手十二井穴刺络放血保护脑缺血的作用与甘露醇相似

doi:10.3969/j.issn.1673-5374.2013.06.006

摘要/Abstract

摘要：

实验用线栓法法建立大鼠大脑中动脉永久性栓塞模型，造模成功后，分别应用手十二井穴刺络放血和（或）尾静脉注射甘露醇进行治疗，2次/d。造模后24，48，72 h取脑，并于取脑前1h眼底静脉取血并尾静脉注射伊文思蓝。结果发现，模型大鼠脑组织伊文思蓝渗出量和血清一氧化氮合酶活性明显升高，而手十二井穴刺络放血和（或）尾静脉注射甘露醇治疗均可不同程度降低模型大鼠脑组织伊文思蓝渗出量和血清一氧化氮合酶活性，且3者比较差异无显著性意义。可见手十二井穴刺络放血可降低大脑中动脉栓塞大鼠血脑屏障通透性和血清一氧化氮合成酶活性，其作用与尾静脉注射甘露醇或两者合用相似。

关键词: 神经再生, 针灸, 脑损伤, 十二井穴, 穴位, 刺络放血, 甘露醇, 大脑中动脉栓塞模型, 脑缺血, 血脑屏障, 一氧化氮合成酶, 脑水肿, 神经保护, 基金资助文章

Abstract:

A rat model of middle cerebral artery permanent occlusion was established using the modified Longa method. Successfully established model animals were treated by blood-letting puncture at twelve Jing-Well points of the hand, and/or by injecting mannitol into the caudal vein twice daily. Brain tissue was collected at 24, 48 and 72 hours after modeling, and blood was collected through the retinal vein before Evans blue was injected, approximately 1 hour prior to harvesting of brain tissue. Results showed that Evans blue leakage into brain tissue and serum nitric oxide synthase activity were significantly increased in model rats. Treatment with blood-letting punctures at twelve Jing-Well points of the hand and/or injection of mannitol into the caudal vein reduced the amount of Evans blue leakage into the brain tissue and serum nitric oxide synthase activity to varying degrees. There was no significant difference between single treatment and combined treatment. Experimental findings indicate that blood-letting punctures at twelve Jing-Well points of the hand can decrease blood-brain barrier permeability and serum nitric oxide synthase activity in rats following middle cerebral artery occlusion, and its effect is similar to that of mannitol injection alone and Jing-Well points plus mannitol injection.

Key words: neural regeneration, brain injury, Jing-Well points of hand, acupoint, blood-letting, mannitol, middle cerebral artery occlusion, cerebral ischemia, cerebral infarction, blood-brain barrier, nitric oxide synthase, cerebral edema, neuroprotection, grants-supported paper, neuroregeneration

. 手十二井穴刺络放血保护脑缺血的作用与甘露醇相似[J]. 中国神经再生研究(英文版), 2013, 8(6): 532-539.

Xuan Lu, Zelin Chen, Yi Guo, Liang Gao, Liyuan Jiang, Zhongzheng Li, Jianqiao Fang. Blood-letting punctures at twelve Jing-Well points of the hand can treat cerebral ischemia in a similar manner to mannitol[J]. Neural Regeneration Research, 2013, 8(6): 532-539.

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引言

材料方法

Design

A randomized, controlled, animal experiment.

Time and setting

Experiments were performed at the Research Center Laboratory of Experimental Acupuncture, Tianjin University of Traditional Chinese Medicine, China, from September 2010 to July 2011.

Materials

Altogether, 140 male Wistar rats weighing 230–250 g, aged 3 months were selected from the Animal Center of Tianjin University of Traditional Chinese Medicine, China [license No. SCXK (Tianjin) 2009-0001]. Animals were allowed to acclimatize for 1 week prior to the experiment, and were housed under a 12-hour light-dark cycle subsisted with a standard laboratory diet and tap water ad libitum. Animal treatments were performed in a manner to minimize pain or discomfort in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China^[48].

Methods

Establishment of middle cerebral artery occlusion models

A modified Longa method was applied to prepare the model of middle cerebral artery embolism in rats^[49]. Briefly, rats were anesthetized by abdominal injection with 10% chloral hydrate at a dose of 3.3 mL/kg and then fixed in a supine position. A midline incision was made at the neck to separate the common carotid artery, the external carotid artery and the internal carotid artery on the left side. Arteries were marked by ligatures on the external carotid artery and the proximal end of the common carotid artery. A 0.285-mm nylon thread was inserted from the distal end of the common carotid artery to the internal carotid artery, about 18.0 ± 0.5 mm in depth to the anterior cerebral artery. This thread gave rise to the blockage of the middle cerebral artery to induce focal cerebral ischemia (Figure 1). After animals recovered, deficits in neurological function were scored according to the method described by Longa^[49]. Rats with a score of 1–3 were considered to have undergone successful cerebral ischemic injury. If rats did not score 1–3, they were not used for further experimentation. The nylon thread was not applied in the sham operation group.

Blood-letting puncture at twelve Jing-Well points of the hand

After model establishment, a 21 gauge blood lancet (Suzhou Sterilance Medical Equipment Co., Ltd., Suzhou, Jiangsu Province, China) was used to prick the acupoints, in sequence, for blood-letting at Shaoshang (LU11), Shangyang (LI1), Zhongchong (PC9), Guanchong (TE1), Shaochong (HT9) and Shaoze (SI1) at a depth of 1 mm^[49] (Figure 2). The needle first penetrated vertically into the left foreleg and was not retained. This method was then repeated on the right side, at analogous Jing-Well points of the human hand^[50]. Squeezing of the finger was done until bleeding stopped. Rats underwent surgery twice daily at 9:00–10:00 a.m. and 4:00–5:00 p.m. Rats from the control and sham operation groups were similarly grasped with equal strength minus the blood-letting puncture.

Mannitol injection

After model establishment, 0.5 g/kg of 20% mannitol (Tianan Pharmaceutical Co., Ltd., Tianjin, China) was injected into the caudal vein of rats twice a day. In the Jing-Well points plus mannitol group, blood-letting puncture and mannitol injection were performed simultaneously.

Determination of blood-brain barrier permeability with Evans blue

At 23, 47, and 71 hours after model establishment, 4 mL/kg of 2% Evans blue (Sigma, St. Louis, MO, USA) in physiological saline was injected into the caudal vein of rats. One hour later, the rats were anesthetized with 10% chloral hydrate and were perfused with physiological saline. Dyed brain samples (0.1 g) were weighed and homogenized in 3 mL formamide (Sigma), incubated for 24 hours at 54°C, and centrifuged at 3 000 r/min for 15 minutes. The supernatants were analyzed at 620 nm using a 722-type spectrophotometer (Shanghai Third Analytical Instrument Factory, Shanghai, China).

Evans blue was made into a series of concentrations (0.05, 0.025, 0.012 5, 0.006 3, 0.003 1, 0.001 6, 0.000 7 μg/mL), and then absorbance was determined. A standard curve was plotted and linear regression revealed the equation y = 7.846x + 0.002 7, r = 0.999. These findings showed that Evans blue content was closely related to Evans blue absorbance.

Brain tissue Evans blue content (μg/g) was calculated using the equation: A × formamide quantity (mL)/brain tissue wet weight (g), where A is obtained by substituting the Evans blue absorbance into the linear regression equation.

Determination of nitric oxide synthase activity using spectrophotometry

Blood (2–3 mL) was collected from the retinal vein of rats before Evans blue was injected, and was centrifuged at 3 000 r/min for 15 minutes. Serum was analyzed at 530 nm using the 722-type spectrophotometer. Determination of nitric oxide synthase activity, using a kit, was strictly based on the instructions provided by the Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu Province, China).

Total nitric oxide synthase activity (U/mL) was determined using the following equation: (absorbance value of detected tube – absorbance value of blank tube)/molar extinction coefficient × (volume of reaction solution/sampling amount)/(1/chromatic light diameter × reaction time)/1 000 ≈ 141.57 × (absorbance value of detected tube – absorbance value of blank tube).

Statistical analysis

All results were expressed as mean ± SD for each group. Statistical analyses were performed with SPSS 11.0 software (SPSS, Chicago, IL, USA). One-way analysis of variance was used to compare mean differences in Evans blue leakage and nitric oxide synthase activity among groups. A P value less than 0.05 was considered to be a significant difference.