【作者】 李星儿；

【导师】 靳瑞； 唐纯志；

【作者基本信息】 广州中医药大学 ， 针灸推拿学， 2012， 博士

【摘要】 目的新生儿缺氧缺血性脑病(hypoxic-ischemic encephalopathy, HIE)严重危害新生儿的健康,是导致脑性瘫痪(cerebral palsy, CP)、癫痫(epilepsy)等儿童脑病的直接因素。对此类疾病的治疗至今仍为世界性医学难题。如何有效改善HIE所致的病理状况一直是医学界研究的重要内容之一。近十几年来,针灸在治疗新生儿缺氧缺血性脑病后遗症之一的脑瘫方面,取得了很大突破,针灸在脑瘫领域展现出空前的繁荣景象。随着研究的深入,一些学者发现患儿接受针灸治疗的年龄越小,疗效越显著,因此萌生了在新生儿期即对缺氧缺血性脑损伤的患儿进行针灸治疗的想法。但由于HIE的危险性和严重性、现代医学目前在我国医学系统尤其是新生儿急救领域的主导地位、中医针灸对该病作用机制的实验研究尚属空白、中医“科学性”受现代医学质疑、针灸作为有创治疗在新生儿领域的安全性等多种原因,决定了目前中医针灸难以进入该领域一展所长。因此,先在动物实验上验证中医针灸的有效性和安全性,并深入探索和揭示针灸治疗HIE的作用机制,将有助于推动针灸治疗进入新生儿HIE领域的进程。靳三针疗法是已故广州中医药大学治疗弱智儿童的首席教授靳瑞教授所创立的学术体系,被誉为“岭南针灸新流派”。靳瑞教授及其带领的靳三针团队,以“靳三针头穴”为主治疗小儿脑病二十余年,疗效肯定,国家中医药管理局已将“靳三针治疗儿童精神发育迟滞和儿童自闭症临床规范化研究”两项成果作为临床适宜疗法向全国推广。目前对“靳三针疗法”治疗小儿脑病的临床观察研究很多,但缺乏对其作用机理的实验基础研究。本论文目的有二：一方面,以“靳三针头穴”为切入点,旨在探索针刺对HIBD新生大鼠的作用机制是否与减少新生神经细胞凋亡有关,并进一步寻求其起效通路,为针灸治疗HIE寻找可靠的实验依据；另一方面,以针刺介入时间为切入点,比较HIBD发生后不同时间开始针刺对新生神经细胞凋亡相关指标的影响,为临床研究提供必要的理论支持。方法文献研究本部分回顾了现代医学对新生儿HIE的认识和治疗进展、古代中医对新生儿HIE认识和现代中医治疗进展、神经干细胞凋亡基因的研究进展等三大内容。在此基础上对新生儿HIE的中西医治疗现状进行了归纳总结。实验研究本部分共包括六个实验项目。实验一采用延迟5min剖宫产术模拟宫内窘迫制造HIBD新生大鼠模型,设立正常组(自然分娩的新生大鼠)和假手术组(正常剖宫产术分娩的新生大鼠)作为对照组。根据新生大鼠出生时神经行为学表现、脑组织HE染色鉴定造模成功。将实验一中获得的各组新生大鼠随机分为正常组、假手术组、模型组、针刺1组和针刺2组,每组再根据处死观察的时间分为21d、28d、35d、42d、49d亚组,每组雌雄各半,饲养到第14天时,用苦味酸标记后,进入后续实验。实验二观察各组新生大鼠14～49天的体重变化,研究针刺对HIBD新生大鼠体重的影响。实验三～六研究针刺对HIBD新生大鼠大脑皮层缺血灶中的新生神经细胞凋亡的影响。其中：实验三采用Tunel法检测大脑皮质中凋亡的神经细胞数量；实验四采用免疫组织化学法检测大脑皮质中促凋亡基因Caspase-3蛋白的表达；实验五采用免疫组织化学法检测大脑皮质中BrdU.Nestin蛋白的表达,来反映大脑皮质中新生神经干细胞的数量和细胞增殖状况；实验六采用免疫组织化学法检测大脑皮质中抗凋亡基因14-3-3蛋白的表达。结果实验研究1、建立模型1)新生大鼠死亡率：出生21天时各组死亡率为正常组5.13%(4/78),假手术组6.82%(3/44),模型组14.66%(28/191),卡方检验p=0.049。2)新生大鼠出生时表现：正常组甫出生即全身呈现较深的粉红色,在雌鼠的舔舐下可扭动身体、挥舞四肢、发出叫声,并可迅速吃乳。假手术组和造模组甫从子宫中取出时,均全身青紫或苍白,四肢伸展,没有呼吸,心跳过速,对触觉刺激无反应。3)新生大鼠出生第2天翻正反射：最短翻正时间假手术组<正常组<模型组,其中模型组与正常组和假手术组的差异均有统计学意义(P<0.01)。10次翻正中的成功次数正常组>假手术组>模型组,其中模型组与正常组和假手术组的差异均有统计学意义(P<0.01)。4)新生大鼠出生第14天悬吊试验：正常组和假手术组完成试验的时间差别无统计学意义(P>0.05),造模组完成试验的时间均短于正常组(P<0.01)和假手术组(P<0.01)。5)新生大鼠出生第14天斜坡试验：正常组和假手术组完成试验的时间差别无统计学意义(P>0.05),造模组完成试验的时间长于正常组(P<0.01),而和假手术组的差别无统计学意义(P>0.05)。其中,正常组参加斜坡试验的新生大鼠27只,成功25只(92.6%),不成功2只(7.4%)；假手术组参加斜坡试验的新生大鼠15只,成功13只(86.7%),不成功2只(13.3%)；造模组参加斜坡试验的新生大鼠84只,成功55只(65.5%),不成功29只(34.5%)。卡方检验P<0.01,可以认为斜坡试验成功与否的结果与组别有关。6)脑组织HE染色：正常组大脑皮质的神经元分布由表及里可见分子层、外颗粒层、外锥体细胞层、内颗粒层、内锥体细胞层、多形细胞层,神经元的6层结构完整。神经元圆形或锥形,细胞质内见嗜碱性均匀分布的尼氏体,细胞核位于胞体中央,核膜明显。在一片正常细胞中间或出现个别固缩细胞。假手术组大脑皮质神经元的6层结构基本完整,局灶区域可见3层(外锥体细胞层)锥体细胞胞浆红染,胞核浓缩深染,结构消失。模型组大脑皮质的3层和5层(外锥体细胞层和内锥体细胞层)锥体细胞数量减少,细胞体积缩小,变成三角形,胞浆呈均匀的嗜伊红染色,胞核浓缩深染,结构消失。部分海马椎体细胞层细胞亦见上述变化。2、针刺对体重的影响：新生大鼠14-49天体重：35天龄前,模型组、针刺1组、针刺2组和正常组或假手术组的体重差异有统计学意义,42天龄后,各组的体重差异均无统计学意义。3、针刺对细胞凋亡的影响：针刺1组凋亡细胞个数21-35天龄时和模型组的差异均无统计学意义(P>0.05)。针刺2组凋亡细胞个数35-49天龄时均少于模型组(P<0.01)。35天龄时,针刺2组凋亡细胞个数少于针刺1组(P<0.05)。4、针刺对Caspase-3的影响：针刺1组Caspase-3的表达21天龄时和模型组的差异无统计学意义(P>0.05),28天龄时低于模型组(P<0.01),35天龄时高于模型组(P<0.01)。针刺2组Caspase-3的表达35天龄时高于模型组(P<0.01),42和49天龄时和模型组的差异无统计学意义(P>0.05)。35天龄时,针刺1组和针刺2组Caspase-3的表达差异无统计学意义(P>0.05)。5、针刺对BrdU的影响：针刺1组BrdU的表达21-35天龄时和模型组的差异无统计学意义(P>0.05)。针刺2组BrdU的表达35-42天龄时和模型组的差异无统计学意义,49天龄时低于模型组(P<0.01)。35天龄时,针刺1组和针刺2组BrdU的表达差异无统计学意义(P>0.05)。6、针刺对Nestin的影响：针刺1组Nestin的表达21和35天龄时和模型组的差异无统计学意义(P>0.05),28天龄时高于模型组(P<0.01)。针刺2组Nestin的表达35-42天龄时和模型组的差异无统计学意义(P>0.05),49天龄时高于模型组(P<0.01)。35天龄时,针刺2组Nestin的表达低于针刺1组(P<0.01)。7、针刺对14-3-3的影响：针刺1组14-3-3的表达21天龄时高于模型组(P<0.01),28天和35天龄时,表达急剧降低,和模型组的差异无统计学意义(P>0.05)。针刺2组14-3-3的表达35天龄时低于模型组和正常组(P<0.01),42天和49天龄时,表达略有上升,而模型组和正常组在此阶段的大脑皮质中已经没有表达。35天龄时,针刺2组14-3-3的表达低于针刺1组(P<0.01)。结论文献研究1、新生儿缺氧缺血性脑病的发病机制和病理生理过程复杂,中西医多种方法综合治疗是未来的发展方向。目前国内外对该病的治疗仍以支持疗法和对症处理为主,高压氧、亚低温、神经营养因子、神经干细胞移植等多种方法仍以动物实验研究为主,未大规模应用于临床。2、古代中医对该病的理论认识和临床经验相当丰富,古人已经发展出一套处理新生儿缺氧缺血性脑病类似症状的辨证论治方案。迄今为止,尚未有学者对古代中医治疗该病的方法进行过系统总结,也未与现代中医的临床和实验研究成功对接。这是一个巨大的宝库,值得广大中医研究者去挖掘和应用。3、现代中医在新生儿缺氧缺血性脑病治疗领域以数种单一中药提取物制剂为主,临床应用也未体现中医的辨证论证精神。针灸推拿对该病的治疗集中于其神经后遗症——脑性瘫痪上,真正在新生儿期介入治疗的临床报道非常少。实验研究1、延迟5min剖宫产术在短期内可以制备大量长期存活的HIBD新生大鼠,高度模拟胎儿宫内窘迫的发病机制,且操作简便,是一种值得推广的研究缺氧缺血性脑损伤的造模方法。该方法可造成相对温和的缺氧缺血性脑损伤,新生大鼠出生后21-49天存在神经细胞“延迟凋亡”现象,是研究中枢神经细胞凋亡的理想模型。2、获得正常新生大鼠神经系统发育和细胞凋亡的生理规律：(1)正常新生大鼠21-42天龄时大脑皮质的神经干细胞持续存在,42-49天年龄段突然下降,表明42-49天年龄段,新生大鼠神经系统发育接近成熟。(2)正常新生大鼠42天龄大脑皮质的神经细胞出现凋亡高峰,随后逐渐下降。42天以前,大脑皮质中可能存在某些抑制Caspase-3促凋亡作用的因素,抑制神经细胞过度凋亡,14-3-3可能是其中一个因素。42天以后,14-3-3表达消失,大量神经细胞进入凋亡程序,但Caspase-3可能与此无关。3、获得模型组新生大鼠细胞凋亡相关的病理规律：(1)该模型42天后大脑皮质的神经细胞开始大量凋亡,新生神经干细胞减少,同时伴随异常的细胞增殖,这些增殖的细胞很可能不是正常成熟的神经元,而是修复损伤的其他神经细胞。(2)42-49天年龄段,大脑皮质神经细胞出现“延迟凋亡”的现象,与Caspase-3的表达存在因果关系。4、获得针刺对观察指标的作用规律：(1)针刺具有抑制HIBD新生大鼠大脑皮质中的细胞凋亡和BrdU的表达、提高大脑皮质中Nestin和14-3-3表达水平的作用。其中,针刺2组的上述作用均优于针刺1组。(2)针刺对神经细胞凋亡的抑制作用,可能与针刺提高14-3-3的表达水平有关,并通过抑制凋亡,间接抑制了BrdU的表达,另一方面,针刺具有促进Nestin在大脑皮质中表达的作用,能提高损伤区的新生神经干细胞水平,从而修复受损组织。(3)针刺对Caspase-3的抑制作用不明显,针刺抑制HIBD新生大鼠神经细胞凋亡的作用很可能不是通过Caspase-3途径来实现的。5、针刺介入时间研究对临床应用的启示：(1)决定针刺介入时间的关键,不一定在于越早越好。最佳的针刺介入时机,应取决于受针刺影响的靶蛋白,以及该蛋白在HIBD模型神经系统发育过程中表达发生转折的年龄段。(2)针刺影响靶蛋白表达的维持时间偏短,提示机体对针刺具有耐受性。因此找准关键时间点介入针刺治疗,才能更大限度地提高疗效。在治疗过程中,定时更换穴位进行刺激,可能有利于减少机体对该穴位的耐受性,提高针刺治疗敏感度。更多还原

【Abstract】 ObjectiveNeonatal hypoxic-ischemic encephalopathy seriously endanger the health of the newborn, directly leading to children’s encephalopathy such as cerebral palsy (CP) and epilepsy. The treatment of these diseases so far is still a worldwide medical problem. How to effectively improve the pathological conditions due to HIE has been an important work of medical researches.Over the last decade, acupuncture in the treatment of CP caused by HIE has made so great breakthrough that has showed an unprecedented prosperity in the field of CP. With further research, some scholars found that the younger children receiving acupuncture treatment, the more significant the efficacy being. Therefore, the idea of using acupuncture treatment on children with hypoxic-ischemic brain injury In the neonatal period came out.However, there are a variety of reasons obstruct the development of acupuncture in the field of HIE. These reasons include the risk and severity of HIE, the dominance in the newborn first aid of western medicine especially in China’s medical system, the deficiency of acupuncture experimental study on the mechanism of the disease, the distrust of the "science" of traditional Chinese medicine by the overwhelming majority of modern medical workers, the security of acupuncture as an invasive treatment in the neonatal field, such an so on. Therefore, the first verification in animal experiments on the effectiveness and security of acupuncture, and the revealing of the mechanism of action of acupuncture treatment on HIE by in-depth exploration, will help to promote acupuncture treatment into the field of neonatal HIE. Jin’s three needle therapy which was known as the " new therapy in the acupuncture field in the south of Qiling Mountain " was developed by Professor Jin Rui, who was the chief professor for mentally handicapped children of Guangzhou University of Traditional Chinese Medicine. Professor Jin Rui and his students used points on the head in Jin’s three needle as the main points to treat children with encephalopathy for more than20years and these points have been proved ot be effective. The State Administration of Traditional Chinese Medicine has recognized the Jin’s three needle therapy as the clinically appropriate treatment of children with mental retardation and children with autism. Although there are a great deal of clinical studies on Jin’s three needle in the field of children with encephalopathy, the foundamental studies for its mechanism are still rare.There are two purposes of this paper:on the one hand, is to explore whether the mechanism of Jin’s three needle treating HIBD neonatal rats is relative with the reduction of neonatal neuronal apoptosis, and to further seek its onset pathway in order to look for a reliable experimental foundation for acupuncture treating HIE; on the another hand, is to compare the influence of different starting times of acupuncture intervention after HIBD occurs on the newborn nerve cell apoptosis related indicators to provide the necessary theoretical support of clinical researches.MethodLiterature researchThis section reviews the understanding and treatment progress of neonatal HIE of modern medicine and Chinese medicine. The study progress of the genes relate to neural stem apoptosis is also included. On these basic works, the treatment situation of Chinese and Western medicine on neonatal HIE is summarized.Experimental researchThis section includes a total of six experimental parts.Experiment one established a HIBD pup rat model by a5min-delayed cesarean section which could simulat intrauterine asphyxia. Took the spontaneously delivered pup rats as the normal group and the normal cesarean section delivered pup rats as the sham group. The normal group and the sham group were considered to be control groups. The modal was identified to be successful according to the neurobehavioral performance of the neonatal rats and the results of brain tissue HE staining.The neonatal rats obtained in experiment one were randomly divided into normal group, sham operation group, model group, acupuncture group one and acupuncture group two. Each group was randomly divided into21-day-old,28-day-old,35-day-old,42-day-old,49-day-old subgroups by the sacrifice time. Male and female neonatal rats were equally taken into each group. The rats were marked with the picric acid on their14days old and then came into the following experiments.Experiment two observed the weight of neonatal rats of each group from14to49days and researched the influence of acupuncture on the weight of the HIBD neonatal rats.Experiment three to six studied the influence of acupuncture on the newborn nerve cell apoptosis in the ischemic lesions of the cerebral cortex of the HIBD neonatal rats.In experiment three, the number of apoptotic nerve cells in the cerebral cortex were detected by the Tunel assay. In experiment four, the expression of apoptosis related gene Caspase-3protein in the cerebral cortex was detected by immunohistochemistry. In experiment five, the expression of BrdU and Nestin protein in the cerebral cortex was detected by immunohistochemistry in order to reflect the number of newborn neural stem cells in the cerebral cortex and the proliferation of cells. In experiment six the expression of the anti-apoptotic gene14-3-3protein in the cerebral cortex was detected by immunohi stochemistry.ResultExperimental research 1. The establishment of animal model1) The mortality rate of neonatal rats:On the age of21days old, the mortality rate of the normal group was5.13%(4/78), the sham operation group was6.82%(3/44), and the model group was14.66%(28/191).2) Performance of the neonatal rat pups:The body of the normal group rats presented deep pink just after birth. The rat pups twisted their body, waved their limbs, were able to cry when licked by their mother and could quickly eat milk from the female rats. In contrast, rat pups of the sham group and the model group became cyanosis or pale the whole body when they were removed from the uterus, and were limbs extended, no breathing, tachycardiac and unresponsive to tactile stimulation.3) The righting reflex test of the neonatal rat pups on P2d:The order of each group about the shortest righting time was:the sham group<the normal group<the model group. The difference between the model group and the normal group was statistically significant (P<0.01), and the same was the model group and the sham group. The order of each group about the successful times in10times righting reflex test was:the normal group> the sham group> the model group. The difference between the model group and the normal group was statistically significant (P<0.01), and the same was the model group and the sham group.4) The rope suspension test of neonatal rats on P14d:There was no statistically significant difference between the normal group and the sham group on completing the test (P>0.05). But it took the model group shorter time to stay on the rope than the normal group (P<0.01) and the sham group (P<0.01).5) The slope test of neonatal rats on P14d:There was no statistically significant difference between the normal group and the sham group on completing the test (P>0.05). The time the model group took to complete the test was longer than the normal group (P<0.01), but had no significant difference with the sham group (P>0.05). There were27,15,84neonatal rats separately came from the normal group, the sham group and the model group that participated in the test. Among them, there were25(92.6%),13(86.7%),55(65.5%) winners who completed the test successfully that separately came from the normal group, the sham group and the model group, contrast with2(7.4%),2(13.3%),29(34.5%) losers who fail to pass the test. The P value of Chi-square test was less than0.01, so it was believed that whether to complete the slope test was relate with the groups.6) HE staining of the brain:The cerebral cortex of the brain of the normal group was divided into six layers from outside to inside according to the distribution of neurons:molecular layer, external granular layer, external pyramidal cell layer, internal granular layer, internal pyramidal cell layer and polymorphonuclear cell layer. The neurons presented round or conical with basophilic Nissl bodies uniformly distributing in the cytoplasm. The nucleus was located in the center of the cell body with significant nuclear membrane. There were also several individual pyknotic cells among the normal cells. The six layers of the cerebral cortex of the brain of the sham group were integrity in a rough way. But the cytoplasm of the pyramidal cells in some areas of the external pyramidal cell layer was stained red. The nucleus concentrated, deeply stained and had the structure disappeared. The number of pyramidal cells in the external and internal pyramidal cell layers of the cerebral cortex of the model group decreased. The cells were reduced into a triangle and the cytoplasm was homogeneous eosinophilic staining. The nucleus concentrated, deeply stained and had the structure disappeared. Part of pyramidal cells in the hippocampal also present the above changes.2. The influence of acupuncture on weight1) The weight of neonatal rats from P14d to P49d:There were statistically significant difference among the normal group, the sham group, the model group, acupuncture group one and acupuncture group two before35days old. But the difference disappeared after42days old.2) Body weight growth rate of neonatal rats:There was no statistically significant difference among the five groups on the body weight growth rate of21,28,35,49days to14days (P>0.05). The body weight growth rates of42days to14days of the model group were lower than the sham group (P<0.05). There was no statistically significant difference among the five groups on the body weight growth rate of per seven days in the period of14-21,21-35, 42-49days old (P>0.05). But the body weight growth rates of the two acupuncture groups were lower than the normal group in the period of28-35days old (P<0.05) and the ones of the model group and acupuncture group two were lower than the sham group in the period of35-42days old (P<0.01). The body weight growth rats of each group in the period of21to35days old were higher than the other periods.3) The influence of acupuncture on apoptosis:There was no statistically significant difference of the number of apoptotic cells in the cerebral cortex between acupuncture group one and the model group in the period of2135days old (P>0.05). The number of apoptotic cells in the cerebral cortex of acupuncture group two was less than the model group in the period of35-49days old (P<0.01). The number of apoptotic cells of acupuncture group two was less than acupuncture group one in theie35days old (P<0.05).4) The influence of acupuncture on caspase-3:There was no statistically significant difference of the expression of caspase-3between acupuncture group one and the model group on21days old (P>0.05). But the expression of caspase-3of acupuncture group one was lower than the model group on28days old (P<0.01) and then higher than the model group on35days old (P<0.01). The expression of caspase-3of acupuncture group two was higher than the model group on35days old (P<0.01). There was no statistically significant difference of the expression of caspase-3between acupuncture group two and the model group on42-49days old (P>0.05). There was no statistically significant difference of the expression of caspase-3between the two acupuncture groups on35days old (P>0.05).5) The influence of acupuncture on BrdU:There was no statistically significant difference of the expression of BrdU between acupuncture group one and the model group on21-35days old (P>0.05). The same result was found between acupuncture group two and the model group on35-49days old (P>0.05).There was no statistically significant difference of the expression of BrdU between the two acupuncture groups on35days old (P>0.05).6) The influence of acupuncture on Nestin:There was no statistically significant difference of the expression of Nestin between acupuncture group one and the model group on21and35days old (P>0.05), but the expression of acupuncture group one was higher than the model group on28days old (P<0.01). There was no statistically significant difference of the expression of Nestin between acupuncture group two and the model group on35-42days old (P>0.05), but the expression of acupuncture group two was higher than the model group on49days old (P<0.01). The expression of Nestin of acupuncture group two was lower than group one on35days old (P<0.01).7) The influence of acupuncture on14-3-3:The expression of14-3-3in the cerebral cortex of acupuncture group one was higher than the model group on21days old (P<0.01), and then drastically reduced in the period of28-35days old that had no statistically significant difference from the model group (P>0.05). The expression of14-3-3in the cerebral cortex of acupuncture group two was lower than the model group and the normal group on21days old (P<0.01), but slightly increased in the period of42-49days old so that higher than the other groups which had no expression of14-3-3at all. The expression of14-3-3of acupuncture group two was lower than group one on35days old (P<0.01).ConclusionLiterature research1. The pathogenesis and pathophysiology process of neonatal hypoxic-ischemic encephalopathy is so complex that comprehensive treatment of a variety of methods of Chinese and Western medicine is the development direction in the future. The present treatment of the disease still takes supportive therapy and symptomatic treatment as the main methods. Main new methods such as hyperbaric oxygen, hypothermia, neurotrophic factor, neural stem cell transplantation and so on are still under animal study that may take a long time to be used as the common methods in clinical application.2. The theory and clinical experience of the disease from ancient Chinese Medicine are so rich that the ancients have developed a set of diagnosis and treatment program for the symptoms which are similar to neonatal HIE. However, no scholars have never tried to systemly summarize the ancient treatments of such symptoms about HIE and never successfully mapped the clinical practice of ancient Chinese Medicine with the experimental reseaches of modern Chinese medicine to date. Chinese Medicine is so a great treasure that worthy to study by Chinese medicine researchers.3. Modern Chinese medicine only takes several kinds of extract of single Chinese medicine as the main medicine to treat neonatal HIE without embodying dialectical argumentation which is the spirit of traditional Chinese medicine. Acupuncture and massage focus on treating cerebral palsy, the neurological sequelae of neonatal HIE, and fail to have a try on treating the disease in very early neonatal period.Experimental research1. Delay-five-minutes cesarean section can simulate the pathogenesis of intrauterine asphyxia to a high degree, prepare a large number of long-term survival HIBD neonatal rats in a short term and is easy to operate. So this method is worthy of promoting for establishing hypoxic-ischemic brain injury model. It can cause relatively mild hypoxic-ischemic brain injury that induce delayed apoptosis of nerve cells in the cerebral cortex of the neonatal rats during21to49days old after birth. So this model is also good for study apoptosis of the central nervous.2. The physiological rules of nervous system development and apoptosis of the normal neonatal rat are following below:(1) The normal neonatal rats had neural stem cells persisted in the cerebral cortex during21to42days old, and then suddenly droped on42days old. This phenomenon indicates that the42-day-old neonatal rat’s nervous system development is nearing completion.(2) The normal neonatal rats had nerve cells apoptosis in the cerebral cortex to reach the peak on42days old, then decrease gradually. It might due to some factors existed in the cerebral cortex before42days old that could inhibite the promoting effect on apoptosis of caspase-3.14-3-3may be one of the factors because the expression of14-3-3disappeared after42days old, just when the expression of casapse-3activated and then followed a large number of nerve cells getting into the apoptotic program.3. The pathological rules of apoptosis of the HIBD neonatal rat are following below:(1) The apoptosis of nerve cells in the cerebral cortex began in large scale after42days old, accompanied by new neural stem cells decreasing and abnormal cells proliferating at the same time. These proliferating cells were likely not normal mature neurons, but other nerve cells to repair the damage.(2) Physiological apoptosis ultimately depends on caspase-3pathway. Pathological apoptosis that induced by HIBD doesn’t depend on the activation of caspase-3, but may be endoplasmic reticulum stress pathway instead4. The influence rules of acupuncture on apoptosis-related indexes are following bellow:(1) Acupuncture could inhibit nerve cells apoptosis and BrdU expression and increase Nestin and14-3-3expressions in the cerebral cortex of the HIBD neonatal rats. The performance of acupuncture group two was better than acupuncture group one.(2) Acupuncture may inhibit neuronal apoptosis by promoting the expression level of14-3-3. The inhibition of BrdU expression may due to the inhibition of neuronal apoptosis. On the other hand, acupuncture can promote Nestin expression in the damage area of the cerebral cortex which marks the newborn neural stem cells, so it was suggested that acupuncture can repair damaged tissue by promoting the increase of newborn neural stem cells.(3) The inhibition of caspase-3by acupuncture was not obvious, so acupuncture may not inhibit the apoptosis of newborn neuronal in HIBD neonatal rats by caspase-3pathway.5. The revelation of the time when acupuncture begins to intervent for clinical application:(1) The best time to have acupuncture is not the early the better. It is best to depend on the target protein which can be changed by acupuncture and the age when the expression of this protein changes physiologically during nervous system development in HIBD model.(2) It’s suggested that organism is easy to tolerate for acupuncture as the significant changes of target proteins expression induce by acupuncture maintained only a short time. Therefore, it’s important to identify the best time to have acupuncture treatment in order to improve efficacy. In the course of treatment, changing acupuncture points regularly may help reduce the tolerance of the body for the points and improve the sensitivity of acupuncture treatment.更多还原