大鼠深低温停循环模型的建立

目的建立大鼠深低温停循环复苏模型。方法采用体外插管法建立大鼠闭胸式深低温停循环复苏模型，并在深低温停循环时，用冷脑保护液间断给予脑组织灌流。结果用大鼠建立深低温停循环复苏模型：呼吸的自主恢复率为80％，心跳恢复率为100％。结论大鼠深低温停循环复苏模型的建立，对研究脑组织低温状态下的病理生理学变化和临床应用低温进行脑保护提供了基本条件。     

局部深低温停循环对大鼠血清NSE的作用

目的通过研究深低温停循环和顺行性灌注大鼠脑复苏对大鼠神经元特异性烯醇化酶（NSE）的影响，探讨深低温停循环和顺行性灌注对脑的治疗及保护效果。方法制造大鼠损伤模型，将大鼠随机分为对照组（9只）；实验组（9只），用酶联免疫吸附法（ELISA）测定血浆中的NSE活性。结果实验组血清NSE活性在损伤后3、6、24h监测明显低于对照组（P〈0．01）。结论深低温停循环和顺行性灌注脑复苏技术通过降低NSE活性来增强脑细胞的自身保护作用可能是其抗神经细胞损伤的分子机制之一。     

顺行性灌注二磷酸果糖对深低温停循环大鼠脑组织c-fos mRNA表达的影响

目的研究大鼠深低温停循环后脑组织中c-fos mRNA表达的时效关系。方法建立大鼠深低温停循环模型，用RT-PCR方法检测不同时间点大鼠海马组织内c-fos mRNA表达的强弱。结果c-fos mRNA的表达增强，在复温后即刻达高峰，与对照组相比，差异无统计学意义；在复温1h后开始减弱，6h最低，但均强于对照组，P〈0.05。结论（1）深低温停循环引起c—fos mRNA的表达；（2）含有1．6二磷酸果糖的冷脑保护液间断灌流，可以增强c—fos基因的表达。     

深低温对全脑缺血大鼠海马即早基因组表达的影响

目的筛选出深低温对全脑缺血大鼠海马影响的即早差异表达基因。方法建立大鼠体外循环模型，实验分成两组，常温缺血组（n=3）：常温条件下停循环全脑缺血5min；低温缺血组（n=3）：深低温条件下停循环全脑缺血5min。采用Affymetrix大鼠全基因组芯片检测两组动物海马基因表达的变化，获取差异表达基因。结果筛选出差异表达基因共有75个，其中39个基因表达有统计学意义（P〈0．01），上调33个，下调42个。结论深低温对全脑缺血大鼠海马即早基因表达有明显影响，这些差异表达基因可能与深低温脑保护作用相关。     

深低温脑保护的差异蛋白质组学研究

目的应用差异蛋白质组学技术，研究深低温和常温停循环后大鼠海马组织蛋白质的变化。方法采用深低温停循环模型，取大鼠海马组织，通过双向电泳、分离蛋白，然后通过胶内酶切、生物质谱分析差异的蛋白质点，鉴定出变化的蛋白质。结果通过Image Master软件分析报告发现有差异的Ratio值大于1．5的蛋白考染点14个。通过对这些蛋白考染点进行质谱鉴定，鉴定出28个蛋白质，其中4个为同一种蛋白，实际的蛋白数为24个。它们是细胞骨架蛋白、介导代谢的酶类、参与核酸合成、参与氧化应激反应的蛋白质及未知蛋白。结论鉴定出的差异蛋白质可能与深低温脑保护作用有关，某些蛋白质在低温脑保护中的作用尚未报道。     

深低温对全脑缺血大鼠海马线粒体细胞色素C释放及细胞凋亡影响

目的 观察深低温对全脑缺血大鼠海马线粒体细胞色素C(Cyt-C)释放和细胞凋亡的影响,探讨深低温的脑保护作用机制. 方法 24只SD大鼠建立大鼠体外循环模型后按随机数字表法分成3组:对照组(建立大鼠体外循环模型5 min后迅速处死取海马组织)、常温缺血组[大鼠于(37±0.3)℃下停循环5 min后迅速处死取海马组织)和低温缺血组(热交换器配合体外冰屑诱导使大鼠肛温降至(18±0.5)℃,停循环5 min后迅速处死取海马组织),每组各8只.Western blotting检测3组大鼠海马神经细胞胞浆Cyt-C的表达,TUNEL法检测3组大鼠海马神经细胞凋亡率. 结果 常温缺血组海马神经细胞胞浆Cyt-C浓度(3.57±0.82)较对照组(0.48±0.14)明显升高,而低温缺血组Cyt-C浓度(1.24±0.53)较常温缺血组明显降低,差异均有统计学意义(P＜0.05).常温缺血组海马神经细胞凋亡率(50.45％±3.71％)较对照组(2.56％±0.43％)明显升高,而低温缺血组海马神经细胞凋亡率(16.51％±2.65％)较常温缺血组明显降低,差异均有统计学意义(P＜0.05). 结论 深低温可抑制全脑缺血大鼠海马线粒体Cyt-C释放和细胞凋亡,是其脑保护作用的重要机制之一.     

猴脑选择性超深低温阻断血流复苏的脑电图观察

缺血性脑损伤的程度通常采用大体标本、组织病理、生化代谢变化等指标进行评定．而同时应用脑电图进行评定则较少。脑电图是一项可早期检测脑缺血的良好方法，对急性缺血性改变早期生物电活动高度敏感。深低温停循环时脑组织酸中毒、大脑低灌注等易造成脑梗死，使手术后中枢神经系统并发症增多，     

亚低温对心肺复苏术后缺血缺氧性脑病的影响

目的探讨亚低温治疗对院内心脏停搏心肺复苏(CPR)后缺血缺氧性脑病者神经功能和预后的影响。方法收集惠州市第一人民医院ICU的心肺复苏术后50例患者的临床资料,在心肺复苏成功自主循环恢复后,30例(亚低温组)接受亚低温治疗(中心体温降至32～34℃,并维持24h),20例(对照组)常规治疗,7d后比较2组患者复苏后神经功能良好率和病死率。结果在心肺复苏后第7天,亚低温组患者神经系统转归率23%(7/30)明显高于常规组15%(3/20,P<0.05);亚低温组病死率50%(15/30),常规组为55%(11/20,P>0.05)。结论亚低温对心肺复苏术后缺血缺氧性脑病患者的病死率无明显影响,但可提高神经系统转归率。     

猪深低温停循环长程时间窗脑保护机制研究

目的研究深低温停循环长程时间窗90min行间歇性一侧颈动脉顺行灌注脑保护液的辅助脑保护作用。方法实验小猪(10～15kg)22只分三组:空白对照组6只,18℃不停循环90min,不加灌注脑保护液;阳性对照组8只,18℃停循环90min,不加脑保护液灌注;实验组8只,18℃停循环90min一侧颈动脉顺行灌注保护液。采用改良开胸体外循环法建立猪深低温模型,转流降温至鼻咽温18℃时停循环90min,分别在停循环期间、降温和复温时加用脑保护液间歇灌注,观察停循环长程时间窗及辅助灌注的脑血流量和生理指标变化、神经血红蛋白(Neuroglobin,Ngb)的表达和电镜下海马组织神经元的超微结构变化。结果实验组脑血流量在辅助灌注下由(22.2±2.5)ml.min-1.100g-1上升到(38.5±2.6)ml.min-1.100g-1。空白对照组检测到少量神经元形态学改变,阳性对照组电镜下可以观察到神经元线粒体明显肿胀,而实验组线粒体形态正常,突触有大量囊泡聚集,RT-PCR神经血红蛋白表达上调,c-fos蛋白表达增加。结论1,6-二磷酸果糖 氧和冷晶液是一种较好的脑保护液;停循环90min内辅助灌注有较好的脑保护作用;深低温下的氧耐受可能源于Ngb有较好的携氧脑保护作用。     

超低温断血流对猴脑氨基酸类神经递质的影响

目的 研究猴脑选择性超深低温断血流复苏过程中脑组织细胞外液的谷氨酸(glutamate, Glu)和γ-氨基丁酸(gamma-aminobutyric acid, GABA)的动态变化.方法 恒河猴7只,随机分为两组:四血管(双侧颈总动脉及椎动脉)阻断冷灌注组(简称四血管组,n=4),两血管(双侧颈总动脉)阻断冷灌注组(简称两血管组,n=3).将微透析管置入右顶叶脑组织内,自缺血前30min开始收集细胞外液,用高效液相色谱-紫外法测定脑选择性超深低温断血流复苏前后脑组织细胞外液Glu及GABA的浓度.结果 四血管组Glu在常温断血流缺血后即迅速升高(P<0.05),低温冷灌注后较常温缺血时明显下降至低于缺血前水平(P<0.05);恢复血流后行再灌注复温,早期即开始升高,迅速达到并超过灌注前水平.GABA则变化不大.两血管组Glu在常温断血流预缺血后迅速升高(P<0.05),低温冷灌注后较常温缺血时明显下降(P<0.05),下降幅度较四血管组明显;恢复血流后行再灌注复温,和行低温冷灌注时相比,Glu变化不明显.GABA在常温断血流预缺血、低温冷灌注及恢复血流复温时均变化不大.结论 脑选择性超深低温断血流复苏通过抑制谷氨酸等兴奋性神经递质的释放而抑制其细胞毒性作用.     

Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination

We have examined the incidence and size of infarction after occlusion of different portions of the rat middle cerebral artery (MCA) in order to define the reliability and predictability of this model of brain ischemia. We developed a neurologic examination and have correlated changes in neurologic status with the size and location of areas of infarction. The MCA was surgically occluded at different sites in six groups of normal rats. After 24 hr, rats were evaluated for the extent of neurologic deficits and graded as having severe, moderate, or no deficit using a new examination developed for this model. After rats were sacrificed the incidence of infarction was determined at histologic examination. In a subset of rats, the size of the area of infarction was measured as a percent of the area of a standard coronal section. Focal (1-2 mm) occlusion of the MCA at its origin, at the olfactory tract, or lateral to the inferior cerebral vein produced infarction in 13%, 67%, and 0% of rats, respectively (N = 38) and produced variable neurologic deficits. However, more extensive (3 or 6 mm) occlusion of the MCA beginning proximal to the olfactory tract--thus isolating lenticulostriate end-arteries from the proximal and distal supply--produced infarctions of uniform size, location, and with severe neurologic deficit (Grade 2) in 100% of rats (N = 17). Neurologic deficit correlated significantly with the size of the infarcted area (Grade 2, N = 17, 28 +/- 5% infarction; Grade 1, N = 5, 19 +/- 5%; Grade 0, N = 3, 10 +/- 2%; p less than 0.05). We have characterized precise anatomical sites of the MCA that when surgically occluded reliably produce uniform cerebral infarction in rats, and have developed a neurologic grading system that can be used to evaluate the effects of cerebral ischemia rapidly and accurately. The model will be useful for experimental assessment of new therapies for irreversible cerebral ischemia.     

Regional dependence of cerebral reperfusion after circulatory arrest in rats.

The severity of neurologic dysfunction after circulatory arrest depends on cerebral reperfusion during and after resuscitation. The objective of current study was to investigate the temporal and spatial patterns of the cerebral perfusion immediately after resuscitation. Precise control of circulatory arrest was achieved in rats by combination of asphyxia and transient blockage of cardiac-specific beta-adrenergic receptors with esmolol, an ultra-short-acting beta-blocker. Animals were randomized into 3 groups with resuscitation starting 0.5 (sham group, no asphyxia, n = 5), 4 (Group 2, n = 5), or 12 minutes (Group 3, n = 8) later by retrograde intraarterial infusion of donor blood along with a resuscitation mixture. Cerebral perfusion was measured by magnetic resonance imaging (MRI) using arterial spin labeling. The average perfusion before arrest was 163 +/- 27 mL 100 g(-1) min(-1) under isoflurane anesthesia. Resuscitation led to transient perfusion increase, which started from thalamus and hypothalamus and later shifted to the cortex. Severe hypoperfusion to as low as 6% to 20% of the normal level developed in the first 10 to 20 minutes of reperfusion and lasted for at least 2 hours. On the fifth day after circulatory arrest, all animals showed a normal level of perfusion (159 +/- 57 mL 100 g(-1) min(-1) ) and minimal neurologic deficit. Nevertheless, histologic examination revealed extensive changes in the CA1 region of the hippocampus consistent with global ischemia and reperfusion damage. The combination of an improved circulatory arrest model and noninvasive MRI cerebral perfusion measurements provides a powerful tool for investigations of circulatory arrest and resuscitation, allowing for evaluation of therapies aimed at modulating cerebral reperfusion.     

左旋精氨酸对犬深低温停循环脑氧代谢的影响

目的 利用犬深低温停循环(DHCA)动物模型,研究左旋精氨酸(L-arg)预处理对DHCA不同时期犬脑氧代谢及超微结构的影响.方法 健康成年杂种犬15只,分为3组,即对照组;L-arg预处理组:停循环前60min给予L-arg100mg/kg预处理;联合处理组:停循环前60min给予L-arg100mg/kg、7-硝基吲唑(7-Ni)25mg/kg联合处理,每组各5只.按临床方法建立体外循环转流降温至鼻咽部温18℃停循环,90min后恢复循环复温.分别于停循环前30min、停循环时(0min)、停循环45min、停循环90min、复温再灌注60min抽取颈静脉、颈动脉血测颈静脉血氧饱和度(SjvO2)、一氧化氮(NO).实验犬处死时颈动脉灌注4%多聚甲醛固定,取脑皮质、海马行透射电镜观察脑组织超微结构.结果 L-arg预处理和联合处理均可增加血浆N0含量、提高DHCA期SjvO2,改善脑氧代谢,减轻脑超微结构损害.停循环前血浆NO与停循环后SjvO2呈正相关(r=0.679,P=0.005).结论 DHCA前给予L-arg预处理有较好的脑保护效果.     

慢性脑低灌注动物模型再探讨

目的 建立一种新的慢性脑低灌注动脉模型，研究动脉和静脉循环对脑灌注压（CPP）的影响及正常灌注压恢复时脑组织病理变化。方法 24只SD大鼠随机分为模型组、动静脉瘘（AVF）组和假手术组。模型组动物行右侧颈外静脉－颈总动脉端侧吻合，同时结扎左侧横窦引流静脉及双侧颈外动脉。分别于术前、术后即刻和术后3个月检测各组动物平均动脉压（MAP）、颅内引流静脉压（DVP）和CPP。3个月后阻断AVF，定量检测血脑屏障（BBB）破坏程度和脑水含量。透射电镜观察脑组织超微结构。结果 动静脉分流术后MAP明显下降，DVP明显升高，CPP明显降低。术后3个月，模型组动物DVP仍明显升高，CPP明显降低，与其他两组动物相比差别有显著性。AVF阻断后模型组动物BBB明显破坏，脑水含量明显增加。电镜证实脑组织中有不同程度的血管源性脑水肿和（或）出血，并与脑部分毛细血管周围星形胶质细胞足突消失有关。结论 颈动静脉端侧吻合可导致CPP降低，静脉引流障碍进一步加重脑低灌注状态，并与灌注压突破密切相关。该动物模型符合人脑动静脉畸形的基本血流动力学特征。     

双侧深低温停循环顺行性脑灌注对主动脉弓替换术患者脑损伤的影响

目的 探讨双侧深低温停循环(Deep hypothermia and circulatory arrest,DHCA)+顺行性脑灌注(Anterograde cerebral perfusion,ACP)对主动脉弓替换术患者脑损伤的影响。方法 选择2018年1月-2020年1月本院实施主动脉弓替换术的DebakeyⅠ型主动脉夹层患者96例,根据不同的脑保护技术将患者分为单侧ACP组和双侧ACP组,各48例; 2组均进行DHCA,单侧ACP组经右侧腋动脉进行单侧ACP,双侧ACP组经右侧腋动脉和左颈总动脉进行双侧ACP。结果 全部患者痊愈出院,出院前CT复查显示主动脉弓和升主动脉的人工血管血流畅通,无人工血管扭曲和造影剂渗漏等状况。双侧ACP组术后短暂性脑损伤发生率为2.08%(1/48),明显低于单侧ACP组的16.67%(8/48)(P<0.05)。双侧ACP组术后苏醒时间为(13.18±3.42)h,明显短于单侧ACP组的(16.98±4.18)h(P<0.05)。体外循环开始后2组血清神经元特异性烯醇化酶(Neuron-specific enolase,NSE)、中枢神经特异性蛋白 100-β亚型(Specific protein 100-β,S100-β)水平均逐渐升高,且在体外循环结束时达到峰值,随后开始降低; 脑灌注5min后双侧ACP组血清NSE,S-100β水平明显低于单侧ACP组(P<0.05)。结论 双侧深低温停循环顺行性脑灌注对主动脉弓替换术患者脑损伤的影响程度更小,其机制可能是通过降低血清NSE,S-100β水平,进而有效保护脑组织。     

不开颅的大脑中动脉供血区梗塞动物模型

用不开颅的方法将血管内凝集剂从Ｗｉｓｔａｒ鼠颈内动脉注入，制成左大脑中动脉供血区局灶性脑缺血模型，并对鼠的血压，心电，体温，呼吸，脑电，血氧饱和度和血糖进行了监测，记录了这一模型的神经功能缺失程度，梗塞灶的体积和病理变化，实验结果表明局灶性缺血鼠的神经功能缺失评分多为２或３级，其梗塞灶主要位于第一感觉运行区，第二感觉运动区，外侧中央前区，内侧中央前区和基底节区，病理上表现为典型的缺血性改变，这一模     

Effects of 2-deoxy-d-glucose on focal cerebral ischemia in hyperglycemic rats.

The authors examined the effects of pretreatment with 2-deoxy-d-glucose (2DG) on the middle cerebral artery occlusion-reperfusion (MCAO/R) model in hyperglycemic rats. Proton magnetic resonance imaging and spectroscopy were used to measure the lesion size, the level of cerebral perfusion deficit, and ratio of lactate to N-acetyl aspartate (NAA) in brain regions. By performing sequential diffusion weighted imaging, gradient echo bolus tracking, steady-state spin echo imaging, and water-suppressed proton magnetic resonance spectroscopy techniques, the time course of the early changes of the lactate/NAA peak ratio and perfusion deficit was examined in hyperglycemic rats undergoing 90-minute MCAO followed by 24-h reperfusion. Compared with the saline-treated hyperglycemic rats, 2DG treatment at 10 minutes before MCAO significantly reduced diffusion weighted imaging hyperintensity by approximately 60% and the lactate/NAA peak ratio by approximately 70% at 4 h after MCAO/R. Both spin echo-measured cerebral blood volume and dynamic gradient echo-relative cerebral blood flow showed that the restoration of blood supply recovered and remained at approximately 80% of baseline during reperfusion in 2DG-treated hyperglycemic rats. These data suggest that inhibition of glucose metabolism by 2DG has a beneficial effect in reducing brain injury and minimizing the production of brain lactate during MCAO/R in hyperglycemic rats.     

Effects of tissue type plasminogen activator in embolic versus mechanical models of focal cerebral ischemia in rats.

Tissue type plasminogen activator (tPA) can be effective therapy for embolic stroke by restoring cerebral perfusion. However, a recent experimental study showed that tPA increased infarct size in a mouse model of transient focal ischemia, suggesting a possible adverse effect of tPA on ischemic tissue per se. In this report, the effects of tPA in two rat models of cerebral ischemia were compared. In experiment 1, rats were subjected to focal ischemia via injection of autologous clots into the middle cerebral artery territory. Two hours after clot injection, rats were treated with 10 mg/kg tPA or normal saline. Perfusion-sensitive computed tomography scanning showed that tPA restored cerebral perfusion in this thromboembolic model. Treatment with tPA significantly reduced ischemic lesion volumes measured at 24 hours by >60%. In experiment 2, three groups of rats were subjected to focal ischemia via a mechanical approach in which a silicon-coated filament was used intraluminally to occlude the origin of the middle cerebral artery. In two groups, the filament was withdrawn after 2 hours to allow for reperfusion, and then rats were randomly treated with 10 mg/kg tPA or normal saline. In the third group, rats were not treated and the filament was not withdrawn so that permanent focal ischemia was present. In this experiment, tPA did not significantly alter lesion volumes after 2 hours of transient focal ischemia. In contrast, permanent ischemia significantly increased lesion volumes by 55% compared with transient ischemia. These results indicate that in these rat models of focal cerebral ischemia, tPA did not have detectable negative effects. Other potentially negative effects of tPA may be dependent on choice of animal species and model systems.     

复方白术对实验性局灶性脑缺血损伤的保护作用

目的 观察复方白术颗粒对大鼠局灶性脑缺血的保护作用。方法 热凝阻断法制成大鼠局灶性脑缺血模型,观察药物对其神经症状、脑电图、脑含水量、脑组织形态学和ＬＰＯ的影响。结果 用药组可以显著减轻缺血性脑卒中动物的神经症状,改善阻断侧额顶区脑电图波幅降低和频率减慢,减轻脑水肿,改善缺血区和病理组织学改变及降低ＬＰＯ含量。结论 复方白术颗粒对大鼠缺血性脑损伤有保护作用。