针灸对HIBD模型幼鼠细胞凋亡及相关蛋白表达的影响
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摘要
新生儿缺氧缺血性脑损伤(hypoxic-ischemic brain damage, HIBD)是由于新生儿窒息,引起脑血供和气体交换障碍所致的一种全脑性损伤,是严重危害新生儿生命健康的疾病,有较高的发病率和死亡率,也是造成儿童智能低下、脑瘫等神经系统残疾的重要原因之一。HIBD的发病原因及发病机制较为复杂,越来越多的证据表明,细胞凋亡(Apoptosis)也参与HIBD,并在其中发挥独特的作用。神经元细胞凋亡的发生与患儿的预后有着密切的联系。在分子水平上,细胞凋亡过程受到多种基因及细胞因子的调控,Bcl-2家族是研究的热点,其中Bax和Bcl-2的相对比值在细胞凋亡中起着重要作用；本实验通过建立新生鼠缺氧缺血性脑损伤模型,对损伤后的各组动物进行针刺、温和灸、针刺结合温和灸后,利用HE染色和免疫组织化学手段观察脑组织中凋亡细胞及其细胞调控因子的动态表达规律,探讨新生鼠缺氧缺血性脑损伤神经元凋亡的形态学动态变化、凋亡调控蛋白动态表达规律,为针灸对新生儿缺氧缺血性脑损伤神经元凋亡及调控蛋白的影响机制研究提供动物实验依据。为推广早期针灸改善新生儿学习记忆能力、提高针灸临床疗效提供实验依据。
(一)研究目的
通过观察针灸HIBD模型幼鼠行为学、学习记忆能力的改变和脑细胞凋亡及相关基因表达的影响,研究针灸对HIBD模型幼鼠治疗作用,为小儿脑瘫早期介入提供实验依据。
(二)研究方法
本实验分为两部分,首先模拟围产期窒息建立HIBD模型,利用悬吊试验、斜坡试验、Y迷宫试验来测试受干预后HIBD模型鼠的学习记忆能力并从细胞水平上观察HIBD模型鼠海马组织正常神经细胞计数及脑皮质组织细胞凋亡情况；随后利用免疫组织化学方法观察HIDB模型幼鼠凋亡调控蛋白动态表达规律来探讨针灸是否可以对抗此效应,以探讨HIBD学习记忆障碍的可能机理和针灸改善HIBD学习记忆障碍的机制。
将动物随机分成①正常组②假手术组③模型组④单纯针刺组⑤温和灸组⑥针刺+温和灸组,制定了治疗的时间表后穴位定位参照《实验针灸学》,结合人与动物骨度类比：顶骨正中定百会；外耳道口直上0.8cm处模拟颞Ⅰ针；桡骨近端的关节外侧,离腕关节约3mm左右的尺桡骨缝间取内关；曲池,桡骨近端的关节外侧前方的凹陷中；足三里(即后三里),在膝关节后外侧,腓骨小头下约5mm处；后肢掌心前正中取涌泉穴进行诊疗,治疗两个阶段后,分别进行HIBD模型幼鼠的行为运动能力及学习记忆能力测试,然后处死取材,分别检测模型幼鼠的脑细胞病理形态、凋亡及相关的基因表达指标。
(三)结果1.体重指标：多重比较结果显示：①正常组,②假手术组,⑤艾灸组,⑥针刺+艾灸组这四组之间均数无统计学差异,③模型组与④单纯针刺组之间也无统计学差异。而前四组与后两组相比,P=0.000<0.01,体重增加都有统计学意义,可以认为前四组比后两组体重增加更多。区组间F=16.670,P=0.149>0.05,可认为区组因素没有统计学意义。以上结果说明：与模型组相比,灸组、针刺+艾灸组对HIBD幼鼠的体重增加有促进作用,它们两者之间没差异,使其体重增加速度和幅度与正常和假手术幼鼠相当。单纯针刺在这次实验中没发现有作用,原因需进一步研究。
2.悬吊试验、斜坡试验及Y迷宫试验结。肌力(悬吊试验),②假手术组>①正常组>⑥针刺+温和灸组>⑤温和灸组>③模型组>④单纯针刺组；平衡能力(斜坡试验)①正常组>②假手术组>⑥针刺+温和灸组>⑤温和灸组>④单纯针刺组>③模型组。因素time和处理方法的交互作用方面,肌力(悬吊试验)增长趋势：②假手术组>①正常组>⑥针刺+温和灸组>⑤温和灸组>③模型组>④单纯针刺组；平衡能力(斜坡试验)增长趋势：①正常组>②假手术组>⑥针刺+温和灸组>④单纯针刺组>⑤温和灸>③模型组。以上结果说明：与模型组相比,艾灸对HIBD模型幼鼠的肌力和平衡能力增长得更好,而针刺结合温和灸后效果更佳；同样的,与模型组相比,温和灸对HIBD幼鼠的肌力和平衡能力的增长有更好的促进作用,而针刺结合温和灸后效果更佳；但都比不上正常组和假手术组。针刺结果不稳定,原因需进一步研究。Y迷宫实验结果显示：不同处理组间,F=2.3753,P=0.000<0.01。不同窝别间,F=40.404,P=0.000<0.01。可以认为不同处理组间的Y迷宫正确次数不完全相等。多重比较结果显示：①正常组>②假手术组>⑥针刺+温和灸组>⑤温和灸组>④单纯针刺组>③模型组。说明在智力和记忆力方面,针刺、温和灸对HIBD模型幼鼠都有促进作用,而两者结合后效果最佳。
3.脑组织病理形态学观察及细胞数
(1)海马HE染色观察：普通光镜10×20倍下随机观察每张切片5个视野内脑缺血缺氧后病理变化情况。各组实验大鼠脑组织病理形态学改变：脑组织常规石蜡切片,HE染色后,神经细胞胞浆呈淡红色,核呈蓝黑色。正常对照组及假手术组皮层脑细胞结构正常,胞核清晰,胞浆丰富,间质无水肿表现,海马锥体细胞2-3层,排列紧密,细胞核圆而大,核仁清晰。模型组可见皮层变薄,部分神经元变性,体积变小,胞核与胞浆界限模糊,核固缩成三角形或不规则形,核仁消失。海马锥体细胞层数减少,排列稀疏、不规则,细胞体积变小,可见核固缩现象。针刺组、温和灸组及针刺结合温和灸治疗组仍有不同程度的神经细胞变性,但变性神经细胞数量显著减少,零星可见一些损伤细胞,海马锥体细胞层细胞形态、排列基本正常。
(2)各组幼鼠脑皮质正常神经细胞数目的比较结果示：假手术组与正常对照组比较(P>0.05)差距无统计学意义；假手术组与模型组对照(P<0.01),差距有显著的统计学意义；治疗组与模型组对照差距(P<0.05)有统计学意义,且针刺结合艾温和灸组在所有的治疗组中的效果更优。结果表明,针刺、温和灸、针刺结合温和灸对HIBD模型幼鼠的治疗效果均有统计学意义。且针刺结合温和灸组疗效更优。
(3)实验各组脑皮质组织细胞凋亡数比较结果示：假手术组与正常对照组比较(P>0.05)差距无统计学意义；假手术组与模型组对照(P<0.001),差距有极显著的统计学意义；治疗组与模型组对照差距(P<0.01)有显著统计学意义,且针刺结合温和灸组在多有的治疗组中的效果更明显。结果表明,针刺、温和灸、针刺结合温和灸对HIBD模型幼鼠的治疗效果均有统计学意义。且针刺结合温和灸组疗效更优。
4.脑细胞超微结构
正常组：神经元胞核大而圆,双层核膜有清晰的核周隙,其宽度均匀而明显；胞质内有较多核糖体、内质网、高尔基氏体和线粒体；线粒体呈圆或卵圆形,嵴较多而明显。
假手术组：神经细胞胞体基本完整,细胞核呈圆形或卵圆形,膜结构完整、规则,核周间隙均匀,核内染色质分布均匀,偶见异染色质；胞质内细胞器丰富,可见高尔基体呈扁平囊泡平行排列；线粒体增多,呈圆形、椭圆形,基质均匀,偶有空泡；内质网核糖体丰富。
模型组：神经细胞核膜模糊,多呈不规则形状,核内异染色质增多、电子密度增高,核膜及核周隙模糊不清,局部核质分界不清；核仁呈均一电子密度,网状结构消失；细胞器减少或变形,结构不清；线粒体呈气球样肿胀,粗面内质网几乎见不到；质膜结构不清或消失断裂。
针刺组：神经细胞核膜尚规则,核内染色质仍有凝集,胞质内可见大量线粒体,形态基本正常,偶有肿胀,空化；胞质内可见粗面内质网、高尔基体有空泡样改变,分布散乱。
温和灸组：神经细胞核膜尚规则,胞质内常染色质均匀,偶见异染色质,胞质内可见大量线粒体,形态基本正常,偶有肿胀、空化；胞质内可见粗面内质网、高尔基体有空泡样改变,分布散乱。针灸结合组：神经细胞核膜尚规则,胞质内常染色质丰富,可见大量线粒体,基质较均匀,偶有肿胀；胞质内粗面内质网、高尔基体囊腔少数略有扩张。
5.实验各组幼鼠大脑皮质bcl-2、bax蛋白表达
(1)各组幼鼠大脑皮质bcl-2蛋白表达的比较结果示：假手术组与正常对照组比较(P>0.05)差距无统计学意义；假手术组与模型组对照(P<0.05),差距有统计学意义；治疗组与模型组对照(P<0.05)差距有统计学意义,且针刺结温和灸组在所有的治疗组中的效果更明显。结果表明,针刺、温和灸、针刺结合温和灸对HIBD模型幼鼠的治疗效果均有统计学意义。且针刺结合温和灸组疗效更优。
(2)各组实验幼鼠大脑皮质bax蛋白表达的比较结果示：假手术组与正常对照组比较(P>0.05)差距无统计学意义；假手术组与模型组对照(P<0.05),差距有统计学意义；治疗组与模型组对照(P<0.05)差距有统计学意义,且针刺结合温和灸组在多有的治疗组中的效果更明显。结果表明,针刺、温和灸、针刺结合温和灸对HIBD模型幼鼠的治疗效果均有统计学意义。且针刺结合温和灸组疗效更优。
(四)结论
1.从实验一结果看,假手术组、模型组及治疗组都有不同程度的细胞凋亡,从细胞超微结构的结果来观察,干预组对HIBD脑组织细胞都有保护的治疗效应,且除了能减轻神经元损伤外,更重要的是应该能改善脑细胞凋亡造成的远期功能改变。
2.本实验选用百会、颞1针、内关、曲池、足三里、涌泉给予针刺激并温和灸幼鼠的头顶区,发现慢性应激大鼠的空间学习和记忆能力明显提高。同时可明显减轻缺血缺氧对神经元和神经胶质细胞超微结构的损害程度。
3.本实验二通过对HIBD模型幼鼠在同一基线特征的基础上随机分配成四组,分别是：模型组、针刺组、针刺结合温和灸组和温和灸组,并设立了假手术组和正常对照组。在针刺、温和灸、针灸结合温和灸干预下,结果发现：Bcl-2蛋白表达增加,Bax蛋白表达增下降,细胞凋亡发生过程得到控制。且针刺结合温和灸的效果更优,
4.从实验的结果看,灸法对HIBD模型幼鼠早期介入的疗效值得肯定,这对脑瘫患儿早期治疗提供了一个简便易行的独特疗法打下基础,值得推广和应用。因细胞凋亡主要发生在缺氧缺血相对较早的时期,如果在达到凋亡高峰后仍不能得到足够的血液灌注或抑制凋亡措施的干预,其结果造成局部脑组织进一步发展为坏死,若能得到及时有效的治疗,就有可能阻断、甚至逆转细胞凋亡,从而挽救神经元。
5.在缺氧缺血神经元凋亡的过程中,存在着若干神经元保护的机制,如果这些机制能早期在积极而有效的各种适当的干预下,则有可能可使细胞凋亡的进程得到控制,甚至重新建立新的细胞生长机制。
Neonatal hypoxic-ischemic brain damage (HIBD) is due to asphyxia caused cerebral blood supply and gas exchange impairment caused by a brain injury, a serious disease endangering the health of newborn lives, have a higher morbidity and mortality, also one of the important reasons contributed to children's mental retardation, cerebral palsy and other neurological disabilities. The causes and pathogenesis of HIBD are more complex; more and more evidence has showed that apoptosis is also involved and plays a unique role. The occurrence of neuronal apoptosis and prognosis in children is closely linked. At the molecular level, apoptosis is affected by many genes and the regulation of cytokines, Bcl-2 family is the research focus, Bax and Bcl-2 in which the relative ratio of apoptosis plays an important role. This experiment establishes of hypoxic ischemic brain injury model and after injury all mouses in each group apply the corresponding intervention, using TUNEL staining and immunohistochemical methods to examine brain tissue and cell apoptosis regulation factors of dynamic expression pattern, explore HIBD in neuronal apoptosis dynamic morphological changes, apoptosis regulatory proteins in the dynamic expression pattern, for acupuncture on neonatal HIBD and providing evidence for neuronal apoptosis mechanism of regulation of protein via animal experiment. This promotes early acupuncture treatment for improving neonatal's learning and memory ability and to improve the clinical efficacy of acupuncture by providing experimental evidence.
research purposes
Though observing HIBD model on the changes in learning and memory in mouses and brain cell apoptosis and related gene expression, to further study acupuncture on the treatment of mouse HIBD model and lay the groundwork for molecular mechanism, to provide a new scientific information on the mechanism on acupuncture treatment to HIBD model, to promote early acupuncture treatment for improving learning and memory ability of neonatal and improve the clinical efficacy of acupuncture by providing experimental evidence.
research methods
The experiment was divided into two parts:firstly to establish a simulation model of perinatal asphyxia HIBD, using suspension tests, the slope test, Y maze test to test the model by the intervention HIBD learning and memory in mice and observed from the cell level model of hippocampal HIBD normal tissue cell and cerebral cortex neural cell apoptosis; then used immunohistochemistry to observe the HIDB mouse dynamic expression of apoptosis regulatory proteins to investigate whether acupuncture can combat this effect, to explore the possible mechanism of learning and memory impairment of HIBD and how acupuncture could improve learning and memory impairment caused by HIBD. The animals were randomly divided into (1)normal group, (2) sham group, (3) model group, (4) simple acupuncture group, (5) moxibustion group, (6) acupuncture+moxibustion group, developed a treatment timetable with acupoints reference from (Study of experimental acupuncture), combining human and animal bones analogy:acupoints taken for treatment include parietal bone center for'Baihui'; external ear canal, straight 0.8cm simulate take 'Temporal 1 needle'; radial bone proximal lateral aspect from the wrist off 3cm or so take'Neiguan'; radius proximal lateral joint when elbow is flexed take'Quchi'; anterior aspect of lower leg under the fibular head and about 5mm take'Zusanli'; hind legs before the middle of the sole take'Yongquan' After treatment for two stages, separately carry out tests for HIBD mouse model on behavior motor ability and on learning and memory ability, and then after mice were sacrificed, examine the model mouse brain cell apoptosis and related gene expression targets.
Results
1. Weight:multiple comparisons showed that:for the four groups (1) the normal group, (2) sham group, (5) moxibustion group, (6) acupuncture+moxibustion group, there was no statistical significance between the mean, there was also no statistical significance on (3) model group and (4) simple acupuncture group. The first four groups compared with the later two groups, P= 0.000 <0.01, both statistically significant in weight gain can be considered, the first four group weight gain more than the other two groups. Area group F= 16.670, P= 0.149> 0.05, can be considered that area group was not statistically significant. These results suggest that:compared with the model group, moxibustion group, acupuncture+moxibustion group can promote weight gain in HIBD mouse, without differences between them same as the speed and magnitude of weight gain in normal and sham group mouse. Simple acupuncture in this experiment did not find useful and further study is needed.
2. Over hanging test (OHT), inclined plane test (IPT) and the Y maze test node. Strength (suspension test), (2) sham group>(1) normal group>(6) acupuncture +moxibustion group>(5) moxibustion group>(3) model group>(4) simple acupuncture group; balance ability (inclined plane test) (1) normal group>(2) sham group>(6) acupuncture+moxibustion group>(5) moxibustion group>(4) simple acupuncture group>(3) model group. Factors time and treatment interaction, the strength (suspension test) trend growth:(2)sham group>(1) normal group)(6) acupuncture+moxibustion group)(5) moxibustion group>(3) model group>(4) simple acupuncture group; balance (inclined plane tests) growth trends:(1) normal group>(2)sham group>(6) acupuncture+NGF group>(4) simple acupuncture group> (5) acupuncture+moxibustion group> (3) model group. These results suggest that:compared with model group, moxibustion on HIBD mice increased muscle strength and balance ability, the effect of acupuncture combined with moxibustion were even better; similarly compared with the——group, moxibustion on HIBD mice increased muscle strength and balance balance ability, the effect of acupuncture combined with moxibustion were even better; but could not compare with the normal group and sham group. Acupuncture results were unstable and suggested for further study. Y maze test results showed that: different treatment groups, F= 2.3753, P= 0.000<0.01. Between different group, F= 40.404, P= 0.000<0.01. That suggested the correct numbers for the different treatment groups in the Y maze is not totally equal. Multiple comparison results showed that:(1) normal group>(2) sham group>(6) acupuncture +moxibustion group>(5) moxibustion group>(4) simple acupuncture group>(3) model group. That suggested both acupuncture and moxibustion on HIBD mice could promote the intelligence and memory ability, combination would be even better.
3. Brain pathology and cell morphology
(1) HE staining:under normal light microscope 10 X 40 times for each section were observed stochastically each slice 5 field pathological changes after cerebral ischemia. Pathological changes in mice brain for each group were examined:brain tissue of convention paraffin slice, after HE staining, nerve cells showed light red, with blue-black nucleus. The cerebral cortex brain cell structure was normal for the normal group and the sham group, clear nucleus, cytoplasm-rich, interstitial edema-free, hippocampal pyramidal cells in 2 to 3 layers, closely arranged, round and large nucleus, clear nucleolus. For the model group, cortical thinning was observed, part of the neuronal degeneration and smaller, the boundary of nucleus and cytoplasm was not clear, nuclear shrunk into triangles or irregular shapes, nucleoli disappear. Reduced hippocampal pyramidal cell layer, arranged in sparse, irregular, smaller cell volume, nuclear shrunk phenomenon can be seen. For the acupuncture group, acupuncture combined with moxibustion and moxibustion group, there are varying degrees of nerve cell denatured, but the quantiy of denatured nerve cells was significantly reduced, occasionally observed some damaged cells, hippocampal pyramidal cell layer, cell morphology and arrangement were normal.
Experiment on individual groups on their normal hippocampus nerve cell count
(2) comparison of normal mice cerebral cortex nerve cells:the sham group and the normal group (P> 0.05) difference was not statistically significant; sham group and model group (P<0.01), the difference was statistically significant; treatment group and model group difference (P<0.05) was statistically significant, and acupuncture combined with moxibustion group was more significant. The results showed that acupuncture, moxibustion, acupuncture combined with moxibustion treatment on HIBD model were statistically significant. And the effect of acupuncture combined with moxibustion group were better.
(3) cerebral cortex in experimental groups compared with the number of apoptosis results showed that:the sham group and the normal control group (P> 0.05) difference was not significant; sham operation group and model group (P<0.001), there were major statistical significancy; treatment group and model group, the difference (P<0.01) are statistically significant, and acupuncture combined with moxibustion group were amongst the treatment group more effective. The results show that acupuncture, moxibustion, acupuncture combined with moxibustion treatment on HIBD model were statistically significant. The effect of acupuncture combined with moxibustion group was even better.
4. Ultrastructure of brain cells
(1) Normal group:neuronal cell body integrity, the nuclei were round, smooth nuclear membrane without folds, with nuclear pore, nucleus space uniform, evenly distributed nuclear chromatin, euchromatin-rich nucleus, occasionally nucleolus, cell quality rich in various organelles, normal morphology and structural integrity.
(2) Sham group:number of neurons and more occasional interstitial cell gap, the nuclei were round or oval, clear membrane structure, rules, uniform perinuclear space, nuclear chromatin distribution, occasionally heterochromatin; abundant organelles, structural integrity, takes the shape of flattened golgi vesicles can be seen parallel; mitochondria were round, oval, film rules, uniform matrix; ribosome-rich endoplasmic reticulum.
(3) Model group:number of neurons and more nuclei with irregular shape, decreased organelles, cell shrinkage, chromatin condensing different integrated block; mitochondria decreased, swelling into a spherical, transparent into the vacuole; rough endoplasmic reticulum expansion, surface ribosomes fall off. Apoptotic state of cells in more condensed cytoplasm, the whole cell electron density is higher, more irregular nuclei, chromatin highly condensed, marginalized.
(4) Acupuncture group:neuronal nuclear membrane more clear and complete, uniform chromatin, the cytoplasm shows a large number of mitochondria, were essentially normal, occasional swelling, cavitation; occasionally Golgi; endoplasmic reticulum rich in ribosomes, the cell has micro-swelling.
(5) Moxibustion group:a little fuzzy neuron membrane, cytoplasm, euchromatin-rich, occasionally heterochromatin, shows a large number of mitochondria within the cytoplasm, were essentially normal, small amount of swelling and cavitation; endoplasmic reticulum ribosome rich cells a micro-swelling.
(6) Acupuncture combined with moxibustion group:neuronal nuclear membrane nuclear membrane is smooth, without wrinkles, euchromatin-rich cytoplasm, showing that a large number of mitochondria were round, oval, film rules, matrix is uniform, with occasional swelling; endoplasmic reticulum ribosomes than rich in cells micro-swelling.
5. Experiment on each group of big mouse on cerebral cortex bcl-2 protein expression situation
(1) comparison results of the cerebral cortex bcl-2 protein showed:the sham group and the normal control group (P> 0.05) difference was not significant; sham operation group and model group (P<0.05), the difference was statistically significant; treatment groups and model group (P<0.05) difference was statistically significant, and the acupuncture combined moxibustion treatment group obviously performed better than other groups. The results show that acupuncture, moxibustion, acupuncture combined with moxibustion on HIBD model were statistically significant and the effect of acupuncture combined with moxibustion group was even better.
(2) The set of experimental cerebral cortex the expression of bax protein comparison results showed:the sham group compared with the control group (P> 0.05) difference was not significant; the sham group compared with model group (P<0.05), difference was statistically significant; treatment groups and model group (P<0.05) difference was statistically significant, and the acupuncture combined moxibustion group performed even better. The results show that acupuncture, moxibustion, acupuncture combined with moxibustion treatment on HIBD model were statistically significant and the effect of acupuncture combined with moxibustion group was even better.
Conclusion
1. The results showed the sham group, the model group and the treatment groups have different degrees of apoptosis. It was observed that the intervention groups had protection treatment effect to HIBD brain cells, reduced the nerve cell damage and, more importantly be able to improve the eventual condition caused by brain cells apoptosis. Recent medical acupuncture reports in China indicated that acupuncture and moxibustion are effective to hypoxic ischemic brain damage and can improve learning and memory disorder caused by various reasons. The experiment used acupoints Baihui','Temporal one pin','Neiguan','Quchi','Zusanli' and 'Yongquan'and found that spatial learning and memory ability of mice could be significantly enhanced, while activity in the hippocampus Nos down, NO reduce hippocampal formation significantly reduced neuronal apoptosis, indicating that acupuncture on chronic stress-induced spatial learning and memory impairment have significant protective effect, probably related to the decrease on hippocampal neurons No toxicity. Acupuncture to neonatal hypoxic-ischemic brain damage mice at acupoints'Baihui','Temporal one pin','Neiguan','Quchi', 'Zusanli'and'Yongquan'can significantly reduce ischemia on neurons and glial ultrastructure damage. All mammals have a brain growth and development of a critical period, critical period of mice brain development is 5 weeks before and 3 weeks after, cerebrum development and maturation is a month or so. During this period the structure of brain tissue, biochemical and physiological functions are rapidly mature, most plasticity, compensatory ability is the best. If HIBD occurs, this could lead to edema, necrosis, hemorrhage which can affect brain development, but suitable stimulation at the above acupoints can relieve spasm, and promote nerve conduction and functional recovery of lesions and improve the function of brain cortex such that damaged brain cell could be regenerated, thereby improving the clinical symptoms. In this study, I applied the above acupoints to the neonatal mouse, one week after its birth.'Baihui'in lots of ancient acupuncture literature made reference to mental function, nerves and mind; a number of modern clinical research shows that Baihui can regulate central nervous system, the brain function, improve brain disorders, cerebrovascular circulation and increase cerebral blood flow, systolic and diastolic blood vessels, qi and blood, and organs disease with lower function. Temporal one pin correspond the temporal three-pin position to follow the line of intersection shaoyang meridian, and also lies in the temporal cortex projection area, and related to learning and memory. The Temporal three pins can be encouraged by the Shaoyang temporal germinal of the machine, which will help improve the physical motor function, at the same time promote the improvement of intelligence. Yongquan were first seen in "Lingshu" Meridian" chapter, for the Shao Yin Kidney Meridian of the well points, located in foot and toe plantar flexion was depression time, power good at yang sedation, Su Jue Stasis, Jiangni qi through the meridians, will help to improve motor function of limbs at the same time can promote the improvement of intelligence.'Neiguan'for the pericardium Meridian is for indications and mind-related diseases, to accompany 'Quchi' attending upper limb; both'Zusanli','Quchi'are main acupoints for a strongand balanced body which have been used in the treatment of paralyzed limbs, brain paralysis and improve coordination and balance of the body.
2. The second experiment on HIBD model based on the same baseline characteristics were randomly assigned into four groups, namely:the model group, acupuncture, acupuncture combined with moxibustion group and moxibustion group and the establishment of the sham group and normal control group. Measures in the different treatment intervention was found:Bcl-2 protein expression, Bax expression decreased growth, cell apoptosis process control. Apoptosis occurs mainly in relatively early stage in hypoxic-ischemic, if, after reaching the peak of apoptosis cannot get enough blood perfusion or measures of intervention to inhibit apoptosis, which resulted in the further development of local brain tissue necrosis. If timely and effective treatment can be exercised, it may control or even reverse the apoptosis, and thus save the neurons. Therefore, in the process of hypoxic ischemic neuronal apoptosis there exists a number of neuroprotective mechanisms, if these mechanisms are active triggered with appropriate and effective intervention, this will likely control the process or speed of apoptosis, or even build a new mechanism of cell growth.
(一)研究目的
通过观察针灸HIBD模型幼鼠行为学、学习记忆能力的改变和脑细胞凋亡及相关基因表达的影响,研究针灸对HIBD模型幼鼠治疗作用,为小儿脑瘫早期介入提供实验依据。
(二)研究方法
本实验分为两部分,首先模拟围产期窒息建立HIBD模型,利用悬吊试验、斜坡试验、Y迷宫试验来测试受干预后HIBD模型鼠的学习记忆能力并从细胞水平上观察HIBD模型鼠海马组织正常神经细胞计数及脑皮质组织细胞凋亡情况；随后利用免疫组织化学方法观察HIDB模型幼鼠凋亡调控蛋白动态表达规律来探讨针灸是否可以对抗此效应,以探讨HIBD学习记忆障碍的可能机理和针灸改善HIBD学习记忆障碍的机制。
将动物随机分成①正常组②假手术组③模型组④单纯针刺组⑤温和灸组⑥针刺+温和灸组,制定了治疗的时间表后穴位定位参照《实验针灸学》,结合人与动物骨度类比：顶骨正中定百会；外耳道口直上0.8cm处模拟颞Ⅰ针；桡骨近端的关节外侧,离腕关节约3mm左右的尺桡骨缝间取内关；曲池,桡骨近端的关节外侧前方的凹陷中；足三里(即后三里),在膝关节后外侧,腓骨小头下约5mm处；后肢掌心前正中取涌泉穴进行诊疗,治疗两个阶段后,分别进行HIBD模型幼鼠的行为运动能力及学习记忆能力测试,然后处死取材,分别检测模型幼鼠的脑细胞病理形态、凋亡及相关的基因表达指标。
(三)结果1.体重指标：多重比较结果显示：①正常组,②假手术组,⑤艾灸组,⑥针刺+艾灸组这四组之间均数无统计学差异,③模型组与④单纯针刺组之间也无统计学差异。而前四组与后两组相比,P=0.000<0.01,体重增加都有统计学意义,可以认为前四组比后两组体重增加更多。区组间F=16.670,P=0.149>0.05,可认为区组因素没有统计学意义。以上结果说明：与模型组相比,灸组、针刺+艾灸组对HIBD幼鼠的体重增加有促进作用,它们两者之间没差异,使其体重增加速度和幅度与正常和假手术幼鼠相当。单纯针刺在这次实验中没发现有作用,原因需进一步研究。
2.悬吊试验、斜坡试验及Y迷宫试验结。肌力(悬吊试验),②假手术组>①正常组>⑥针刺+温和灸组>⑤温和灸组>③模型组>④单纯针刺组；平衡能力(斜坡试验)①正常组>②假手术组>⑥针刺+温和灸组>⑤温和灸组>④单纯针刺组>③模型组。因素time和处理方法的交互作用方面,肌力(悬吊试验)增长趋势：②假手术组>①正常组>⑥针刺+温和灸组>⑤温和灸组>③模型组>④单纯针刺组；平衡能力(斜坡试验)增长趋势：①正常组>②假手术组>⑥针刺+温和灸组>④单纯针刺组>⑤温和灸>③模型组。以上结果说明：与模型组相比,艾灸对HIBD模型幼鼠的肌力和平衡能力增长得更好,而针刺结合温和灸后效果更佳；同样的,与模型组相比,温和灸对HIBD幼鼠的肌力和平衡能力的增长有更好的促进作用,而针刺结合温和灸后效果更佳；但都比不上正常组和假手术组。针刺结果不稳定,原因需进一步研究。Y迷宫实验结果显示：不同处理组间,F=2.3753,P=0.000<0.01。不同窝别间,F=40.404,P=0.000<0.01。可以认为不同处理组间的Y迷宫正确次数不完全相等。多重比较结果显示：①正常组>②假手术组>⑥针刺+温和灸组>⑤温和灸组>④单纯针刺组>③模型组。说明在智力和记忆力方面,针刺、温和灸对HIBD模型幼鼠都有促进作用,而两者结合后效果最佳。
3.脑组织病理形态学观察及细胞数
(1)海马HE染色观察：普通光镜10×20倍下随机观察每张切片5个视野内脑缺血缺氧后病理变化情况。各组实验大鼠脑组织病理形态学改变：脑组织常规石蜡切片,HE染色后,神经细胞胞浆呈淡红色,核呈蓝黑色。正常对照组及假手术组皮层脑细胞结构正常,胞核清晰,胞浆丰富,间质无水肿表现,海马锥体细胞2-3层,排列紧密,细胞核圆而大,核仁清晰。模型组可见皮层变薄,部分神经元变性,体积变小,胞核与胞浆界限模糊,核固缩成三角形或不规则形,核仁消失。海马锥体细胞层数减少,排列稀疏、不规则,细胞体积变小,可见核固缩现象。针刺组、温和灸组及针刺结合温和灸治疗组仍有不同程度的神经细胞变性,但变性神经细胞数量显著减少,零星可见一些损伤细胞,海马锥体细胞层细胞形态、排列基本正常。
(2)各组幼鼠脑皮质正常神经细胞数目的比较结果示：假手术组与正常对照组比较(P>0.05)差距无统计学意义；假手术组与模型组对照(P<0.01),差距有显著的统计学意义；治疗组与模型组对照差距(P<0.05)有统计学意义,且针刺结合艾温和灸组在所有的治疗组中的效果更优。结果表明,针刺、温和灸、针刺结合温和灸对HIBD模型幼鼠的治疗效果均有统计学意义。且针刺结合温和灸组疗效更优。
(3)实验各组脑皮质组织细胞凋亡数比较结果示：假手术组与正常对照组比较(P>0.05)差距无统计学意义；假手术组与模型组对照(P<0.001),差距有极显著的统计学意义；治疗组与模型组对照差距(P<0.01)有显著统计学意义,且针刺结合温和灸组在多有的治疗组中的效果更明显。结果表明,针刺、温和灸、针刺结合温和灸对HIBD模型幼鼠的治疗效果均有统计学意义。且针刺结合温和灸组疗效更优。
4.脑细胞超微结构
正常组：神经元胞核大而圆,双层核膜有清晰的核周隙,其宽度均匀而明显；胞质内有较多核糖体、内质网、高尔基氏体和线粒体；线粒体呈圆或卵圆形,嵴较多而明显。
假手术组：神经细胞胞体基本完整,细胞核呈圆形或卵圆形,膜结构完整、规则,核周间隙均匀,核内染色质分布均匀,偶见异染色质；胞质内细胞器丰富,可见高尔基体呈扁平囊泡平行排列；线粒体增多,呈圆形、椭圆形,基质均匀,偶有空泡；内质网核糖体丰富。
模型组：神经细胞核膜模糊,多呈不规则形状,核内异染色质增多、电子密度增高,核膜及核周隙模糊不清,局部核质分界不清；核仁呈均一电子密度,网状结构消失；细胞器减少或变形,结构不清；线粒体呈气球样肿胀,粗面内质网几乎见不到；质膜结构不清或消失断裂。
针刺组：神经细胞核膜尚规则,核内染色质仍有凝集,胞质内可见大量线粒体,形态基本正常,偶有肿胀,空化；胞质内可见粗面内质网、高尔基体有空泡样改变,分布散乱。
温和灸组：神经细胞核膜尚规则,胞质内常染色质均匀,偶见异染色质,胞质内可见大量线粒体,形态基本正常,偶有肿胀、空化；胞质内可见粗面内质网、高尔基体有空泡样改变,分布散乱。针灸结合组：神经细胞核膜尚规则,胞质内常染色质丰富,可见大量线粒体,基质较均匀,偶有肿胀；胞质内粗面内质网、高尔基体囊腔少数略有扩张。
5.实验各组幼鼠大脑皮质bcl-2、bax蛋白表达
(1)各组幼鼠大脑皮质bcl-2蛋白表达的比较结果示：假手术组与正常对照组比较(P>0.05)差距无统计学意义；假手术组与模型组对照(P<0.05),差距有统计学意义；治疗组与模型组对照(P<0.05)差距有统计学意义,且针刺结温和灸组在所有的治疗组中的效果更明显。结果表明,针刺、温和灸、针刺结合温和灸对HIBD模型幼鼠的治疗效果均有统计学意义。且针刺结合温和灸组疗效更优。
(2)各组实验幼鼠大脑皮质bax蛋白表达的比较结果示：假手术组与正常对照组比较(P>0.05)差距无统计学意义；假手术组与模型组对照(P<0.05),差距有统计学意义；治疗组与模型组对照(P<0.05)差距有统计学意义,且针刺结合温和灸组在多有的治疗组中的效果更明显。结果表明,针刺、温和灸、针刺结合温和灸对HIBD模型幼鼠的治疗效果均有统计学意义。且针刺结合温和灸组疗效更优。
(四)结论
1.从实验一结果看,假手术组、模型组及治疗组都有不同程度的细胞凋亡,从细胞超微结构的结果来观察,干预组对HIBD脑组织细胞都有保护的治疗效应,且除了能减轻神经元损伤外,更重要的是应该能改善脑细胞凋亡造成的远期功能改变。
2.本实验选用百会、颞1针、内关、曲池、足三里、涌泉给予针刺激并温和灸幼鼠的头顶区,发现慢性应激大鼠的空间学习和记忆能力明显提高。同时可明显减轻缺血缺氧对神经元和神经胶质细胞超微结构的损害程度。
3.本实验二通过对HIBD模型幼鼠在同一基线特征的基础上随机分配成四组,分别是：模型组、针刺组、针刺结合温和灸组和温和灸组,并设立了假手术组和正常对照组。在针刺、温和灸、针灸结合温和灸干预下,结果发现：Bcl-2蛋白表达增加,Bax蛋白表达增下降,细胞凋亡发生过程得到控制。且针刺结合温和灸的效果更优,
4.从实验的结果看,灸法对HIBD模型幼鼠早期介入的疗效值得肯定,这对脑瘫患儿早期治疗提供了一个简便易行的独特疗法打下基础,值得推广和应用。因细胞凋亡主要发生在缺氧缺血相对较早的时期,如果在达到凋亡高峰后仍不能得到足够的血液灌注或抑制凋亡措施的干预,其结果造成局部脑组织进一步发展为坏死,若能得到及时有效的治疗,就有可能阻断、甚至逆转细胞凋亡,从而挽救神经元。
5.在缺氧缺血神经元凋亡的过程中,存在着若干神经元保护的机制,如果这些机制能早期在积极而有效的各种适当的干预下,则有可能可使细胞凋亡的进程得到控制,甚至重新建立新的细胞生长机制。
Neonatal hypoxic-ischemic brain damage (HIBD) is due to asphyxia caused cerebral blood supply and gas exchange impairment caused by a brain injury, a serious disease endangering the health of newborn lives, have a higher morbidity and mortality, also one of the important reasons contributed to children's mental retardation, cerebral palsy and other neurological disabilities. The causes and pathogenesis of HIBD are more complex; more and more evidence has showed that apoptosis is also involved and plays a unique role. The occurrence of neuronal apoptosis and prognosis in children is closely linked. At the molecular level, apoptosis is affected by many genes and the regulation of cytokines, Bcl-2 family is the research focus, Bax and Bcl-2 in which the relative ratio of apoptosis plays an important role. This experiment establishes of hypoxic ischemic brain injury model and after injury all mouses in each group apply the corresponding intervention, using TUNEL staining and immunohistochemical methods to examine brain tissue and cell apoptosis regulation factors of dynamic expression pattern, explore HIBD in neuronal apoptosis dynamic morphological changes, apoptosis regulatory proteins in the dynamic expression pattern, for acupuncture on neonatal HIBD and providing evidence for neuronal apoptosis mechanism of regulation of protein via animal experiment. This promotes early acupuncture treatment for improving neonatal's learning and memory ability and to improve the clinical efficacy of acupuncture by providing experimental evidence.
research purposes
Though observing HIBD model on the changes in learning and memory in mouses and brain cell apoptosis and related gene expression, to further study acupuncture on the treatment of mouse HIBD model and lay the groundwork for molecular mechanism, to provide a new scientific information on the mechanism on acupuncture treatment to HIBD model, to promote early acupuncture treatment for improving learning and memory ability of neonatal and improve the clinical efficacy of acupuncture by providing experimental evidence.
research methods
The experiment was divided into two parts:firstly to establish a simulation model of perinatal asphyxia HIBD, using suspension tests, the slope test, Y maze test to test the model by the intervention HIBD learning and memory in mice and observed from the cell level model of hippocampal HIBD normal tissue cell and cerebral cortex neural cell apoptosis; then used immunohistochemistry to observe the HIDB mouse dynamic expression of apoptosis regulatory proteins to investigate whether acupuncture can combat this effect, to explore the possible mechanism of learning and memory impairment of HIBD and how acupuncture could improve learning and memory impairment caused by HIBD. The animals were randomly divided into (1)normal group, (2) sham group, (3) model group, (4) simple acupuncture group, (5) moxibustion group, (6) acupuncture+moxibustion group, developed a treatment timetable with acupoints reference from (Study of experimental acupuncture), combining human and animal bones analogy:acupoints taken for treatment include parietal bone center for'Baihui'; external ear canal, straight 0.8cm simulate take 'Temporal 1 needle'; radial bone proximal lateral aspect from the wrist off 3cm or so take'Neiguan'; radius proximal lateral joint when elbow is flexed take'Quchi'; anterior aspect of lower leg under the fibular head and about 5mm take'Zusanli'; hind legs before the middle of the sole take'Yongquan' After treatment for two stages, separately carry out tests for HIBD mouse model on behavior motor ability and on learning and memory ability, and then after mice were sacrificed, examine the model mouse brain cell apoptosis and related gene expression targets.
Results
1. Weight:multiple comparisons showed that:for the four groups (1) the normal group, (2) sham group, (5) moxibustion group, (6) acupuncture+moxibustion group, there was no statistical significance between the mean, there was also no statistical significance on (3) model group and (4) simple acupuncture group. The first four groups compared with the later two groups, P= 0.000 <0.01, both statistically significant in weight gain can be considered, the first four group weight gain more than the other two groups. Area group F= 16.670, P= 0.149> 0.05, can be considered that area group was not statistically significant. These results suggest that:compared with the model group, moxibustion group, acupuncture+moxibustion group can promote weight gain in HIBD mouse, without differences between them same as the speed and magnitude of weight gain in normal and sham group mouse. Simple acupuncture in this experiment did not find useful and further study is needed.
2. Over hanging test (OHT), inclined plane test (IPT) and the Y maze test node. Strength (suspension test), (2) sham group>(1) normal group>(6) acupuncture +moxibustion group>(5) moxibustion group>(3) model group>(4) simple acupuncture group; balance ability (inclined plane test) (1) normal group>(2) sham group>(6) acupuncture+moxibustion group>(5) moxibustion group>(4) simple acupuncture group>(3) model group. Factors time and treatment interaction, the strength (suspension test) trend growth:(2)sham group>(1) normal group)(6) acupuncture+moxibustion group)(5) moxibustion group>(3) model group>(4) simple acupuncture group; balance (inclined plane tests) growth trends:(1) normal group>(2)sham group>(6) acupuncture+NGF group>(4) simple acupuncture group> (5) acupuncture+moxibustion group> (3) model group. These results suggest that:compared with model group, moxibustion on HIBD mice increased muscle strength and balance ability, the effect of acupuncture combined with moxibustion were even better; similarly compared with the——group, moxibustion on HIBD mice increased muscle strength and balance balance ability, the effect of acupuncture combined with moxibustion were even better; but could not compare with the normal group and sham group. Acupuncture results were unstable and suggested for further study. Y maze test results showed that: different treatment groups, F= 2.3753, P= 0.000<0.01. Between different group, F= 40.404, P= 0.000<0.01. That suggested the correct numbers for the different treatment groups in the Y maze is not totally equal. Multiple comparison results showed that:(1) normal group>(2) sham group>(6) acupuncture +moxibustion group>(5) moxibustion group>(4) simple acupuncture group>(3) model group. That suggested both acupuncture and moxibustion on HIBD mice could promote the intelligence and memory ability, combination would be even better.
3. Brain pathology and cell morphology
(1) HE staining:under normal light microscope 10 X 40 times for each section were observed stochastically each slice 5 field pathological changes after cerebral ischemia. Pathological changes in mice brain for each group were examined:brain tissue of convention paraffin slice, after HE staining, nerve cells showed light red, with blue-black nucleus. The cerebral cortex brain cell structure was normal for the normal group and the sham group, clear nucleus, cytoplasm-rich, interstitial edema-free, hippocampal pyramidal cells in 2 to 3 layers, closely arranged, round and large nucleus, clear nucleolus. For the model group, cortical thinning was observed, part of the neuronal degeneration and smaller, the boundary of nucleus and cytoplasm was not clear, nuclear shrunk into triangles or irregular shapes, nucleoli disappear. Reduced hippocampal pyramidal cell layer, arranged in sparse, irregular, smaller cell volume, nuclear shrunk phenomenon can be seen. For the acupuncture group, acupuncture combined with moxibustion and moxibustion group, there are varying degrees of nerve cell denatured, but the quantiy of denatured nerve cells was significantly reduced, occasionally observed some damaged cells, hippocampal pyramidal cell layer, cell morphology and arrangement were normal.
Experiment on individual groups on their normal hippocampus nerve cell count
(2) comparison of normal mice cerebral cortex nerve cells:the sham group and the normal group (P> 0.05) difference was not statistically significant; sham group and model group (P<0.01), the difference was statistically significant; treatment group and model group difference (P<0.05) was statistically significant, and acupuncture combined with moxibustion group was more significant. The results showed that acupuncture, moxibustion, acupuncture combined with moxibustion treatment on HIBD model were statistically significant. And the effect of acupuncture combined with moxibustion group were better.
(3) cerebral cortex in experimental groups compared with the number of apoptosis results showed that:the sham group and the normal control group (P> 0.05) difference was not significant; sham operation group and model group (P<0.001), there were major statistical significancy; treatment group and model group, the difference (P<0.01) are statistically significant, and acupuncture combined with moxibustion group were amongst the treatment group more effective. The results show that acupuncture, moxibustion, acupuncture combined with moxibustion treatment on HIBD model were statistically significant. The effect of acupuncture combined with moxibustion group was even better.
4. Ultrastructure of brain cells
(1) Normal group:neuronal cell body integrity, the nuclei were round, smooth nuclear membrane without folds, with nuclear pore, nucleus space uniform, evenly distributed nuclear chromatin, euchromatin-rich nucleus, occasionally nucleolus, cell quality rich in various organelles, normal morphology and structural integrity.
(2) Sham group:number of neurons and more occasional interstitial cell gap, the nuclei were round or oval, clear membrane structure, rules, uniform perinuclear space, nuclear chromatin distribution, occasionally heterochromatin; abundant organelles, structural integrity, takes the shape of flattened golgi vesicles can be seen parallel; mitochondria were round, oval, film rules, uniform matrix; ribosome-rich endoplasmic reticulum.
(3) Model group:number of neurons and more nuclei with irregular shape, decreased organelles, cell shrinkage, chromatin condensing different integrated block; mitochondria decreased, swelling into a spherical, transparent into the vacuole; rough endoplasmic reticulum expansion, surface ribosomes fall off. Apoptotic state of cells in more condensed cytoplasm, the whole cell electron density is higher, more irregular nuclei, chromatin highly condensed, marginalized.
(4) Acupuncture group:neuronal nuclear membrane more clear and complete, uniform chromatin, the cytoplasm shows a large number of mitochondria, were essentially normal, occasional swelling, cavitation; occasionally Golgi; endoplasmic reticulum rich in ribosomes, the cell has micro-swelling.
(5) Moxibustion group:a little fuzzy neuron membrane, cytoplasm, euchromatin-rich, occasionally heterochromatin, shows a large number of mitochondria within the cytoplasm, were essentially normal, small amount of swelling and cavitation; endoplasmic reticulum ribosome rich cells a micro-swelling.
(6) Acupuncture combined with moxibustion group:neuronal nuclear membrane nuclear membrane is smooth, without wrinkles, euchromatin-rich cytoplasm, showing that a large number of mitochondria were round, oval, film rules, matrix is uniform, with occasional swelling; endoplasmic reticulum ribosomes than rich in cells micro-swelling.
5. Experiment on each group of big mouse on cerebral cortex bcl-2 protein expression situation
(1) comparison results of the cerebral cortex bcl-2 protein showed:the sham group and the normal control group (P> 0.05) difference was not significant; sham operation group and model group (P<0.05), the difference was statistically significant; treatment groups and model group (P<0.05) difference was statistically significant, and the acupuncture combined moxibustion treatment group obviously performed better than other groups. The results show that acupuncture, moxibustion, acupuncture combined with moxibustion on HIBD model were statistically significant and the effect of acupuncture combined with moxibustion group was even better.
(2) The set of experimental cerebral cortex the expression of bax protein comparison results showed:the sham group compared with the control group (P> 0.05) difference was not significant; the sham group compared with model group (P<0.05), difference was statistically significant; treatment groups and model group (P<0.05) difference was statistically significant, and the acupuncture combined moxibustion group performed even better. The results show that acupuncture, moxibustion, acupuncture combined with moxibustion treatment on HIBD model were statistically significant and the effect of acupuncture combined with moxibustion group was even better.
Conclusion
1. The results showed the sham group, the model group and the treatment groups have different degrees of apoptosis. It was observed that the intervention groups had protection treatment effect to HIBD brain cells, reduced the nerve cell damage and, more importantly be able to improve the eventual condition caused by brain cells apoptosis. Recent medical acupuncture reports in China indicated that acupuncture and moxibustion are effective to hypoxic ischemic brain damage and can improve learning and memory disorder caused by various reasons. The experiment used acupoints Baihui','Temporal one pin','Neiguan','Quchi','Zusanli' and 'Yongquan'and found that spatial learning and memory ability of mice could be significantly enhanced, while activity in the hippocampus Nos down, NO reduce hippocampal formation significantly reduced neuronal apoptosis, indicating that acupuncture on chronic stress-induced spatial learning and memory impairment have significant protective effect, probably related to the decrease on hippocampal neurons No toxicity. Acupuncture to neonatal hypoxic-ischemic brain damage mice at acupoints'Baihui','Temporal one pin','Neiguan','Quchi', 'Zusanli'and'Yongquan'can significantly reduce ischemia on neurons and glial ultrastructure damage. All mammals have a brain growth and development of a critical period, critical period of mice brain development is 5 weeks before and 3 weeks after, cerebrum development and maturation is a month or so. During this period the structure of brain tissue, biochemical and physiological functions are rapidly mature, most plasticity, compensatory ability is the best. If HIBD occurs, this could lead to edema, necrosis, hemorrhage which can affect brain development, but suitable stimulation at the above acupoints can relieve spasm, and promote nerve conduction and functional recovery of lesions and improve the function of brain cortex such that damaged brain cell could be regenerated, thereby improving the clinical symptoms. In this study, I applied the above acupoints to the neonatal mouse, one week after its birth.'Baihui'in lots of ancient acupuncture literature made reference to mental function, nerves and mind; a number of modern clinical research shows that Baihui can regulate central nervous system, the brain function, improve brain disorders, cerebrovascular circulation and increase cerebral blood flow, systolic and diastolic blood vessels, qi and blood, and organs disease with lower function. Temporal one pin correspond the temporal three-pin position to follow the line of intersection shaoyang meridian, and also lies in the temporal cortex projection area, and related to learning and memory. The Temporal three pins can be encouraged by the Shaoyang temporal germinal of the machine, which will help improve the physical motor function, at the same time promote the improvement of intelligence. Yongquan were first seen in "Lingshu" Meridian" chapter, for the Shao Yin Kidney Meridian of the well points, located in foot and toe plantar flexion was depression time, power good at yang sedation, Su Jue Stasis, Jiangni qi through the meridians, will help to improve motor function of limbs at the same time can promote the improvement of intelligence.'Neiguan'for the pericardium Meridian is for indications and mind-related diseases, to accompany 'Quchi' attending upper limb; both'Zusanli','Quchi'are main acupoints for a strongand balanced body which have been used in the treatment of paralyzed limbs, brain paralysis and improve coordination and balance of the body.
2. The second experiment on HIBD model based on the same baseline characteristics were randomly assigned into four groups, namely:the model group, acupuncture, acupuncture combined with moxibustion group and moxibustion group and the establishment of the sham group and normal control group. Measures in the different treatment intervention was found:Bcl-2 protein expression, Bax expression decreased growth, cell apoptosis process control. Apoptosis occurs mainly in relatively early stage in hypoxic-ischemic, if, after reaching the peak of apoptosis cannot get enough blood perfusion or measures of intervention to inhibit apoptosis, which resulted in the further development of local brain tissue necrosis. If timely and effective treatment can be exercised, it may control or even reverse the apoptosis, and thus save the neurons. Therefore, in the process of hypoxic ischemic neuronal apoptosis there exists a number of neuroprotective mechanisms, if these mechanisms are active triggered with appropriate and effective intervention, this will likely control the process or speed of apoptosis, or even build a new mechanism of cell growth.
引文
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