| 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 purposesThough 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 methodsThe 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.Results1. 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.Conclusion1. 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.
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