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ORIGINAL ARTICLE Cluster Analysis for Acupoint Specificity of Acupuncture Effect Based on Cerebral Infarction Rat Model CHANG Xiao-bo (常晓波)1,2, FAN Xiao-nong (樊小农)3,4, WANG Shu (王 舒)2,3, MENG Zhi-hong (孟智宏)3, YANG Xue (杨 雪)5, and SHI Xue-min (石学敏)3,6 ABSTRACT Objectives: To study the acupoint specificity through the comprehensive evaluation of the acupuncture effect on rat model with the middle cerebral artery occlusion (MCAO). Methods: Duplicated MCAO model by Zea-longa's thread ligation was assessed by Zausinger's six-point method, and rats with 1–3 scores were chosen to be grouped. According to the randomized and controlled principle, rats were divided into 6 basic control groups (including normal group, sham group, model control group, model group without intervention, Nimodipine group, lateral-to-Renzhong group) and 6 acupuncture groups [including Neiguan (PC6) group, Weizhong (BL40) group, Chize (LU5) group, Sanyinjiao (SP6) group, Renzhong (DU6) group and non-acupoint group]; in each acupoint or non-acupoint 9 different parameter combinations were respectively set by orthogonal intersection method. There were in total 60 groups, and each group had 12 rats. The rats were treated by acupuncture with the lifting-thrusting manipulation every 12 h, in total 6 times. Indices of neurobehavioral scores, cerebral blood flow, infarction rate, microcirculation, and light microscope, etc. were measured. In order to comprehensively evaluate the acupuncture effect to research the specificity of acupoint, a cluster analysis was made with SPSS17.0 for the comprehensive effect scores of the samples computed according to the comprehensive evaluation models, and then sorting and classification were made for the comprehensive effect scores. Results: In the acupuncture groups every acupoint had different therapeutic effect due to different acupuncture parameter combinations; among the acupuncture groups the orders of curative effect were as followings: Neiguan group was of exceptional result, Weizhong group and Sanyinjiao group were of valid results in upper level, Chize group and Renzhong group were of valid results in lower level, and non-acupoint group was of invalid result; the therapeutic result of acupoint group was superior to non-acupoint group; and the curative effect of acupuncture group was also superior to basic control group. Conclusion: The acupoint has the specificity of acupuncture effect, and the acupoint specificity is relative. KEYWORDS model rats of middle cerebral artery occlusion, acupuncture effect, comprehensive evaluation, acupoint specificity, cluster analysis

The acupoint is not only the special spot where qi and blood from viscera and meridians effuse and infuse in the body surface, but also the reflecting spot of the disease and stimulating spot of treatment. The acupoint has relatively specificity, which can be sufficiently demonstrated from the ancient classical documents and the modern scientific researching results. The Chinese medicine thinks that, the acupoint of the body is attributed to the meridian, but every meridian is also attributed to a certain viscera, thus makes an interrelation of the acupointmeridian-viscera, an inseparable relationship. Just like Spiritual Pivot (Ling Shu • Hai Lun) said: "Twelve meridians are connected with the viscera internally, and associated with limbs and skeleton externally". The human body is a complex network system, it is the relationships among acupoints, meridians and

visceras, limbs-and-skeleton and the five sense organs that make human body into a complete ©The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag Berlin Heidelberg 2013 Supported by the National Program on Key Basic Research Project (973 Program, No. 2006CB504504, 2010CB530500 and 2012 CB 518505) 1. College of Acupuncture-Moxibustion and Tuina, Tianjin University of Traditional Chinese Medicine, Tianjin (300193), China; 2. Critical Research Room of Encephalopathy Acupuncture Therapy, Tianjin (300193), China; 3. The First Hospital Affiliated to Tianjin University of Traditional Chinese Medicine, Tianjin (300193), China; 4. Class Ⅲ Laboratory of Acupuncture Quantity-Effect Relationship Affiliated to the State Administration of Traditional Chinese Medicine, Tianjin (300193), China; 5. Mathematics College of Tianjin Polytechnic University; Tianjin (300387), China; 6. Tianjin Key Laboratory of Acupuncture and Moxibustion Science, Tianjin (300193), China Correspondence to: Prof. WANG Shu, Tel: 86-22-27432011, E-mail: [email protected] DOI: 10.1007/s11655-013-1530-7

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system. The correlation of acupoint-meridian-viscera is just the theory basis of the meridian specificity. But with the acupuncture therapy constantly heading for the world, the acupuncture point specificity is being questioned and challenged by the international and domestic researchers.(1) Some research institutions in Germany, Holland, and the United Kingdom have drawn an invalid conclusion in the clinical evaluation of acupuncture treatment of chronic asthma, lowback pain, smoking cessation and thought there was no significant difference between acupuncture and pseudo-acupuncture,(2-4) which is an undoubted negation to our meridians and collaterals doctrine of Chinese medicine. Whereas domestic acupuncture studies put most emphases on the acupuncture effect, few importance on the acupoint specificity. On account of this, our hospital has undertaken a great task, "Research on the specificity of the meridian acupoint and its key influencing factors based on cerebral infarction". In this article the multiple-index weight coefficients determined by the comprehensive evaluation model of the acupuncture effect which was established according to the principalcomponent-factor analysis, were applied to compute comprehensive scores of all cases, then classified through cluster analysis by SPSS 17.0 to carry on comprehensive evaluation of the acupuncture effect and to research the acupoint specificity.

METHODS Reagents and Instruments Hydrated chlorine aldehydes (analytically pure), 4% 2,3,5-triphenyltetrazolium chloride (TTC) and 1 mol/L potassium di-hydrogen phosphate (K2HPO4), were provided by Tianjin Regent Chemicals Co., Ltd., China). DRT4 laser doppler flow meter was produced by American HP Company, and provided by Swedish Perimed Medical Instrument and Chinese Company. XW-4 Color Microcirculation Detector was provided by Hefei Xiangshi Electronic Technology Co., Ltd., China. Filiform needles with the brand of "Huatuo" were purchased from Suzhou Medical Products Factory, China, 1.5 inches in length, 0.32 mm in diameter.

Animal Sources and Groups A total of 720 adult male specific pathogen free Wistar rats, with the weight of around 250–300 g, were provided by the Laboratory Animal Center of

the Chinese Academy of Medical Sciences, with No.050275 of the animal certificate number for (Jinghai) animal epidemic prevention. According to randomized and controlled principle, the rats were divided into basic control group (including normal control group, sham group, model control group, model group without intervention, Nimodipine group, lateral-to-Renzhong group), and acupuncture group [including Neiguan (PC6) group, Weizhong (BL40) group, Chize (LU5) group, Sanyinjiao (SP6) group, Renzhong (DU6) group and non-acupoint group]. Moreover, in the acupuncture groups every acupoint or non-acupoint group was respectively set 9 different parameter combinations by orthogonal intersection method. There were 12 rats in each group, in total 60 groups.

Middle Cerebral Artery Occlusion Rat Model

According to Zea-longa thread ligation (5) to duplicate middle cerebral artery occlusion (MCAO) model. The main steps: the rats were fasting with water for 12 h, received intraperitoneal injection for anesthesia with 10% hydration chlorine aldehyde (350 mg/kg) until animal pain stimuli was disappeared, the rats were fixed on the back to the surgery board. After local disinfection skin preparation, a left incision in the middle of the neck of 2–2.5 cm was taken, the thyroids on both sides were separated, the triangle area between the sternocleidomastoid muscle and the sternohyoid muscle on the left was exposed, the left common carotid artery and external carotid artery were blunt separated, and the external carotid artery was ligatured with 0 operation line. Sections of carotid artery in the arterial bifurcation/ fork and near the heart was respectively clipped with small arteryclamp, then a small hole near the heart was pierced with 1 mL syringe needle, a fish line with 0.26 mm was slowly inserted into the internal carotid, until resistance was met, with the intracranial fish line depth of 18–20 mm, now fish line was plugged to close the brain artery openings, and the blood flow was blocked in the middle artery of the brain. After the success of the carotid artery ligation, cephalic artery and pinhole were ligatured, open another small artery clamp. At last the incision was steralized and layered sutures.

Acupuncture Intervention Methods Acupoint Location On the basis of the acupoints location of commonused experimental animals in the Experimental

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Acupuncture Science,(6) Neiguan, Weizhong, Chize, Sanyinjiao, Renzhong and non-acupoint, in total 6 points were chosen to be undertaken acupuncture with thrusting-lifting manipulation. Among them, Neiguan is located in the forefoot inside, between the ulna and the radius, about 3 mm near the wrist, 3 mm with peripendicular insertion; Weizhong is located in the right rear indentation of the knee-joint, 5 mm with peripendicular insertion; Chize is located in indentation of elbow horizontal grain near the radial side, 5 mm with peripendicular insertion; Sanyinjiao is located in the medial side of the hind leg, 10 mm directly above the tip of the medial malleolus, 3 mm with peripendicular insertion; Renzhong is located in the philtrum, 1 mm directly below nasolabial sulcus, 2 mm with the oblique insertion to the direction of the nasal septum; non-acupoint as a comparison, is located between iliac crest and ribs on involved side and 10 mm on the top of iliac crest, also 2 mm with the oblique insertion.

the operation group, and the animals were with neurobehavioral score at 5 assessed by Zausinger six-score method. Nimodipine group: after model duplication was successful, in the corresponding period, the model rats were treated by intraperitoneal injection of Nimodipine at 0.15 mg/kg, once daily . Lateral-to-Renzhong group: after model duplication was successful, in the corresponding period, the model rats were acupunctured at 2 mm with peripendicular insertion in the left side (suffered side) of philtrum line, 5 mm lateral to the Renzhong acupoint.

Intervention of Parameters The acupuncture groups were intervened by acupuncture according to the orthogonal intersection method of acupuncture parameters [2 factors (frequency and time) and 3 levels (180, 120, and 60 cycles/min of the frequency and 5, 60, and 180 s of the time)], see Table 1.

Therapeutic Methods Model control group: after model duplication was successful and the model rats were completely awake, they were not performed any acupuncture, and the indices of cerebral blood flow, micro-circulation were immediately tested and then indices of light microscope and infarction rate were also tested after the rat model was sacrificed and the cerebral tissues were taken.

Intervention Time After model duplication was successful, the model rats were completely awake for 1 h and then acupunctured for the first time, once every 12 h, in total 6 times.

Effective Indices Neurobehavioral Score

Model group without intervention group: after model duplication was successful, in the corresponding period, the model rats were grabbed without any implement of acupuncture intervention for 6 times in the same conditions as the acupuncture group.

When the rats were completely awake after successful model duplications, their nerve function were assessed by Zausinger 6-score method(7) and the model rats with the scores of 4 and 5 were removed. The evaluation standard of Zausinger 6-score method: 0 score means the rat does not spontaneously walk; 1 score means the rat rotates contralateral lesions under the condition of freely walking; 2 scores mean the rat rotates contralateral lesions when its tail being seized; 3 scores mean the resistance to the lateral pressure in the opposite side of the lesion descends;

Normal control group: the rats were not implemented the model-duplication and the acupuncture intervention, but were grabbed for 6 times in the same conditions. Sham group: besides not being inserted the fish line, the other measures were the same as Table 1. Parameter Frequency (cycles/min) Time (s)

Nine Different Acupuncture Parameter Combinations

2

3

4

5

6

7

8

9

10

60

120

180

60

120

180

60

120

180

5

60

180

60

180

5

180

5

60

Note: in this paper, the influence of the acupunctural duration of frequency and time on the acupuncture effect was mainly discussed; parameter 1 was used for the needle-keeping group which was not discussed here

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4 scores mean the rat can't unbend front paws in contralateral lesions, even the whole body buckling to the contralateral lesions; 5 scores mean the rat has no neurological function defect.

number, microvascular number, inflammatory cell number, positive cells (necrosis cell) counts and neuronophage number were counted.

Statistical Methods Determination of Cerebral Blood Flow and Circulation (8)

According to the method of Bao, et al, the rat was fixed in the stereotaxic instrument, a midline incision on the scalp was taken, the periosteum was cut, the bregma would be exposed, then a bone window was opened, centered on 3 mm lateral to the left of the middle line after the bregma 1 mm, with the depth of breakthrough sense as a degree. DRT4 laser doppler flow meter was used to continuously and dynamically monitor the blood flow in the local ischemia brain. And the 4-XW color microcirculation instrument was applied to measure the microcirculation on the soft meninges.

Determination of Cerebral Ischemia Area (Infarction Rate) After the 3rd day of the treatment, the head of the model rat was cut and its brain was obtained, the olfactory bulb, the cerebellum and the low brain stem were separated and removed, the rest of the cerebrum was equally cut into 5 slices, which were then put into the liquid of 4% TTC and 1 mol/L K2HPO4, on the condition of light-free and warm hatching at 37 ℃ temperature for 30 min; sections were scanned with the digital camera; after the image analysis with IPWIN60, the infarction rate was calculated with the ratio of infarction area mean and total area mean.

Observation of Light Microscope After the 3rd day of the treatment, the head of the model rat was cut and the brain was obtained and washed by normal saline; cerebral tissue in the 3 different locations of cortex, hippocampus and striatum were made into light specimens respectively, at 400 times in high magnification normal nerve cell Table 2.

When data processing, the concept of negative index was introduced, namely, the indices of infarction rate, necrosis nerve cell, inflammatory nerve cell, neuronophage and necrosis nerve cell rate were changed into negative indices, then the mathematical model of comprehensive evaluation on acupuncture effect was established by the principal component-factor analysis, the weight coefficients were ascertained, comprehensive effect score of each sample was computed, by which a cluster analysis was made by the statistical software of SPSS 17.0.

RESULT Classified Comparison in the Acupuncture Groups Classified comparison in acupuncture groups showed that for the Neiguan group, the parameter 7, 8, 9, and 10 were of the powerful therapeutic result of acupuncture comprehensive effect; the parameter 2, 3, and 4 were of the effective therapeutic result and the parameters 5 and 6 were of invalid. The rest of the groups were just like the Neiguan group. Variance analysis showed that there was a significant difference in comparison among 3 effect levels of comprehensive effect in every acupoint group (P

Cluster analysis for acupoint specificity of acupuncture effect based on cerebral infarction rat model.

To study the acupoint specificity through the comprehensive evaluation of the acupuncture effect on rat model with the middle cerebral artery occlusio...
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