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Acupuncture
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18
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33.05±2.11*†
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53.08±3.01*†§
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26.98±2.13*†‡¶
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Compared with control: *P<.01; compared with model: †P<.01; compared with that after 24 hours: ‡P<.05, §P<.01; compared with that after 72 hours: ¶P<.01.
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In the 3 phases, compared with the control group, content of LPO both in the model group and in the acupuncture group was significantly elevated (P,.01) (Table 2). Twenty-four hours after cerebral hemorrhage, LPO was further increased at 72 hours in the model and acupuncture groups (P,.01). One week after hemorrhage, there were statistically significant differences between the acupuncture group and model group (P,.01); compared with LPO at 72 hours, it was decreased in the model group and acupuncture group (P,.01). LPO in the model and acupuncture groups was markedly lower than that at 24 hours (P,.05 and P,.01, respectively).
DISCUSSION
SOD and LPO are 2 indexes that have been proven to have a close relationship with the metabolism of free radicals, which could affect the injury and repair of neurocytes. The majority of free radicals in vivo are induced by oxygen, so they are called oxygen-derived free radicals, and they consist of superoxide anion free radical (O2), hydroxy radical (OH), and hydrogen peroxide (H2O2), etc. The unstable oxygen-derived free radicals are apt to react with protein, nucleic acid, lipids, and other molecules such as hyaluronic acid. They can destroy the structure of these molecules. Membrane phospholipids, being rich in unsaturated fatty acid, are often attacked by free radicals.12
Lipid peroxidation resulting from the attack of free radicals on cellular membranes leads to continuous generation of LPO; the latter is resolved into a stable metabolite that brings about changes of cellular structure and function. In physiological conditions, the body can prevent and terminate lipid peroxidation by regulating and controlling reactions induced by free radicals through means of enzyme reaction. Consequently, LPO is decreased and damage caused by free radicals is prevented. In pathological conditions, the cellular membrane is attacked and spoiled by free radicals due to overgeneration of free radicals or reduction of such free radical scavengers as SOD. Nerve tissues are especially sensitive to free radicals because they contain a large quantity of phospholipids, lowly active catalase, low SOD content, abundant ferric ion, and lysosomes.13-15
This study revealed an obvious elevation of free radicals and reduction of their endogenetic clearance at ACH. Massive generation of free radicals will damage cellular membranes, resulting in dysfunction of the blood-brain barrier. While aggravating cerebral edema, injury of cellular membrane leads to calcium overloading, damage of neurocytes, vasospasm, thickened blood viscosity, and decreased cerebral blood flow. All these aggravate brain injury.16-18
In this experiment, we observed that within 1 week after cerebral hemorrhage, activity of SOD in brain tissue of the model group was markedly decreased in a trend of descending in the earlier stage and rising again in the late stage; however, activity of SOD in the acupuncture group increased steadily. We also observed that 1 week after hemorrhage, there was markedly increased content of LPO in the model group in a trend of increasing in the earlier stage and descending in the late stage; markedly reduced content of LPO in the acupuncture group was observed, indicating that acupuncture could facilitate endogenetic clearance of free radicals and suppress lipid peroxidation. Therefore, acupuncture treatment on cerebral hemorrhage at 24 hours may be reliable and helpful to patients' recovery.
CONCLUSION
We found that acupuncture is useful for ACH in rabbits, which is an important foundation for research on humans. If these acupoints could be used to treat ACH with good effect in humans very early, it would be beneficial for patients' rehabilitation in the late stage. As a result, patients could be proactive in their therapy and daily routines instead of remaining predominantly dormant.
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AUTHORS' INFORMATION
Dr Luo Song graduated from Chengdu University of Traditional Chinese Medicine, People's Republic of China. He is an attending physician at Sichuan Provincial People's Hospital in China (1,000 patient beds), and performs research in cerebrovascular disorders and Alzheimer disease. Dr Song is experienced in acupuncture, moxabustion, Chinese herb therapy, and neurology.
Luo Song, MD*
2 Xi'an Bei Rd
Chengdu, Sichuan 610075
P R China
E-mail: cdsuosong@hotmail.com
Liao Fang Zheng, BS, is Professor, Chengdu University of Traditional Chinese Medicine, People's Republic of China
Professor Liao Fang Zheng
E-mail: zhangfarong@21cn.com
*Correspondence and reprint requests
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