【作者】 徐辉；

【导师】 郭永章； 陈训如；

【作者基本信息】 昆明医学院 ， 普通外科学， 2006， 博士

【摘要】 目的：随着腹腔镜技术的迅速发展，基础和临床工作中同时也对气腹下正常机体脏器的功能变化作了大量研究。随着微创外科的深入发展，腹腔镜手术适应症范围的拓宽、手术难度的增加，由人工气腹所带来的一系列病理生理变化日益受到重视，一些研究表明：气腹状态下，引起腹腔室间隔综合症，作为实质性器官的中枢神经系统发生低血流量损害及再灌注损伤，解除气腹后中枢神经系统功能受到不同程度的损害。气腹对于机体呼吸、循环、免疫及重要脏器影响的实验研究得以重视与深入，但有关预存有功能不全时气腹对脏器功能的不良影响及其机制的报道尚少。本课题旨在探讨不同CO₂气腹压及不同时段下正常及缺氧缺血性脑损伤大鼠血、脑脊液及脑组织S-100蛋白（S-100）、神经元特异性烯醇化酶（NSE）水平的变化，并进而探讨CO₂气腹对中枢神经系统的影响。方法：采用健康成年Wistar大鼠建立气腹模型和缺氧缺血性脑损伤模型，确定成模大鼠80只，随机分组，闭合法建立CO₂气腹，分别施加5mmHg（0.67Kp）、1小时气腹和10mmHg（1.33KPa）、2小时气腹，并分别于气腹解除后48小时处死各组动物。用光镜和电镜观察大鼠脑组织胶质细胞和神经元细胞组织学变化；应用抗体夹心法检测气腹模型后血液和脑脊液中S-100、NSE水平变化，用逆转录-聚合酶链式反应（RT-PCR）技术和免疫组织化学的方法观察脑组织中S-100、NSEmRNA和蛋白水平表达的变化；TUNEL法检测石蜡组织块切片中脑组织细胞凋亡，分别随机计脑组织胶质细胞和神经元细胞凋亡细胞数。结果：（1）CO₂气腹后，A_5-1组与对照组比较，海马区胶质细胞和神经元细胞均无明显变化，A_10-2组胶质细胞和神经元细胞在光镜下呈局轻度水样变，电镜下线粒体轻度肿胀；缺氧缺血性脑损伤组（B）与对照组比较，海马区胶质细胞和神经元细胞均呈水样变，无核坏死，电镜下线粒体、内质网中度水肿，B_5-1组与B组比较，海马区胶质细胞和神经元细胞水肿加重，B_10-2组胶质细胞和神经元细胞在光镜下呈弥漫性水样变，部分胶质细胞核浓缩和细胞皱缩，神经元细胞胞浆酸性细胞增多，电镜下胶质细胞水样变，线粒体肿胀、脊断裂，胶质细胞内髓鞘样结构形成，神经元细胞线粒体、内质网肿胀变性，核膜破裂。（2）CO₂气腹后血液中S-100在A_5-1组（0.415±0.096）ug/L和A_10-2组（0.445±0.086）ug/L，NSE分别为（0.511±0.022）ng／ml和（0.531±0.041）ng/ml，对照组S-100和NSE分别为（0.393±0.100）ug/L和（0.493±0.045）ng/ml；脑脊液中S-100在A_5-1组（0.440±0.082）ug/L和A_10-2组（0.458±0.127）ug/L，NSE分别为（0.538±0.059）ng/ml和（0.574±0.048）ng/ml，对照组S-100和NSE分别为（0.431±0.124）ug/L和（0.531±0.048）ng/ml，血液和脑脊液中S-100、NSE随CO₂气腹压力及时间增加而逐渐增加，但差异无显著性意义（P＞0.05）；缺氧缺血性脑损伤组（B）与对照组比较，血液和脑脊液中S-100、NSE水平均明显增高，差异有显著性意义（P＜0.05），B_5-1组与B组比较水平均有增加，差异无显著性意义，在B_10-2组差异有显著性意义（P＜0.05）。S-100和NSE在血液和脑脊液中水平变化分别呈正相关（P＜0.05）。（3）S-100 mRNA在A_5-1组（64.215±10.404）和A_10-2组（67.020±13.339），NSE分别为（8.976±2.379）和（9.183±3.365），对照组S-100、NSE mRNA分别为（59.340±11.378）和（8.500±2.940），S-100、NSE mRNA在CO₂气腹后逐渐增加，但与对照组比较，差异无显著性意义（P＞0.05），B组与对照组或B_10-2组与B组比较均有显著性差异（P＜0.05）。（4）CO₂气腹后脑组织S-100蛋白、NSE表达阳性面积随时间及压力增加而逐渐增加，在A_5-1点和A_10-2点与对照组比较无显著性差异（P＞0.05），B组与对照组或B_10-2组与B组比较均有显著性差异（P＜0.05）。（5）细胞凋亡检测显示低气腹压时脑组织胶质细胞和神经元细胞凋亡数无显著变化，高气腹压凋亡细胞数增多。结论：（1）在5mmHg压力、1小时位点，CO₂气腹对正常大鼠中枢神经系统组织学、生化标志物、蛋白转录水平、蛋白水平表达的变化及脑组织胶质细胞和神经元细胞凋亡细胞数无显著影响，CO₂气腹在这一范围应用对中枢神经系统是安全的。（2） 10mmHg压力、2小时位点，CO₂气腹对正常大鼠中枢神经系统组织学、生化标志物、蛋白转录水平、蛋白水平表达的变化及脑组织胶质细胞和神经元细胞凋亡细胞数无显著影响，CO₂气腹在这一范围应用对中枢神经系统是安全的。（3）在5mmHg压力、1小时位点，CO₂气腹对缺血缺氧性脑损伤大鼠中枢神经系统组织学、生化标志物、蛋白转录水平、蛋白水平表达的变化及脑组织胶质细胞和神经元细胞凋亡细胞数有一定影响，CO₂气腹在这一范围应用对中枢神经系统仍是安全的。（4）在10mmHg压力、2小时位点，CO₂气腹对缺血缺氧性脑损伤大鼠中枢神经系统组织学、生化标志物及蛋白转录水平、蛋白水平表达的变化及脑组织胶质细胞和神经元细胞凋亡细胞数有显著影响，CO₂气腹在这一范围应用应充分考虑中枢神经系统并发症的危险，应尽量缩短手术时间或采取相应的调控措施。更多还原

【Abstract】 Objective With the rapid development and renovation of laparoscopic technology, many experimental and clinical researches concerned on the function changes of normal organs under carbon dioxide pneumoperitoneun which was largely used expend manipulative working space during laparoscopic procedure. With the rapid development of minimally invasiving surgery, the spectrum of laparoscopic operation have been growing wider and the difficulty of them also have been the focus in the field. Some studies have indicated that the brain ischemic and reperfusive injury developed during pneumoperitoneun and the function of Central nerous system were impaired after pneumoperitoneun. The reason for the abdominal compartment syndrome is an important factor for the injury of parenchymatous organ. The researches about the influence of pneumoperitoneun on the respiration, circulation, immunology and the vital abdominal organs are spring up. Howerver, the studies on the influence of pneumoperitoneun on the vital parenchymal organs in the pathological situation were very few and superficial. In healthy patients, carbon dioxide pneumoperitoneun effect on the body is transient and without any apparent sequelae. however, few articles in the literature were reported about the influence of CO₂ PP on the body with dysfunctional organ , such as heart failure、chronic liver dysfunction and so on. The purpose of this study was to evaluate the changes of blood ,cerebrospinal fluid（CSF） and brain tissue S-100 protein （S-100）,neuron specific enolse （NSE） levels and the expression of the mRNA and protein level for S-100 and NSE following pneumoperitoneun with carbon dioxide （CO₂ PP） at different of time and pressuer,and to explore whether CO₂ PP produces effect on central nerous system（CNS） in normal and hypoxic-ischemic brain damage rats. Methods Healthy adult Wistar rats were used to establish models of CO₂ PP and of hypoxic-ischemic brain damage .80 model rats were randomly divided into group s.The abdominal cavity of these rats were gradually insufflated CO₂ gas by closeing method and maintained at the pressure of 5mmHg （0.67Kp） for 1hour and 10mmHg （1.33Kp） for 2 hours.animals in each group were sacrificed at 48 hours individually after deflation .Astrocytes and neurons in the hippocamps of brian tissue of rat were observed by light microscope and electron microscope. Their serial blood and CSF S-100 and NSE were measured by ELISA way. By using the RT-PCR technique the expression of mRNA for S-100 and NSE in the brain tissue were tested .Immunohistochemical assay was used to investigate the changes of the expression of S-100 and NSE at the protein level. Detected by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick and labeling （TUNEL）,apoptosis of the astrocytes and neurons in the hippocampus of brian tissue of rat was counted on each slices.Results （1） There were no any histologic different between A_5-1 group and control group after pneumoperitoneun with carbon dioxide. The astrocytes and neurons in the hippocampus of brian tissue of rat in A_10-2 group showed local hydropic degeneration under light microscope , mitochondrion swelling under electron microscope. There were significantly histologic difference between B group and control group . The astrocytes and neurons in the hippocampus of brian tissue of rat in B group showed hydropic degeneration, no nuclear necrosis under light microscope , mitochondrion swelling under electron microscope. Astrocytes in the hippocampus of the B_10-2 group showed diffuse hydropic degeneration, nuclear pyknosis, and cell shrinkage under light microscope.mitochondrion and glycogen swelling , fragmentation of ridge myelinogenesis in the astrocytes .Neuron in the B_10-2 group showed diffuse hydropic degeneration, cytoplasmic hyperosinophilia , local nuclear pyknosis and cell shrinkage under light microscope .Nuclear membrane disruption under electron microscope. （2） The values of S-100, NSE were （0.415±0.096）ug/L and （0.511±0.022） ng/ml respectively in A_5-1 group and （0.445±0.086）ug/L, （0.531±0.04）ng/ml respectively in A_10-2 group in the blood, were （0.440±0.082） ug/L and （0.538±0.059） ng/ml respectively in A_5-1 group and （0.458±0.127）ug/L, （0.574±0.048）ng/ml respectively in A_10-2 group in the CSF after CO₂ PP. The values of S-100 ,NSE were increased in both blood and CSF samples in A_5-1 group and A_10-2 group after CO₂ PP than the values of control group （0.393±0.100ug/L,0.493±0.045 ng/ml and 0.431±0.124ug/L,0.531±0.048ng/ml） respectively. Nevertheless there were no significance between A_5-1 group and control group or A_10-2 group and control group （ P>0.05）. The findings showed a significantly increased levels of S-100 and NSE in the B group compared with the control group （P<0.05） or the B_10-2 group compared with the B group （P<0.05）respectively. The values of S-100, NSE in the blood were found to be positively correlated with the values in the CSF （P<0.05） )respectively.（3） The values of expression of mRNA for S-100 and NSE were （64.22±10.40） and （8.98±2.38） in A_5-1 group and （67.02±13.34）, （9.18±3.37） respectively in A_10-2 group after CO₂ PP,were （59.34±11.38） and （8.50±2.94） in control group .The expressions of S-100 and NSE increased with the increase of pressure and time of CO₂ PP. There were no significance between them （ P>0.05）. The protein s of mRNA for S-100 and NSE increased progressively in B group ,B_5-1 group and B_10-2 group than the expressions of control group. There were significance between B group and control group （P<0.05） or B_10-2 group and B group （P<0.05）.（4） The positive areas of the expression of S-100,NSE in the brian tissue increased progressively with the increase of pressure and time of CO₂ PP. There were no significance between A_5-1 group and control group （ P>0.05） or A_10-2 group and control group （ P>0.05）. There were significance between B group and control group （ P<0.05） or B_10-2 group and Bgroup （ P<0.05）.（5） Apoptosis of astrocytes and neurons in the hippocampus of brian tissue of rat had the similar amount to the control group at low IAP. Dramatical increase of apoptosis of astrocytes and neurons in per high visual field were demonstrated increased significantly after CO₂ PP pressure at 10mmHg （1.33Kp） for 2 hours .There were significance between B_10-2 group and control group. Apoptosis cells in astrocytes and neurons in the hippocampus of brian tissue had slightly but not significantly enhanced in normal rat, significantly enhanced in hypoxic-ischemic brain damage rats at 10mmHg （1.33Kp） for 2 hours.Conclusion （1） CO₂ PP at pressure of 5 mmHg and within 1 hour does not produce any effect on histology, biochemical marker, protein transcriptase level, protein expression level and apoptosis of astrocyte and neuron of central nervous system in normal rat. CO₂ PP is used during this extent is safe to central nervous system.（2） CO₂ PP at pressure of 10mmHg and within 2 hours does not produce significantly effect on histology, biochemical marker, protein transcriptase level, protein expression level and apoptosis of astrocyte and neuron of central nervous system in normal rat. CO₂ PP is used during this extent is safe to central nervous system . （3） CO₂ PP at pressure of 5 mmHg and within 1 hour produce a some effect on histology, biochemical marker, protein transcriptase level, protein expression level and apoptosis of astrocyte and neuron of central nervous system in hypoxic-ischemic brain damage rats. CO₂ PP is used during this extent is safe to central nervous system （4） CO₂ PP at pressure of 10 mmHg and within 2 hours produce significantly effect on histology, biochemical marker, protein transcriptase level, protein expression level and apoptosis of astrocyte and neuron of central nervous system in hypoxic-ischemic brain damage rats. CO₂ PP is used during the extent is injurious to central nervous system, and trying to short PP time or use control way.更多还原