| IntroductionCerebral vascular diseases (CVD) are common in current societies. Among which, intracerebral hemorrhage(ICH) is an emergent disease endangering the people's life and work because of its high mortality and serious disability. The most important pathological changes after ICH were the hematoma itself and the secondary edema, which caused hematoma-related death and severe neurological deficits. Although lots of improvements have been made on the prevention and treatment of ICH, the mechanism of brain edema formation and secondary neuron injury were not clear now. So the researches on ICH become the focus of clinical and primary studies.This experiment applied collagenaseⅣinjection to install animal model of Intracerebral hemorrhage, TdT mediated dUTD gap end labeling and detected apoptosis associated protein. This experiment approaches hematoma perienchyma of experiment cerebral hemorrhage apoptosis and injury and analysis after intracerebral hemorrhage TUNEL positive cell, apoptosis associated protein Bcl-2, Bax and Caspase-3 expressed the rule of phase diversity and the changes of apoptosis expression and apoptosis' condut ive signal Cytc,Fas with time in lateral cerebral striatum of rats caused by intracerebral hemorrhage (ICH). Finding time window of anti apoptosis curing and finding influence of early treatment with hematoma aspiration on signal conduction of apoptosis will be a new target that preventing and curing hemorrhagic brain injury. Section A Establishment and Evaluation for the model of Intracerebral Hemorrhage with AspirationObjectiveTo establish a simple, provident rodent model of intracerebral hemorrhage, which is reproducibilitical and can model clinical course; and to evaluate of hematoma aspiration.Methods66 mature male Sprague-Dawley rats were randomly divided into normal control group (n=12), false operation group (n=18), natural recovery group (n=18) and hematoma aspiration group(n=18). In false operation group, normal saline instead of bacterial collage-nase was injected into normal rats caudate nucleus. In normal control group, the animals were normal rats. Intracerebral hemorrhage was induced by injection ofⅣbacterial collagenase into the caudate nucleus of rats, and urokinase was used to lyse the hematoma 10 hours after hemorrhage induction, and then the clot was aspirated. They are used to evaluation of minimally invasive hematoma aspiration, through the measurement of hematoma volume and brain water and the evaluation of behavioral function in three groups including a group of hematoma aspiration, a group of hematoma and a group of sham operation。All data are presented as mean±SEM. Data were analyzed by Student's t test to compare the hematoma and aspiration groups.ResultsHematoma volume of the treated group is smaller than the control group' s (P＜0.01), but there was no significant difference in brain water between the two groups (P＞0.05), and the treated rats performed significantly better than controls on neuro-functional rehabilitation on days 3 and 7 after intracerebral hemorrhage (P＜0.05).ConclusionHematoma aspiration after collagenase-induced hemorrhage in a rat model may eliminate clot better and promote acute neuro-functional rehabilitation.Section B Apoptosis related genetic expression in a model of Intracerebral Hemorrhage with AspirationObjectiveThe appropriate therapy for intracerebral hemorrhage (ICH) to reduce disability rate and mortality rate remains a subject of debate. In the past, it was believed that the primary mechanisms of neural injury in ICH were due to the massive effects of hematoma and secondary brain edema. So, the main therapy for ICH was desiccation to maintain intracranial pressure. However, the curative effect is disappointing. Other mechanisms of neural injury must be invovled in ICH. Yet, in comparison with ischemia, relatetively few experimental investigations have been devoted to studying the pathophysiology of ICH. Apoptosis was involved in the occurence and development of many diseases. Previous studies have demonstrated that in ICH there was a "penumbra" around hematoma similar to that existed in cerebral ischemia and that components in hematoma such as thrombin and hemoglobin could induce apoptosis in cell culture. However, it is poorly understood whether apoptosis was involved in ICH, So, the purpose of this study is:①to investigate whether apoptotic injury exists in perihemotoma in ICH in rats and to explore mechanisms of neural injury else in ICH;②to analyze the rule of apoptosis change and to search " time window" for anti-apoptotic injury therapy after ICH, if apoptotic injury exists in ICH;③The influence of the early aspiration to apoptosis. Materials and Methods1. Animal and Group:150 Male SD rats were adopted, weighting180～220g, 3～5months. The rats were divided into six groups: normal control, Operation groups(6h, 12h, 1d, 3d, 7d, 11d), ICH groups(6h, 12h, 1d, 3d, 7d, 11d), 6h hematoma aspiration(12h, 1d, 3d, 7d, 11d), 12h hematoma aspiration(1d, 3d, 7d, 11d), 1d hematoma aspiration (3d, 7d, 11d), six rats were adopted at each group and each time.2. Equipment and Reductant;Stereotaxis instrument of rat (KOPF company USA.)MicroinjectorMicroanalytical balanceTUNEL kit (Promega company)Immunohistochemistry kit anti-rat rabbit AbCaspase-3 (Lab Vision company)Immunohistochemistry kit anti-rat rabbit Ab Bax (Tian Jin Hao Yang Biological Manufactureco. ltd)Immunohistochemistry kit anti-rat rabbit Ab Bcl-2 (Tian Jin Hao Yang Biological Manufactureco. ltd)3. Methods;①Rats were killed respectively at 6h,12h,1d,3d,7d,11d after injection. TUNEL and immunohistochemistry technology to probe protein Caspase-3 or protein Bcl-2, Bax were performed to detect apoptosis around hematoma at different time Points and the rule of apoptosis change after ICH was analysed.②stereotaxic aspiration was performed respectively at 6h,12h,1d. TUNEL positive cell and apoptosis associated protein Caspase-3 Bax and Bcl-2were detected at different time point.Results1. Tunel changes At 6h, TUNEL positive cells appeared in ICH model(16. 3±2.4), increasing at 12h(24.33±2.5), continue increasing at 1d(60.31±8.96,P＜0.05), peaking at 3d(104.41±21.45,P＜0.05), at 7d (65.12±18.94), 11d(35.17±2.87,P＜0.05).At hematoma aspiration group: After infusing collagenase 1d, 3d, 7d, Tunel positive cells were decreased (1d:30.12±18.94,P＜0.05;3d:60.41±21.45,P＜0.05;7d:32.31±8.96,P＜0.05)2. Immunohistochemistry results of Caspase-3Protein Caspase-3 were expressed at 6h after ICH (2.86±2.44, P＜0.05), increasing at 12h (20.75±5.21,P＜0.05), peaking at 1d (60.15±2.15,P＜0.05), 3d(44.21±4.21,P＜0.05), 7d,11d(35.22±1.22,36.22±4.39,P＜0.05).At hematoma aspiration group: After infusing collagenase 1d, 3d, 7d, Caspase-3 positive cells were decreased (1d:37.12±2.14,P＜0.05;3d; 27.21±2.14,P＜0.05;7d:17.21±2.33,P＜0.05)3. Immunohistochemistry results of BaxProtein Bax were expressed at 6h after ICH(2.55±3.14,P＜0.05), increasing at 12h(18.22±4.55,P＜0.05), peaking at 1d(45.32±5.22,P＜0. 05), 3d(40.21±2.12,P<0.05), 7d,lld(40.01±3.23,38.21±2.33,P< 0.05).At hematoma aspiration group: After infusing collagenase 1d, 3d, 7d, Bax positive cells were decreased (1d:25.12±2.14,P＜0.05;3d:20.15±4.21,P＜0.05;7d:24.23±5.12,P＜0.05)4. Immunohistochemistry results of Bcl-2Protein Bcl-2 were expressed decreased at 6h after ICH(43.22±2.37,P＜0.05), increasing at 12h(45.22±2.02,P＜0.05), peaking at 1d(42.13±2.11,P＜0.05), 3d(37.13±2.15,P＜0.05), 7d,11d(45.23±5.11,47.22±8.89,P＜0.05).At hematoma aspiration group: After infusing collagenase 1d, 3d, 7d, Bcl-2 positive cells were increased(1d:67.12±9.25,P＜0.05;3d:66.12±20. 07,P＜0.05;7d:70.22±4.21,P＜0.05). DiscussionIn this study using rat models of ICH, we have presented evidence suggesting that apoptosis may contribute to brain damage after ICH: (1) there were significant numbers of TUNEL positive cells around blood clot and ipsilateral cortex. (2) most TUNEL positive cells appeared to be neurons, whereas some appeared to be glia. In addition, TUNEL positive endothelial cells may contribute to blood brain barrier disruption and vasogenic edema.However the triggers that responsible for initiating the apoptotic cascade after hemorrhage remain to be defined. It is well known that mild ischemia together with the extravasation of activated cytokines can lead to brain cell apoptosis after ICH. Iron/heme, which is obviously abundant in blood, is another possible trigger for apoptosis after ICH. Finally, the high levels of thrombin present after ICH is likely to contribute to neuronal apoptosis.The neighbouring sections were detected respectively by TUNEL and Caspase-3 Bax,Bcl-2 protein immunohistochemistry.The distribution of TUNEL, Caspase-3,Bax of positive cells are identical, and the time variation is the same. So after ICH, the neuron apoptosis was followed the Caspase-3, Bax expression and the Bcl-2 would be suppressed.Early aspiration group at 6h, 12h, 1d the TUNEL posive cells, Protein Caspase-3, Bax were obviously decreased. Contrary to it, Bcl-2 expression was einforced.ConclusionThe apoptosis induced by ICH is related to the expression of Bcl-2 and Bax,Caspase-3. It proved apoptosis induced by ICH is related to mitochondrion.Early therapy of aspiration can obviously decrease apoptosis induced by ICH. Section C Apoptosis' Signal Conduction of in a model of Intracerebral Hemorrhage with AspirationObjectiveBrain histiooytic death way contains necrosis and apoptosis after intracerebral hemorrhage, and apoptosis is a death way of gene regulation, which participates pathological procedure of secondary brain injury after intracerebral hemorrhage. To study and research apoptosis rule of intracerebral hemorrhage, how moning suppress apoptosis further and to promote neurofunctional recovery this is very important for instruction of clinical treatment, this experiment's purpose is to investigate the changes of apoptosis expression and apoptosis 'condutive signal Cytc,Fas with time in lateral cerebral striatum of rats caused by intracerebral hemorrhage (ICH) and the influence of early treatment with hematoma aspiration on signal conduction of apoptosis.Materials and Methods1. Animal:78 mature male Sprague-Dawley rats were randomly divided into normal control group(n=6), false operation group(n=6), natural recovery group (n=36) and hematoma aspiration group (n=30).2. Equipment and Reductant;Stereotaxis instrument of rat (KOPF company USA.)MicroinjectorMicroanalytical balanceTUNEL kit (Promega company)Immunohistochemistry kit anti-rat goat Ab Fas (Tian Jin Hao Yang Biological Manufactureco. ltd)Immunohistochemistry kit anti-rat mouse Ab Cytc (Santa cruz biotechnology) 3. Methods;①Rats were killed respectively at 6h,12h,1d,3d,7d,11d after injection. TUNEL and immunohisto-chemistry technology to probe protein Fas or Cytc were performed to detect apoptosis around hematoma at different time Points and the rule of apoptosis change after ICH was analysed.②stereotaxic aspiration was performed at 6h. TUNEL positive cell and apoptosis associated protein Fas or Cytc were detected at different time point.Results1. The apoptosis of ICH group, were markedly more than that of both normal control group and false operation group, showing significant difference (P＜0.05).2. The peak time of Cytc,Fas' expression in ICH group appear at 3d-7d; Cytc' s expression of hematoma aspiration group greatly decreased than those of natural recovery group at 12 h-7 d, the difference was significant (P＜0. 05); Fas'expression of hematoma aspiration group greatly decreased than those of natural recovery group at3 d, the difference was significant(P＜0.05).DiscussionDuring the course of apoptosis, the signal transduction has two main access as follows; the first has been called "The death-receptor signal transduction", and the other is named Fas-FasL system mediated apoptosis signal transduntion. The discovery of Fas-FasL system has increased new knowledge of PCD mechanism, and then we connect the PCD endochylema and intranuclear event with membrane signal transduction mechanism. There is evidence that, in some cells the expression of Fas is continuously increasing but Bcl-2, a kind of gene to inhibit apoptosis, is appeared as taper. In the end, apoptosis is generated to complete the metabolic process, in order to balance the cell proliferation and death. Thus apoptosis is under the control of ced-3 and Bcl-2, and the Fas-FasL system.The surface of cell membrane express functional Fas protein, and the a- mount achieves to a certain level is primary condition through Fas-FasL inducing apoptosis. The chondriosome signal transduction pathway: In the existing of dATP, Cytc combines with Apaf-1, to exposure CARD motif on Apaf-1, and then combines CARD of proaspase-9 into apoptosome, The result is the activation of procaspase-9, and the latter activates Caspase-3, 6s, according induces apoptosis.The neighbouring sections were detected respectively by TUNEL and Caspase-3 Bax,Bcl-2 protein immunohisto-chemistry. The distribution of Tunel, Cytc, Fas of positive cells are identical, and the time variation is the same. So after ICH, the neuron apoptosis was followed the Cytc,Fas expression. Early aspiration group at12h,1d,3d the Tunel posive cells,Protein Cytc,Fas were obviously decreased.ConclusionHematoma aspiration may decrease apoptosis expression of ICH's rats significantly through hondriosome (Cytc) channel.
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