Study On The Protective Effects And Its Mechanism On Neuron With TBI Rat Model Treated By Acupuncture In Erent Periods

Objective:1. To study on preparation of TBI rats model by eCCI, and provide a model preparation method of easy operation, the degree of damaged controlled, good repeatability and low mortality for the TBI experimental study.2. To prepare TBI model of SD rat by the method, and study the effects of acupuncture treatment of TBI at different periods. To reveal protective effects and mechanism of the acupuncture to the injured nerve cells of TBI brain, provide experimental basis of acupuncture treatment of acute phase TBI at the best time, and to add the scientific contents of acupuncture treatment of TBI.Methods:1.65SD rats, which were randomly divided into blank control group, model group1, model group2, model group3and sham-operation group, were produced as the animal model of brain injury in different degree by eCCI from selected different fighting parameters. The speed was3m/s and the depth was3mm in model group1. The speed was4m/s and the depth was3mm in model group2. The speed was5m/s and the depth was3mm in model group3. Evaluated the model quality through the following:(1) Evaluation of animal behavior, nerve function, balance ability and walking ability.(2) To observe the morphological changes of brain tissue by HE under light microscope.(3) To observe the ultrastructure changes of brain tissue by electron microscopy.(4)To detect the dynamic changes of S100B and NSE were by ELIASA in animal serum.(5) To detect the contents of NGF and BDNF of brain tissue by immunohistochemical method.(6)To detect the contents of BAX and Bcl-2of brain tissue by immunohistochemical method. 2.70SD rats were randomly divided into blank control group, acupuncture group1, acupuncture group2, acupuncture group3and model control group. The TBI animal models were prepared with the eCCI of the speed of4m/s and the depth of3mm. Than rats were treated by acupuncturing Baihui(DU20), Renzhong(DU26), the left Neiguan((PC6) and the left Zusanli(ST36)) after the brain injured at1day,3days,5days. The treatments of each group were repeated10times. The materials were collected at the15th day and to observe and examine the related index signs:(1) To observe the pathological changes of brain tissue by HE staining light microscope.(2)By immunohistochemical and Western-blot to examine the localization and content of NGF and BDNF in the brain tissue protein, to detect the mRNA changes of NGF and BDNF by Fluorescence metered PCR method.(3) By immunohistochemical and Western-blot to examine the localization and content of Bcl-2and Bax in the brain tissue protein, and detect the mRNA changes of Bcl-2and Bax by Fluorescence metered PCR method.Results:1. Study on preparation of TBI models of rats(1) After the brains of SD rats had been damaged by different speeds of outside force, the amount of bleeding with the stroke strength increasing was on the increase, the breath slowly, the dead animals increasing in model group1,2and3. On the1st day after hit, the nerve function, balance and walking ability were evaluated. The results showed that nerve function, balance and walking ability were decreased with the increase of hit, and there were significant differences in groups(P<0.01). On the third day, each group of nerve function, balance and walk ability were obviously improved than the first day, and there were still differences between the groups after evaluation (P<0.01).(2) Observed by HE under light microscope, the cells of brain tissues were distributed clear and fine,Arranged well, chubbiness, and had clear contour, blue nucleus. The colour of nerve cells been stained was normal in the blank group and sham operation group. In model groups the injured cortex showed that they bled in different degrees. There were infiltration of a large number of inflammatory cells, edema of interstitial brain, wide perivascular space, and they obviously increased with the increase of impact strength. By electron microscopic in normal group and sham operation group, the structure of brain cells was complete and clear. Mitochondria, ribosomes and rough endoplasmic reticulum were rich.The mitochondria had clear inner and outer membrane structure and neat crest. In the model groups, the brain cells showed that there were pyknosis of the nucleus, the nucleus dissolved, and little cytoplasm. The content of organelles was reduced. The mitochondria and endoplasmic reticulum were swelling with the increase of the hit.(3) The content of S100B and NSE in serum was increased on the first day, second day, third day after animal damaged in model group by ELISA. In the model group and sham operation group, there were significantly different (P<0.01).In model group1,2and3, The content of S100B and NSE was increased significantly with the increase of hit (P<0.01).(4) The NGF, BDNF, Bax and Bcl-2protein were detected by immunohistochemistry in brain tissue. The positive expressions were mainly distributed in nerve cells, mainly as small particles and partly as aggregated distribution. The NGF, BDNF, Bax and Bcl-2in the brain tissue of cerebral cortex, hippocampus and cerebellum cytoplasm of neurons showed weak positive reaction in control group and sham operation group. The NGF, BDNF, Bax and Bcl-2of damaged brain in model group showed positive reaction and the expression was markedly increased with the increase of hit (P<0.01).2. Study of time of acupuncture to regulate NGF, BDNF, Bax and Bcl-2of brain tissue(1) Morphological changes of brain tissue by HE. The nerve cells in blank group were dense and the cell morphology can be clearly seen. In model group, the cortical neuron were, little and shrinkage, the cytoplasm deeply colored, illegibility of nuclear, and some died. In acupuncture group, glial cells increased. Apoptotic body and necrotic cells were less obviously reduced. The neurons were close to normal, the area of necrosis reduced, the blood circulation improved.(2) The expression and content of NGF, BDNF, Bax, Bcl-2were detected by immunohistochemistry and Western-blot in brain tissue. Acupuncture can up-regulate NGF, BDNF, Bcl-2, and down-regulation Bax. And by PCR, acupuncture can increase the mRNA of NGF, BDNF, Bcl-2, and decrease the mRNA of Bcl-2. After TBI occurred, while acupuncture treatment was used as soon as possible, the acupuncture was more obvious on the regulation effect. And there was significant differences among the three groups (P<0.01).Conclusion:1. By eCCI TBI rat model can be prepared with regulation of hit speed and depth. The damage of model was related to the hit speed. The method of TBI model preparation has the advantages of easy operation, good control ability, repeatability, low mortality rate, and can according to the need be used to make various animal model for the study.2. Acupuncture treated TBI as early as possible.The results of this study showed that after1day of TBI occurred if acupuncture was used immediately, the curative effect was better than3days and5days latter.