可重复性小鼠局灶性脑缺血/再灌注模型的探讨

目的介绍一种标准的小鼠局灶性脑缺血/再灌注模型的制作方法,并观察不同脑缺血/再灌注时间脑梗死体积和脑水肿的变化。方法用腔内线栓法制作脑缺血/再灌注动物模型,用TTC染色法进行脑大体观察,用甲酚紫染色法观察脑切片梗死灶,用脑血流激光多普勒监测脑血流的变化,用ImageJ软件计算脑梗死体积和脑水肿。结果当线栓封闭大脑中动脉时,脑血流就会急剧下降至最低水平,拔出线栓后脑血流迅速上升至缺血前水平。脑缺血后,脑片上呈现明显的梗死灶,脑梗死体积和脑水肿的大小较恒定。脑缺血90min再灌注24h组梗死体积、脑水肿体积、脑水肿百分数及神经功能缺损程度均显著大于脑缺血30min再灌注24h组(P<0.001)。脑缺血30min/再灌注72h脑水肿非常明显(72.6±4.3)mm3,再灌注7d时脑水肿开始减退,仅为(50.9±4.1)mm3,再灌注30d时脑容积出现萎缩,脑水肿呈负值(-20.1±1.8)mm3。结论该小鼠局灶性脑缺血/再灌注模型具有重复性好、容易操作的优点。脑缺血30min就可造成不可逆性脑损害,脑水肿在再灌注72h即达到高峰。     

Kallikrein基因转导对脑缺血再灌注后局部血流灌注恢复的作用

目的 探讨Kallikrein基因对脑缺血再灌注后梗死灶周围血管增生与局部脑血流灌注恢复的作用.方法 建立大鼠脑缺血再灌注模型,术后将90只大鼠按照随机数字表法分为3组.每组30只,分别是空白对照组、注射生理盐水、注射pAdCMV-人组织激肽释放酶(HTK)组.各组大鼠又分为治疗后12 h、24 h及72 h组,每组各10只.治疗前后行大鼠神经功能缺损评分.TTC染色方法测定脑梗死面积的变化,用免疫组化检测外源性HTK的表达以及局部血管内皮生长因子(VEGF)的表达,并通过14C-iodoantipyrine微示踪技术检测局部脑血流灌注(rCBF)情况.结果 与其他两组相比,pAdCMV-HTK组大鼠脑梗死面积在治疗后24h已有明显减小,72h后这种变化更明显,差异有统计学意义(P<0.05);在治疗后24 h,pAdCMV-HTK组大鼠神经功能缺损评分明显低于生理盐水组及空白对照组,治疗后72h差异更明显,差异有统计学意义(P<0.05).vEGF阳性细胞主要分布于脑梗死灶周边皮质与部分白质;pAdCMV-HTK组VEGF表达在治疗后12h、24h、72h均明显高于生理盐水组及空白对照组,差异有统计学意义(P<0.05).各组缺血再灌注后脑梗死灶周围白质与皮质rCBF均较对侧稍减少:pAdCMV-HTK组治疗后12h,梗死灶周围白质与皮质rCBF较空白对照组与生理盐水组有增高.但不明显,差异无统计学意义(P>0.05),而在治疗24h、72 h后rCBF则明显增高,差异有统计学意义(P<0.05).结论 在脑缺血再灌注后,Kallikrein基因转导可增加梗死灶周围脑组织的血管增生,改善rCBF,减小梗死面积,从而达到保护缺血神经细胞功能的作用.     

吡拉西坦抗短暂性局灶性脑缺血作用观察

目的 观察吡拉西坦的抗局灶性脑缺血作用。方法采用动脉腔内插线大鼠局灶性脑缺血模型及激光多普勒血流计测定半暗带脑血流、湿重-干重法测定缺血半球水含量、3％伊文氏蓝染色结合图像分析测定血脑屏障(BBB)的破坏、HE染色结合图像分析测定缺血后21h的梗死体积。结果100mg／kg体重吡拉西坦对半暗带脑血流无明显影响，200mg／kg体重吡拉西坦可明显升高再灌注期间半暗带脑血流；100mg／kg和200mg/kg体重的吡拉西坦均可明显降低局灶性脑缺血后缺血半球的水含量及BBB的损伤，缩小梗死体积。结论吡拉西坦具有明确的抗局灶性脑缺血作用，它可改善半暗带脑血流、减轻脑组织水肿和BBB的损伤、缩小梗死体积。     

利用激光散斑成像技术观察尤瑞克林对脑梗死大鼠脑血流的影响

目的 利用激光散斑成像技术研究尤瑞克林对大鼠脑梗死后局部脑血流的影响.方法 成年雄性SD大鼠24只,线栓法制备大鼠永久性大脑中动脉梗死模型.激光散斑成像系统观测缺血半球皮质及大脑中动脉供血区血流,2,3,5-三苯基氯化四氮唑(TTC)染色法测定脑梗死体积,并进行神经功能评分.结果 皮质及大脑中动脉供血区血流在大剂量组第1天及第2天给药后均有明显改善,部分大脑皮质血管增粗,血流速度加快,小剂量组及生理盐水组无明显变化,脑缺血48 h后,大、小剂量尤瑞克林组及生理盐水组的梗死体积分别为10.14%±3.02%,25.99%±3.90%,27.10%±3.32%,大剂量组与生理盐水组比较差异有统计学意义(F=61.14,P<0.01),小剂量组与生理盐水组比较差异无统计学意义.缺血后4 h,大剂量组神经功能损伤明显改善,小剂量组及生理盐水组无明显改变,36 h各组间的神经功能评分差异无统计学意义.结论 尤瑞克林可以减少大鼠局灶性脑缺血后梗死体积,延缓神经功能损伤,其作用可能与促进侧支循环的开放,增加大脑皮质和缺血区血流有关.     

3-硝基丙酸预处理对大鼠局灶性脑缺血后促红细胞生成素蛋白和mRNA表达的影响

目的 观察小剂量线粒体毒素3-硝基丙酸(3-nitroppropionic acid，3-NPA)预处理对大鼠局灶性脑缺血后促红细胞生成素(erythropoietin，Epo)蛋白和mRNA表达的影响，揭示3-NPA预处理诱导脑缺血耐受的机制。方法 大鼠腹腔注射3-NPA 3d后制作大鼠局灶性缺血再灌注模型，采用TTC染色、免疫组织化学方法、逆转录聚合酶链式反应技术，观察3-NPA预处理对缺血2h再灌注24h脑梗死体积和Epo蛋白及mRNA表达的影响。结果 脑缺血再灌注后Epo蛋白表达广泛，主要分布于缺血侧基底节区、海马和部分大脑皮质。3-NPA预处理使脑梗死体积减小，Epo蛋白和mRNA的表达增多，与对照组比较有显著性差异。结论 小剂量3-NPA预处理可以诱导脑缺血耐受，脑源性Epo表达增强可能是其形成的机制之一。     

3-硝基丙酸预处理对大鼠缺血-再灌注脑损伤保护作用的研究

目的 研究3-硝基丙酸(3-NPA)预处理对大鼠局灶性脑缺血脑梗死体积,脑缺血半暗带神经细胞凋亡的影响,探讨3-NPA预处理诱导脑缺血耐受的机制.方法 大鼠腹腔注射3-NPA,分别在24h和72h后制作大脑中动脉缺血-再灌注模型,采用TTC染色和TUNEL法,观察3-NPA预处理对缺血2h再灌注24h脑梗死体积、神经细胞凋亡的影响.结果 缺血2h再灌注24h,3-NPA预处理组脑梗死体变小,神经细胞凋亡减少,与对照组及假手术组比较有显著性差异.结论 3-NPA预处理可以诱导脑缺血耐受,抑制神经细胞凋亡可能是其机制之一.     

国产降纤酶对大鼠缺血/再灌注脑损伤的保护作用

目的观察3种国产降纤酶对大鼠缺血/再灌注脑损伤的保护作用。方法采用线栓法大鼠局灶性脑缺血/再灌注模型,观察3种国产降纤酶对缺血/再灌注不同时程动物脑梗死体积、血流量、神经功能缺损评分及梗死灶内肉眼出血率的影响。结果持续缺血3h,降纤酶治疗的各组动物脑梗死体积明显小于生理盐水对照组(P<0.05);缺血3h再灌注3h和72h,降纤酶治疗的各组动物脑梗死体积与生理盐水组相比无明显变化,但缺血3h再灌注6h和24h,降纤酶治疗的各组动物脑梗死体积比生理盐水组明显减少(P<0.05)。缺血3h再灌注6h、24h和72h,降纤酶治疗组动物脑血流量比生理盐水组明显增加(P<0.05)。但治疗组动物行为学评分较生理盐水组无相应改善,梗死灶内有肉眼出血的动物较生理盐水组多,但无统计意义。结论国产降纤酶能明显减小缺血/再灌注脑损伤动物的梗死体积和增加脑血流量,改善损伤后的低灌注状态,对脑组织有一定保护作用。     

粒细胞集落刺激因子对大鼠局灶性脑缺血的治疗作用及研究

目的 探讨粒细胞集落刺激因子(G-CSF)对大鼠局灶性脑缺血的治疗作用.方法 在激光脑血流监测下,采用线栓法制成大鼠大脑中动脉局灶性脑缺血模型,在梗死前3d开始皮下注射G-CSF,持续注射8d,对照组注射同样的生理盐水,并且在手术结束后4h开始监测神经功能评分,直至术后第8天.术后第8天及第22天利用图像分析仪进行梗死面积的测定.结果 神经功能缺损评分,病理上梗死面积用药组及对照组都有明显差异.结论 G-CSF可减轻大鼠脑梗死程度,减少脑梗死体积,促进神经功能恢复.     

脑缺血后适应对基质金属蛋白酶-9表达的影响

目的:探讨缺血后适应对大鼠脑缺血再灌注损伤的保护作用及与MMP-9的关系。方法:应用线栓法制做大鼠脑缺血再灌注损伤模型;对大鼠脑缺血再灌注1h和48h进行神经功能评分;48h后TTC染色测定脑梗死体积和脑水肿程度;4h、8h、24h、48h后免疫组化定位定量MMP-9水平。结果:缺血后适应组大鼠脑梗死体积、水肿程度、术后神经功能评分较对照组明显改善(P<0.05),各时间点后适应组大鼠基底节区MMP-9表达较对照组显著减少(P<0.05),梗死侧皮层MMP-9表达无显著改变。结论:缺血后适应能减少脑缺血后MMP-9的表达,缩小梗死体积,减轻脑水肿程度,改善术后神经功能。MMP-9下调可能是缺血后适应脑保护的分子机制之一。     

丁苯酞预处理对大鼠脑缺血再灌注损伤的神经保护作用

目的研究丁苯酞预处理对大鼠局灶性脑缺血再灌注损伤的神经保护作用。方法健康成年SD雄性大鼠48只,随机分为假手术组、缺血再灌注组、丁苯酞预处理组,每组各16只。各组均灌胃5d后,采用线栓法制作大鼠局灶性脑缺血再灌注(MCAO)模型,缺血2h、再灌注24h,进行神经功能缺损评分,TTC染色及图像分析观察脑梗死体积,免疫组化法检测脑组织caspase-3、bcl-2表达的变化。结果与缺血再灌注组相比,丁苯酞预处理组神经缺损程度改善,梗死灶体积减少,caspase-3阳性细胞数量减少,bcl-2表达上调。结论丁苯酞可减轻缺血性脑血管病的发作,具有一定的神经保护作用。     

Does poly-L-lysine coating of the middle cerebral artery occlusion suture improve infarct consistency in a murine model?

Background and Purpose: Rodent models of stroke that employ an intraluminal suture to cause focal cerebral ischemia are associated with some variability of resultant infarct volumes, thus requiring increased numbers of animals to determine significant differences between experimental groups. A recent modification of the occluding suture by coating with poly-L-lysine has been shown to create more uniform infarct volumes in rats. Methods: To evaluate the utility of this modification in murine models of both transient and permanent focal cerebral ischemia, male C57B16J mice were subjected to reversible middle cerebral artery occlusion (MCAo) for 45 minutes (n=42), or to permanent MCAo (n=25), with an intraluminal monofilament suture. Three types of sutures were used: untreated, partially coated, and completely coated with poly-L-lysine. Relative changes in regional cerebral blood flow, severity of neurological deficits, and infarct volumes were measured 24 hours after the ischemic injury. Results: Animals subjected to 45 minutes of temporary occlusion with completely coated poly-L-lysine sutures had infarct volumes of 13.8%+/-5% compared with infarct volumes of 7.2%+/-4% in those subjected to partially coated sutures and 22.4%+/-6% in the group occluded with untreated sutures (P=ns). Use of completely coated sutures resulted in significantly less reperfusion following suture removal. Control animals undergoing permanent occlusion with untreated sutures had infarct volumes of 17%+/-7% compared with 14.1%+/-5% using completely coated sutures and 6.5%+/-3% in animals with partially coated sutures (P=ns). There were no significant differences in cerebral blood flow between the experimental groups undergoing permanent MCAo. Conclusions: Poly-L-lysine coating of intraluminal sutures does not increase the consistency of infarct volumes in a murine model of temporary/permanent MCAo. These findings are in marked contrast to findings in rats.     

Comparison of silicon-coated nylon suture to plain nylon suture in the rat middle cerebral artery occlusion model

A variety of intraluminal sutures have been used in the middle cerebral artery occlusion model (MCAO) of focal ischemia. In the present study we tested commercially available silicon-coated nylon suture in the MCAO model and compared the results to traditional monofilament nylon suture occlusion. Twelve Sprague-Dawley male rats were randomly divided two groups, MCAO with 4-0 nylon suture (Group N, n=6) and MCAO with silicone-coated 4-0 nylon suture (Group S, n=6). Rats were sacrificed 24 h after reperfusion. Assessment included mortality rates, neurological evaluation, and infarct volume. One rat died in each group from subarachanoid hemorrhage. Neurological evaluation demonstrated that Group S tended to have worse neurological outcomes than Group N, although this difference was not statistically significant. On TTC stain Group S had significantly larger infarct volumes than Group N. We conclude that the commercially available silicone-coated occlusion suture provides better occlusion of the middle cerebral artery than the traditional uncoated nylon suture. Classification: Disease-related neuroscience (Section 6).     

A new model of temporary focal neocortical ischemia in the rat.

BACKGROUND AND PURPOSE: We describe a new rat model of temporary focal ischemia that produces neocortical ischemia without the need for prolonged anesthesia. METHODS: Temporary focal cerebral ischemia was initiated during halothane anesthesia, maintained for varying periods without anesthesia, and reversed by clip removal requiring brief anesthesia. Tandem carotid and middle cerebral artery occlusion for 1-4 hours and permanent occlusion were used to determine the duration and extent of ischemia necessary to produce predictable volumes of neocortical infarction in Wistar and spontaneously hypertensive rats. RESULTS: In Wistar rats, occlusion of the right middle cerebral and both common carotid arteries resulted in cerebral blood flow reductions to approximately 8% of baseline. One hour of transient ischemia with 23 hours of reperfusion did not result in infarction. Three hours of ischemia followed by 21 hours of reperfusion resulted in infarction comparable to that caused by 24 hours of permanent ischemia. In spontaneously hypertensive rats, unilateral right middle cerebral and common carotid artery occlusion reduced cerebral blood flow to approximately 11% of baseline. Minimal damage was seen with 1 hour of reversible ischemia, but intervals of 2 and subsequently 3 hours followed by 22-21 hours of reperfusion produced progressively larger infarcts. Damage indistinguishable from that seen with 24 hours of permanent ischemia was seen with 3 or 4 hours of transient ischemia followed by 21 or 20 hours of reperfusion. CONCLUSIONS: For unanesthetized normothermic rats, cerebral blood flow reductions to 10-20% of baseline resulted in maximal infarction once ischemic durations exceeded 2-3 hours. To be effective, experimental therapies aimed at lessening infarct size or restoring blood flow must be initiated within this critical time interval.     

D1和D2受体拮抗剂对乙灶性脑缺血梗塞体积及脑血流的影响

目的 研究多巴胺（ＤＡ）Ｄ１受体拮抗剂ＳＣＨ－２３３９０和Ｄ２受体拮抗剂Ｅｔｉｃｌｏｐｒｉｄｅ对可逆性乙灶性脑缺血梗塞体积及皮层半暗带脑血流的影响。方法 采用激光多普勒脑血流计测量大鼠可逆性乙灶性脑缺血各时相皮层半暗带脑血流,并于缺血后２４小时断头取脑切片,ＴＴＣ染色,计算机图样分析系统测量脑梗塞体积。结果 Ｄ１受体拮抗剂ＳＣＨ－２３３９０可明显缩小局灶性脑缺血梗塞体积,改善缺血期各时相皮层半暗带     

脑血疏口服液对大鼠脑缺血再灌注损伤后血脑屏障的影响

目的 探讨脑血疏口服液对大鼠脑缺血再灌注损伤后血脑屏障的影响。方法 将120只SD大鼠随机分为3组:假手术组、对照组,脑血疏组; 采用线栓法建立大鼠左侧大脑中动脉闭塞再灌注模型,缺血2 h后拔出线栓,恢复灌注24 h; 采用Longa FZ 5级评分法进行大鼠神经功能缺损评分; TTC染色计算脑梗死体积百分比; 运用干-湿重法测脑含水率; 通过伊文思蓝( EB)含量反映血脑屏障的损伤程度; 免疫组化检测基质金属蛋白酶-9(MMP-9)的表达水平。结果(1)假手术组大鼠在神经功能缺损评分、脑梗死体积、脑含水率均低于对照组(P<0.01); 脑组织中EB含量和MMP-9表达水平较对照组低(P<0.01);(2)脑血疏组大鼠的神经功能缺损评分较低、脑梗死体积较小,脑水肿程度较轻; EB含量和MMP-9表达水平均较对照组明显减少(P<0.01)。结论 脑血疏口服液对大鼠脑缺血再灌注损伤后血脑屏障具有保护作用,其机制可能是通过抑制MMP-9的表达。     

大鼠局灶脑缺血再灌注模型的研究

对尼龙线栓塞大鼠大脑中动脉的局部脑缺血再灌注模型进行研究。观察了大鼠脑缺血不同时间再灌注的成功率、脑水肿、神经病学评分及血流量（ｒＣＢＦ）的动态变化。结果如下：（１）改进实验方法后，使大鼠脑缺血不同时间再灌注的成功率明显提高；（２）脑缺血后早期再灌注，可显著降低脑水肿，改善神经损伤症状；（３）大鼠脑缺血期ｒＣＢＦ降至正常值的１５．７％～１９．１％。再灌后，ｒＣＢＦ恢复至正常值的５９．４％～８４．９％。     

Temporal thresholds for neocortical infarction in rats subjected to reversible focal cerebral ischemia.

We investigated the temporal threshold for focal cerebral infarction in the spontaneously hypertensive rat. The right middle cerebral artery and common carotid artery were occluded for 0, 1, 2, 3, 4, or 24 hours, and all the animals were sacrificed 24 hours after the onset of ischemia. Cortical infarct volumes and edema volumes were quantified in serial frozen sections of hematoxylin and eosin-stained tissue using image analysis. Upon occlusion, blood flow in the core of the ischemic zone, measured with laser-Doppler flowmetry, fell to a mean +/- standard deviation of 21 +/- 7% of the preocclusion baseline value (n = 26). During the first hour of ischemia, blood flow in the densely ischemic zone rose to 27 +/- 8% of baseline (n = 25). Release of the middle cerebral artery and common carotid artery occlusions rapidly restored cortical blood flow to 213 +/- 83% of baseline (n = 21). Focal ischemia of 1 hour's duration caused little or no infarction, while ischemic intervals of 2 and 3 hours produced successively larger volumes of infarcted cortex. Ischemic intervals of 3-4 hours' duration followed by approximately 20 hours of recirculation yielded infarct volumes that were not significantly different from those after 24 hours of permanent focal ischemia. The results indicate that 3-4 hours of focal cerebral ischemia in this rat model is sufficient to attain maximal infarction and suggest that recirculation or pharmacological interventions after this time will provide little benefit.     

Tirilazad reduces cortical infarction after transient but not permanent focal cerebral ischemia in rats.

BACKGROUND AND PURPOSE: We examined the cytoprotective effect of the lipid peroxidation inhibitor tirilazad mesylate (U74006F) in rodent models of neocortical infarction induced by transient and permanent focal cerebral ischemia. METHODS: Wistar rats (experiment 1) and spontaneously hypertensive rats (experiment 2) were subjected to 2 hours of transient middle cerebral artery occlusion followed by 22 hours of reperfusion and pretreated with 10 mg/kg i.p. tirilazad, vehicle, or saline. Repeat doses were given at 4 and 10 hours after reperfusion. Spontaneously hypertensive rats were also subjected to permanent middle cerebral artery occlusion and either pretreated with tirilazad, vehicle, or saline intraperitoneally (experiment 3) or treated with either tirilazad or vehicle intravenously after ischemia (experiment 4). Cortical infarct volumes were measured 24 hours after the onset of either transient or permanent ischemia, and changes in core regional cerebral blood flow were monitored with laser Doppler flowmetry. RESULTS: Tirilazad reduced infarct volume after transient ischemia by 40% in Wistar rats (p = 0.08) (experiment 1) and 23% in spontaneously hypertensive rats (p less than 0.05) (experiment 2) but did not reduce infarction after permanent ischemia whether it was given intraperitoneally (experiment 3) or intravenously (experiment 4). Ischemic core blood flows were not affected during ischemia, nor were they affected during reperfusion after transient ischemia. CONCLUSIONS: Tirilazad reduces cortical infarction in transient but not permanent ischemia, an effect not related to improvement in regional cerebral blood flow. Tirilazad might prove to be useful as an adjuvant therapy after successful thrombolysis in acute stroke patients.     

厄贝沙坦对大鼠局灶性脑缺血再灌注后炎症反应的影响

目的观察厄贝沙坦对大鼠局灶性脑缺血再灌注后脑内及外周炎症反应的影响。方法采用改良Longa方法制备大鼠大脑中动脉阻塞(middle cerebralartery occlusion,MCAO)模型,于缺血90min再灌注后24h和72h进行梗死体积的测量,采用免疫组化和ELISA方法测量脑内和外周血的粘附分子。结果厄贝沙坦可以显著减少局灶性脑缺血再灌注后24h和72h的梗死体积(均P<0.01),改善神经功能(均P<0.01);降低脑内ICAM-1、VCAM-1的表达及其外周血浆中可溶性的形式sICAM-1、sVCAM-1蛋白的水平(均P<0.05)。结论厄贝沙坦可以降低粘附分子的表达,减少梗死体积,改善神经功能,对脑缺血再灌注起保护作用。     

神经节苷脂对大鼠脑缺血再灌注损伤的脑保护作用

目的探讨神经节苷脂对大鼠脑缺血再灌注损伤的脑保护作用。方法采用线栓法制作缺血再灌注大鼠模型,分别用神经节苷脂(治疗组)和生理盐水(对照组)腹腔注射。观察两组大鼠缺血90min、缺血90min再灌注24h的脑梗死面积、神经功能缺损程度、细胞凋亡数、细胞凋亡率。结果治疗组大鼠于相同时间点脑梗死面积较对照组明显减小,仅表现轻度的神经功能缺损,且神经细胞的凋亡数较对照组显著减少(均P<0.01)。结论神经节苷脂能明显减小大鼠实验性脑缺血的脑梗死面积,减轻脑缺血再灌注后神经功能缺损程度,显著减轻缺血区神经元损害,具有显著的脑保护作用。