| 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.
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