by Wu Xuping, Zhou Shuang, Wang Yawen, Liu Youxiang, Feng Yi, and Sun Guojie 

(Hubei College of TCM, Hubei 430061, China)
 

Abstract  Objective: To study the mechanism of acupuncture in improving acute cerebral infarction. Methods: The acute cerebral infarction model was made by linear embolus, and then the contents of cAMP and cGMP in plasma before and after acupuncture were determined by radioimmunoassay (RIA). Results: After acupuncture, the lowered content of cAMP increased obviously, the increased content of c GMP decreased and cAMP/cGMP rose. All of them gradually turned to normal value. Conclusion: Acupuncture may improve the blood supply of the brain tissues in the focus of infarction by adjusting the content of cAMP and cGMP and the ratio between them.

Key words  cAMP  cGMP  Acupuncture  Cerebral infarction

Acute cerebral infarction is one of the most commonly encountered cerebrovascular diseases, accounting for 75%[1]. Resent years' researches indicate that the contents of cyclic nucleotides in plasma and cerebrospinal fluid change obviously when disorders of central nervous system occur. This paper is signed to observe changes of cAMP and cGMP contents after acute cerebral infarction and t o study the effect of acupuncture on them in the rat with acute cerebral infarction.

Material and methods
1.Animal Model
90 healthy SD rats, with male and female in equal proportions, weighing from 260-280 g, were randomly divided into normal group(n=10), model group(n=40) and acupuncture group (n=40), those of the latter two groups were respectively further divided into 4 sub-groups with 10 rats in each subgroup. Anesthetized by intraperitoneal injection of 6% chloral hydrate (0.6 ml/100 g), the rat was put on a thermostat operating table. After the skin on the right part of the neck was incised, the right common carotid artery(CCA), external carotid artery and its branches were separated and ligated. Then the right internal carotid artery(ICA) was separated with the proximal end being ligated using a silk suture and the distal end clamped using an artery clamp. An incision was made on the CCA 0.5 cm from the fork. A piece of 1.5 gauge nylon thread with one end burnt into a glabular tip about 0.25 mm in diameter was inserted into ICA. After the line went through ICA and the beginning of the middle cerebral artery (MCA) to the proximal end of the anterior cerebral artery to obstruct all the blood supply of MCA, the thread was fastened. The surplus part of the thread was cut off. At last the skin was sewn up.
2. Methods of Acupuncture
According to “criteria of acupoint locations of experimental animals�?worked out by Experimental Acupuncture and Moxibustion Institute of Chinese Association of Acupuncture and Moxibustion in 1992, Shuigou (GV 26), bilateral Neiguan(PC 6) and Zusanli(ST 36) were selected. The needle inserted in Shuigou (GV 26) was obliquely advanced towards the nasal septum by 2 mm, twirled clockwise for 9 times, then maintained for 20 min. The needle inserted in Neiguan(PC 6) was perpendicularly pushed on by 2-3 mm, while Zusanli (ST 36) by 6-8 mm. Electroacupuncture Therapeutic Instrument G6805 (made in Shanghai) was used to stimulate these acupoints for 20 min with parameters of sparse-density waves, 1 mA in electric current and 30 Hz in frequency. The animal was treated by acupuncture as soon as it recovered from anesthetized state, followed by once per day.
3. Preparation of Sample
1.5 ml blood of the animal was put into a glass tube with 50μl EDTA·2 Nainit. The tube was shaken to make them mixed thorougly and then put into ice bath for centrifugation (2000 rpm, 10 min, which should be done within 1 hour). 0.1 ml of the supernate was drawn out to put into another tube, added with 2 ml absolute alcohol and shaken for 1 min. After 5 min's vest, the new mixture was centrifugated (3000 rpm, 10 min). The new supernate was drawn out to put into a clean and dry Penicillin bottle, while the left residue was mixed with 1 ml of 75% alcohol and smashed by shaking. And then the mixture was centrifugated (3000 rpm, 10 min). The supernate was added into another bottle. The mixed supernate was preserved in 4oC after oven drying.
4. Determination of Sample
The radioimmunoassay reagent box was offered by Shanghai University of TCM. Contents of cAMP and cGMP of the blood samples were detected by using a radio immunity-γ-counter. All the procedures were carried out following the directions of the radioimmunoassay reagent box.

Results
The data were statistically analysed by a computer with t-test. The results are shown in the following table.
1. Effect of acupuncture on plasma cAMP in rats with acute cerebral infarction (Table 1).
 

Table 1. Effect of Acupuncture on Plasma cAMP in Rats

 with Acute Cerebral Infarction (M±SD, pm/ml) 

Notes: Compared with normal group,*P<0.05, **P<0.01; Compared with model group, #P<0.05 

Table 1  shows  that the contents of plasma cAMP of model group after inducing ischemia for 6h, 24h, 48h, 72h were obviously lower than that of the normal group (6h, 24h, P<0.01; 48h, 72h, P<0.05), indicating significant decline of plasma cAMP after acute cerebral infarction. Compared with those of model group, the content of plasma cAMP of acupuncture group rose gradually to the normal level as the time went on. This indicates that acupuncture may increase the content of cAMP in plasma.       

2. Effect of acupuncture on plasma cGMP in rats with acute cerebral infarction ( Table 2 ).   

Table 2. Effect of Acupuncture on Plasma cGMP in Rats 

with Acute Cerebral Infarction (M±SD,pm/ml) 

 Notes: Compared with normal group,*P<0.05, **P<0.01; Compared with model group, ##P<0.05 

Table 2  shows that contents of plasma cGMP of model group after inducing cerebral ischemia for 6h, 24h, 48h, 72h were higher significantly than that of normal group, indicating an increase of cGMP after acute cerebral infarction . Compared with those of model group, the contents of plasma cGMP of acupuncture group decreased gradually to normal level as the time went on. This indicates that acupuncture may lower the content of plasma cGMP in rats acute cerebral infarction. Figure 2. Effect of Acupuncture on Plasma cGMP in Rats with Acute Cerebral Infarction 
 

3. Effect of acupuncture on plasma cAMP/cGMP in rats with cerebral infarction (T able 3 ).   

Table 3. Effect of Acupuncture on Plasma cAMP/cGMP in Rats

 with Acute Cerebral Infarction (M±SD,pm/ml) 

 Notes: Compared with normal group,*P<0.05, **P<0.01; Compared with model group, ##P<0.05

Table 3  shows that plasma cAMP/cGMP of model group were obviously lower than that of normal group (P<0.01). Compared with those of model group, cAMP/cGMP of acupuncture group increased gradually and turned to the normal level as the time went on. This indicates acupuncture may raise plasma cAMP/cG MP in rats with acute cerebral infarction.

Discussion
Since 1965, when cAMP was discovered by Suther Land, many scholars have conducted a lot of researches. It is proved that several kinds of bio-active cyclic nucleotides exist in many kinds of tissues and cells. Among them, cAMP and cGMP are most important. The domestic and overseas researches demonstrate that cAMP and cGMP adjust the function of vascular smooth muscle (mediating relaxation of vascular smooth muscles) by and activity lowering centration of intracellular Ca 2+ and inhibiting the metabolism of lipositol. Our past experiments indicated that acupuncture of Shuigou (GV 26) may increase the cerebral blood flow, reduce the permeability of the wall of micrangium, clarify the contour of the vessels and fasten the speed of blood flow. The data in this paper indicate that acupuncture can regulate abnormal changes of the contents of cAMP and cGMP after acute cerebral infarction. Therefore, acupuncture may cerebrovascular function after acute cerebral infarction improve by adjusting the content of cAMP and cGMP and their ratio.