| ObjectivesBesides the direct compression of the hematoma to the brain tissue, acute brain hemorrhage can also induce the secondary nerve injury. Cell apoptosis, which is very complicated, is the main style of the cell death in the secondary nerve injury. It includes;①inflammatory reaction after the brain hemorrhage. ② during the procedure of the blood coagulation after brain hemorrhage, a large amount of thrombosin can be relaeased, and there are many thrombosin - binding site in the brain and spinal cord for the thrombosin. While the mechanism how the thrombosin induces the apoptosis is not clear. ③ The toxic effect of the component and degradation products of the hematoma after the brain hemorrhage, such as hemoglobin, bilirubin, ferri ion and CO, are also the important reason. ④ Somatostatin can promote the cell apoptosis. At present, there are many research results reveal that the mechanism of cell apoptosis participate the secondary injury of neurocytes. So cell apoptosis is the important factor of neuro-cyte injury after brain hemorrhage, and the block of the cell apoptosis is seemed to be an effective therapeutic measure.Therefore, the treatment of anti - apoptosis is essential part in the therapeutic measure of acute brain hemorrhage. biochemical indicator of apoptosis includes Bax,Bcl - 2, chondrosome - cytochrome C , Casepase , ribozyme - shearing DNA and so on. The levels of the expressions of Bcl - 2 and Bax protein have close correlation with the secondary injury around the hemorrhage hematoma. Bcl — 2 is the abbreviation of B - celllymphoma/leukemia - 2 gene, and it' s the first gene identified to be anti - apoptotic. Bcl — 2 is the anti - apoptosis gene, while Bax is apoptosis promotion gene, they are both expressed in muturebrain tissue. We can learn from many experiments that the variation of Bel - 2 and Bax is usually the reflect of the degree of cell apoptosis.In our research of treatment to anti - apoptosis, sub - hypothermia is widely used, and the mechanism of sub - hypothermia includes: 1. reduce the metabolism of brain;maintain the blood supply and the energy balance of brain. 2. protect the blood - brain barrier and reduce the vasogenic brain edema. 3. inhibait the release of neurotransmitter. 4. reduce the accumulation of Ca +. 5. remove the linkage of free radical and lipid oxidation. 6. reduce the destruction of brain protein, and promote the recovery of structure and function of brain. 7. reduce other endogenous toxic products. Some research results reveal that the sub - hypothermia has the effection of reducing the brain edema, releasing the intracra-nial pressure and preventing the apoptosis of neurocyte.The whole body sub - hypothermia is not easily manipulated in neurosurgery operation, because there is long — time rewarming after operation, which can also bring a lot of severe systemic complications. So Intro - ventricle hypothermia, which is a kind of selective brain sub - hypothermia, becomes the focus in this research. But it is not clear that whether the hypothermy intervention giving to animal model of acute brain hemorrhage have the exact effect on anti - apoptosis , and can protect the brain tissue. So in this experiment , we examine the effect of intro — ventricle hypothermia on anti - apoptosis of neurocyte by analyzing the different expression of the Bel — 2 ^ Bax gene in hypothermia group and normal temperature group of rabbit model of acute brain hemorrhage. We expect that we can find a new thinking of brain hemorrhage therapy in clinical work.Material and method1. Experimental animal 12 male rabbits of 4 ~6 month old,weight 2. 8 ~3. 2 kg, divided into two groups randomly : normal temperature group ( CH + normal temperature fluid ) N hypothermia group (CH + hypothermic fluid) , each 6 rabbit.2. Anesthesia general anesthesia. Animal should abrosia for 12 hours before the experiment, but the water is available. Using ketamine(7 mg/kg)andnorflox - lidocarine (0. 8 mg/kg) iv during general anesthesia. Using half -mount above when anesthesia maintenance.3. Building the model of brain hemorrhage confirm the right internal capsule of brain according to sawyer rabbit brain mapping atlas. 2 % Lidocaine local anesthesia on the point of posterior border line and median line crossing with each other, cut off the epicranium at about 2. 0cm long , expose the anterior bregmatic bone suture. Drill the cranial bone 6mm right the longitudinal suture, lmm anterior the coronal suture. Get 5 ml artery blood of ear medial artery with lml injection syringe, 14mm vertical needling into the bain tissue with 5# injection syringe, inject 0. 1 ml blood at first, and the other 0.4 ml 2 minutes later. Needling withdrawal after 2 min. Give up all that the hematoma refluxing back the needle.4. Building the model of intro - ventricle hypothermia After building the model of brain hemorrhage, Prick left ventricle with double cavity catheter, fix the catheter. One is entrance and the other is exit. Inject normal temperature (38^ )isotonic saline solution to normal temperature group, while inject hypot-hermic(20T! ) isotonic saline solution to hypothermic group, maintain the fluid supply balance.5. Behavior observation after operation ^ Bax, Bel - 2 testing and data analysis Suture the skin after operation, raising the animal in normal temperature for 24 hours. Observe the limb movement and then sacrifice the animal (ketamine 0. 1 iv). Then open the cranial bone and take the brain tissue,4% paraformal-dehyde fix it. After 72 hours fixation , taking the brain tissue , washing , using gradient alcohol dehydration, dimethyl benzene clearing, dipping wax, making 7 jxm serial sections, Bax, Bel - 2 immunohistochemistry staining according to the introduction on the kit.6. Image collecting: Observe 8 different visual fields randomly on each sections under high power lens (400 times) , count the number of positive cells and negative cells in each visual field, and calculate the positive ratio.7. statistical analysis.- input the number of positive cells in each group into SPSS12.0, using t testing in comparing different groups with same index , P < 0. 05 indicates statistical difference. Calculate the positive ratio of expression ofBax amd Bel - 2 protein in normal temperature group and hypothermia group ( Bax/Bcl -2) ,making the rank sum testing to the results , P <0. 05 indicates statistical difference.Results1. Animal behavior observation: 4 in normal temperature group have complete paralysis of left limb, another 2 can only crawl. 4 in hypothermia group can crawl and another 2 can jump.2. Results of expression of Bel — 2ABax in brian tissue;there is buffy granules in the cytoplasm of Bel -2 and Bax staining positive cell. Bel -2 and Bax staining positive cell in hypothermia group are obviously lower than normal temperature group, It has statistical difference( P < 0.05).DiscussionBax gene possess a function that is exactly opposite to that of Bel -2. Bax can form homodimers and heterodimers with Bel - 2, the former accelerates cell apoptosis and the latter, in contrast, represses it. The expression levels of Bel -2 and Bax genes determine whether cell apoptosis happens, and the ratio of Bax to Bel -2 means life and death to the cells. The higher the ratio is, the higher the possibility of cell apoptosis is. Since the ratio of Bax protein/ Bel -2 protein is lower than that in normal temperature group, and have statistical difference, we may come to a conclusion that intra - cerebral - ventricle can protect cells from apoptosis.ConclusionThe limb paralysis of the animal which is protected by Intro - ventricle hypothermia is less severe, and the number of Bel - 2 and Bax positive cell is less, Bax protein/ Bel - 2 protein ratio is lower than normal temperature group, and have statistical difference. So we can conclude from this experiment that thehypothermia therapy to rabbit brain hemorrhage can reduce the celll apoptosis a-round the hematoma, and improve the syndrome of nervous system, and Intro -ventricle hypothermia is a kind of new method for acute brain hemorrhage.
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