Peripheral and central administration of neuropeptide Y in a rat middle cerebral artery occlusion stroke model reduces cerebral blood flow and increases infarct volume

Recent studies have shown increased immunoreactivity for neuropeptide Y (NPY) within the perilesional cortex following experimental middle cerebral artery occlusion (MCAO) or focal excitotoxic damage. Downregulation of the NPY Y1 receptor gene using an antisense oligodeoxynucleotide produced a doubling of the infarct volume, implying that NPY may mediate neuroprotection against focal ischemia. The effects of treatment with NPY on infarct volume and hemodynamic parameters were investigated in the present study. Adult male Sprague-Dawley rats were anesthetized with sodium pentobarbital to undergo right-sided endovascular MCAO for 2 h. A single dose of NPY was given via intracarotid injection (10 microg/kg) at the beginning of reperfusion, intracisternal injection (10 or 30 microg/kg) at 30 min of ischemia, or intracerebroventricular (i.c.v.) injection (10 or 70 microg/kg) at 30 min of ischemia. Control groups received the vehicle only via the same route. Body temperature was maintained constant, and hemodynamic parameters were monitored during anesthesia. Laser Doppler flowmetry was used to monitor the regional cerebral blood flow (rCBF) during ischemia and reperfusion in some rats. The rats were decapitated on day 3, and their brains were cut into 2-mm thick coronal slices before reaction with a 2% solution of 2,3,5-triphenyltetrazolium chloride to reveal the infarct. Compared to the respective control groups, NPY treatment via any method of administration increased the relative infarct volume. Suppression of rCBF was observed during reperfusion. These results indicate that peripheral or central administration of NPY impairs reperfusion following experimental MCAO and worsens the outcome of focal cerebral ischemia.     

bFGF和PDGF-B蛋白在脑缺血再灌注大鼠模型中的表达和血管形成的研究

目的 探讨脑缺血再灌注大鼠不同时间碱性成纤维细胞生长因子(bFGF)和血小板源性生长因子-B(PDGF-B)的表达水平及其与血管形成的关系。方法 采用线栓法制备大鼠大脑中动脉局灶性脑缺血再灌注(MCAO/R)模型,分为假手术组和MCAO/R组,MCAO/R组缺血2 h后根据再灌注时间窗的不同分为0、6、24 h、3、7、14、21 d共7组,应用HE染色观察病理变化并测定脑梗死体积,用免疫组化法检测CD34蛋白的表达水平并计数微血管密度(MVD),用免疫组化法检测bFGF和PDGF-B蛋白的表达水平。结果 bFGF蛋白表达水平在MCAO/R 6 h后即开始升高,3 d到达高峰,7 d开始降低。PDGF-B蛋白表达水平在MCAO/R 24 h后明显升高,3 d到达高峰,7 d有所下降,持续到14 d左右。MVD表达在MCAO/R 3 d开始升高,7 d到达高峰。bFGF和PDGF-B蛋白表达水平与MVD变化呈正相关(P<0.01)。结论 局灶性脑缺血再灌注损伤可诱导bFGF、PDGF-B和新生血管表达增加,激活内源性脑保护机制。     

一氧化氮供体及前体对大鼠脑缺血再灌注损伤保护机制的探讨

目的 观察介入给药一氧化氮（NO）供体硝酸甘油（Nitroglycerine，NG）及前体L-精氨酸（L-Arginine，ARG）对大鼠脑缺血再灌注后海马区星形胶质细胞表达的胶质纤维酸性蛋白（GFAP）的影响，探讨NG及ARG的脑保护机制。方法 采用大鼠大脑中动脉阻塞（MCAO）法建立局灶性脑缺血模型。将大鼠随机分为假手术组、MCAO组、NG组和ARG组。MCAO组、NG组和ARG组于缺血2 h再灌注同时分别局部介入给予生理盐水、NG和ARG，于再灌注3 h或24 h时，荧光法检测血清NO含量。并在3 h或24 h时处死大鼠，病理分析脑梗死体积以及免疫组织化学法检测海马区GFAP表达情况。结果 缺血再灌注后3 h血清NO升高（P <0.01），治疗组较MCAO组明显（P <0.01），GFAP表达阳性细胞数增加，但治疗组较MCAO组减少（P <0.01），各组大鼠脑组织未出现肉眼可见梗死灶；缺血再灌注后24 h，血清NO治疗组较3 h降低，而MCAO组较3 h升高（P <0.05），GFAP表达阳性细胞数较3 h增加（P <0.01），治疗组较MCAO组减少（P <0.01），TTC染色显示脑梗死体积治疗组较MCAO组减小（P <0.05）。结论 脑缺血再灌注后海马区脑组织GFAP表达增强，通过局部介入给予NG、ARG增加NO合成，抑制GFAP高表达，减小脑梗死体积。提示NG、ARG抗脑缺血性损伤的保护机制可能与抑制星形胶质细胞过度表达有关。     

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。     

神经生长因子对兔局灶性脑缺血再灌注神经功能修复的影响和MR影像学评价

目的:研究神经生长因子(NGF)对兔局灶性脑缺血再灌注损伤神经功能修复的影响,并利用MR成像技术进行评价。方法:采用兔大脑中动脉阻断(MCAO)局灶性脑缺血再灌注模型,分别在缺血2h再灌注损伤后0、1、3和6h应用微量进样器将NGF立体定向导入梗死灶周,于再灌注72h,应用MR影像学、TTC染色和流式细胞术评价兔梗死体积、水肿体积、表观弥散系数(ADC)及神经功能缺损和凋亡状态。结果:缺血再灌注0h、1h、3h和6h灶周给予NGF,梗死体积分别比对照组下降50.1%、48.4%、37.6%和13.7%。同时再灌注3h内应用NGF水肿体积、神经功能缺损评分、灶周凋亡率及caspase-3含量明显下降,梗死中心区及灶周ADC比率(ADCR)升高;再灌注6h后给药,则无明显作用。相关分析显示各组灶周ADCR与灶周凋亡率具有明显负相关。结论:NGF对兔局灶性脑缺血再灌注损伤有保护作用,抑制caspase-3活性的表达和细胞凋亡是其保护机制之一,MR影像学检查可作为定量评价基因疗效的可靠指标。     

The effect of hypothermia on transient middle cerebral artery occlusion in the rat.

We investigated the effect of moderate whole body hypothermia (30 degrees C) on transient middle cerebral artery occlusion (MCAO) in the rat. Male Wistar rats were subjected to 2 h of ischemia by inserting a suture into the lumen of the internal carotid artery and occluding the origin of the MCA. Experimental groups were (a) MCAO induced at 37 degrees C body temperature (n = 15); (b) 30 degrees C body temperature induced prior to ischemia and maintained for 2 h of MCAO and 1 h of reperfusion (n = 12); and (c) MCAO with regional brain and body temperatures measured in normothermic (n = 3) and hypothermic MCAO rats (n = 2). Histopathological evaluation was performed 96 h after reperfusion. All normothermic MCAO animals exhibited ischemic infarct involving the ipsilateral cortex and basal ganglia with infiltration of neutrophils, macrophages, and microvascular proliferation. Hypothermic MCAO animals exhibited minor ischemic damage ranging from selective neuronal injury to small focal areas of infarct with minimal inflammatory response. Our data demonstrate that transient ischemia induced by using the intra-arterial suture method to occlude the MCA results in a reproducible brain lesion and that moderate hypothermia has a profound protective effect on the brain injury after transient MCAO.     

莱菔硫烷对大鼠局灶性脑缺血再灌注损伤的保护作用及机制研究

目的 探讨莱菔硫烷对大鼠局灶性脑缺血再灌注损伤的保护作用及机制.方法 采用线栓法制备大鼠大脑中动脉阻断局灶性脑缺血模型,分别于MCAO后1h腹腔注射莱菔硫烷2.5mg/kg、5mg/kg、10mg/kg.于缺血2h再灌注24h时进行神经行为缺损评分,TTC染色评价脑梗死体积,测定脑组织中超氧化物歧化酶(SOD)活力和丙二醛(MDA)含量.免疫荧光组织化学染色法检测黄核蛋白NQ01和脂质过氧化酶Prx6的表达.结果 莱菔硫烷给药组与对照组相比均能改善大鼠脑缺血再灌注后神经行为缺损评分,减少脑梗死体积.其中5mg/kg组能显著改善大鼠脑缺血再灌注后神经行为缺损评分,减少脑梗死体积,增强SOD活性,降低MDA含量.免疫荧光组织化学染色法提示NQ01和Prx6的表达明显增强.结论 莱菔硫烷对大鼠局灶性脑缺血再灌注损伤有神经保护作用,其机制可能与上调内源性抗氧化蛋白NQ01和Prx6的表达有关.     

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。     

ADAMTS13 gene deletion enhances plasma high-mobility group box1 elevation and neuroinflammation in brain ischemia–reperfusion injury

Highly adhesive glycoprotein von Willebrand factor (VWF) multimer induces platelet aggregation and leukocyte tethering or extravasation on the injured vascular wall, contributing to microvascular plugging and inflammation in brain ischemia–reperfusion. A disintegrin and metalloproteinase with thrombospondin type-1 motifs 13 (ADAMTS13) cleaves the VWF multimer strand and reduces its prothrombotic and proinflammatory functions. Although ADAMTS13 deficiency is known to amplify post-ischemic cerebral hypoperfusion, there is no report available on the effect of ADAMTS13 on inflammation after brain ischemia. We investigated if ADAMTS13 deficiency intensifies the increase of extracellular HMGB1, a hallmark of post-stroke inflammation, and exacerbates brain injury after ischemia–reperfusion. ADAMTS13 gene knockout (KO) and wild-type (WT) mice were subjected to 30-min middle cerebral artery occlusion (MCAO) and 23.5-h reperfusion under continuous monitoring of regional cerebral blood flow (rCBF). The infarct volume, plasma high-mobility group box1 (HMGB1) level, and immunoreactivity of the ischemic cerebral cortical tissue (double immunofluorescent labeling) against HMGB1/NeuN (neuron-specific nuclear protein) or HMGB1/MPO (myeloperoxidase) were estimated 24 h after MCAO. ADAMTS13KO mice had larger brain infarcts compared with WT 24 h after MCAO (p < 0.05). The rCBF during reperfusion decreased more in ADAMTS13KO mice. The plasma HMGB1 increased more in ADAMTS13KO mice than in WT after ischemia–reperfusion (p < 0.05). Brain ischemia induced more prominent activation of inflammatory cells co-expressing HMGB1 and MPO and more marked neuronal death in the cortical ischemic penumbra of ADAMTS13KO mice. ADAMTS13 deficiency may enhance systemic and brain inflammation associated with HMGB1 neurotoxicity, and aggravate brain damage in mice after brief focal ischemia. We hypothesize that ADAMTS13 protects brain from ischemia–reperfusion injury by regulating VWF-dependent inflammation as well as microvascular plugging.     

大鼠大脑中动脉缺血/再灌注模型中Caspase-3的表达

目的 研究Caspase-3在缺血性脑损伤中的作用，进一步探讨缺血性脑血管病的分子机制。方法 用Belayev改良的Longa线栓法制备大鼠局灶性大脑中动脉(MCA)缺血／再灌注模型，TTC染色观察梗死灶的形成，分别用原位杂交及免疫组化技术检测鼠脑中Caspase-3 mRNA与活性蛋白的表达。结果 缺血2小时再灌注24小时，TTC染色见明显的梗死灶形成，正常脑组织、假手术组及MCAO缺血对侧脑中有少量的Caspase-3 mRNA表达，但活性蛋白几无表达；再灌注24小时后，缺血侧脑中Caspase-3 mRNA表达明显增强，蛋白质活化增多，再灌注48小时进一步增加。结论 细胞凋亡机制参与了缺血后迟发性神经元死亡，Caspase-3在其中起重要作用。     

局灶性脑缺血时吡拉西坦对缺血半球能量代谢的影响

目的　观察吡拉西坦对局灶性脑缺血时缺血半球能量代谢的影响。方法　采用动脉腔内插线大鼠局灶性脑缺血模型及测定缺血 2 h/再灌注 2 h后缺血半球内乳酸 ( L A)水平、乳酸脱氢酶 ( L DH)和肌酸激酶 ( CK)活性的变化 ,HE染色结合图像分析测定缺血后 2 4h的梗死体积。结果　吡拉西坦 ( 2 0 0 mg/kg)可明显降低缺血半球内 L A水平 ,增加 L DH的活性 ,对 CK活性无明显影响 ,它可明显降低缺血 2 h/再灌注 2 2 h后的皮质、皮质下结构和半球的梗死体积。结论　吡拉西坦具有明确的抗局灶性脑缺血作用 ,它可明显改善缺血组织的能量代谢状态 ,增加 L DH的活性 ,减轻乳酸堆积及组织酸中毒 ,缩小梗死体积。     

Delayed and multiple hyperbaric oxygen treatments expand therapeutic window in rat focal cerebral ischemic model

Although the brain-protective effect of single, early applications of hyperbaric oxygen (HBO) has been reported in acute ischemic stroke models, few studies have reported the long-term effect—especially after multiple HBO applications. This study employed delayed, multiple HBO treatments and evaluated cerebral infarction and neurological functional recovery for 4 weeks after transient focal ischemia. Adult male Sprague-Dawley rats were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) and were subsequently exposed to HBO (2.5 atmospheres absolute [ATA]) for 2 hours per day. HBO was administered at either 6 or 24 hours after MCAO/R and was repeated daily for 6 days. Rat behavior was scored to evaluate neurological deficits. The brains were removed for histological analysis of the infarct ratio at 1 and 4 weeks. Rats with HBO delayed for 6 or 24 hours following MCAO/R displayed a significant decrease of infarct ratio and amelioration of neurological deficits compared to the untreated group. This study suggests that delayed, but multiple, HBO treatments can improve neurological evaluation and reduce cerebral infarction.     

Neuroprotective effect of hyperbaric oxygen therapy monitored by MR-imaging after embolic stroke in rats

The potential neuroprotective effects of hyperbaric oxygen (HBO) were tested in an embolic model of focal cerebral ischemia with partially spontaneous reperfusion. Rats (n = 10) were subjected to embolic middle cerebral artery occlusion (MCAO) and diffusion weighted MRI (DWI) was performed at baseline, 1, 3, and 6 h after MCAO to determine the ADC viability threshold yielding the lesion volumes that best approximated the 2,3,5-triphenyltetrazolium chloride (TTC) infarct volumes at 24 h (experiment 1). For assessment of neuroprotective effects, rats were treated with 100% oxygen at 2.5 atmospheres absolute (ATA, n = 15) or normobaric room air (n = 15) for 60 min beginning 180 min after MCAO (experiment 2). DWI-, perfusion (PWI)- and T2-weighted MRI (T2WI) started within 0.5 h after MCAO and was continued 5 h, 24 h (PWI and T2WI only), and 168 h (T2WI only). Infarct volume was calculated based on TTC-staining at 24 h (experiment 1) or 168 h (experiment 2) post-MCAO. ADC-lesion evolution was maximal between 3 and 6 h. In experiment 2, the relative regional cerebral blood volume (rCBV) of both groups showed similar incomplete spontaneous reperfusion in the ischemic core. HBO reduced infarct volume to 145.3 +/- 39.6 mm3 vs. 202.5 +/- 58.3 mm3 (control, P = 0.029). As shown by MRI and TTC, HBO treatment demonstrated significant neuroprotection at 5 h after embolic focal cerebral ischemia that lasted for 168 h.     

缺血后处理对局灶脑缺血再灌注大鼠Toll样受体2表达的影响

目的 探讨缺血后处理对大鼠局灶性脑缺血再灌注时Toll样受体2(TLR2)表达的影响。方法 成年雄性SD大鼠90只,分为假手术组、缺血再灌注组、缺血后处理组各30只; 用线栓法建立局灶性大脑中动脉闭塞模型(MCAO),随机分为假手术组(sham)、缺血再灌注组(I/R)、缺血后处理组(IPC),分别于再灌注24、48、72 h后留取大脑皮质组织; 采用Longa的等级评分法进行神经行为学评分,免疫组化和蛋白质印记(Western blot)法检测TLR2蛋白的表达水平,逆转录-聚合酶链反应(RT-PCR)检测TLR2 mRNA表达水平。结果(1)缺血后处理组大鼠神经行为学评分明显改善;(2)缺血再灌注组TLR2蛋白在再灌注24、48、72 h表达水平明显升高(P<0.05); 缺血后处理组TLR2蛋白表达在各时间点均减少(P<0.05); TLR2 mRNA的表达趋势与蛋白表达基本一致。结论 缺血后处理可以降低TLR2表达水平,这可能是其脑保护作用的部分机制之一。     

大鼠局灶性脑缺血损伤后半暗带区锌离子的变化

目的观察大鼠局灶性脑缺血再灌注后半暗带区锌离子的变化,探讨锌离子在脑缺血再灌注损伤中的可能作用。方法将28只SD大鼠随机分为假手术组(n=12)和大脑中动脉梗死(MCAO)组(n=16),以线栓法制作大鼠MCAO模型。分别于再灌注0h、3h、12h和24h时处死大鼠,取脑组织行TTC染色检测梗死体积,并制作脑组织冷冻切片,采用Newport Green(NG)染色法计数半暗带区NG阳性细胞数目并检测其平均荧光强度,分析NG阳性细胞数目与脑梗死体积的相关性。结果 (1)假手术组大鼠脑组织无梗死灶,也未见NG染色阳性细胞。MCAO组大鼠随再灌注时间延长脑梗死体积增大(均P<0.01),脑缺血半暗带区域NG阳性染色细胞数目随再灌注时间延长递增(均P<0.01)。各时间点间NG染色阳性细胞平均荧光强度无统计学差异(P>0.05)。(2)MCAO组大鼠脑切片NG阳性细胞数目与脑梗死体积比率呈正相关(r=0.88,P<0.01)。结论锌离子可能参与了脑缺血再灌注损伤的过程。     

大鼠脑缺血再灌注半暗带β淀粉样前蛋白mRNA表达与梗死体积的关系

为了探讨大鼠局灶性脑缺血再灌注缺血半暗带β淀粉样前蛋白(APP)转录水平与缺血时间及梗死体积的相互关系，用插线法建立大鼠局灶性脑缺血再灌注模型，剥取缺血半暗带皮质组织，采用半定量逆转录－聚合酶链式反应（RT－PCR），测定永久性缺血48h和不同缺血时间再灌注48h后，APPmRNA水平的变化。结果显示，梗死体积随再灌注前缺血时间的延长而增大，皮质半暗带缺血30min再灌注48h APPmRNA表达升高；缺血60min和缺血120min再灌注48h APPmRNA升高明显；缺血180min再灌注48h和永久性缺血48h APPmRNA达到高峰。提示缺血半暗带APPmRNA的表达随再灌注前缺血时间延长而增加并与梗死体积有一定的相关性，早期再灌注可减少其表达。（（GFDA1））。     

Cannabinoid CB(2) receptor activation decreases cerebral infarction in a mouse focal ischemia/reperfusion model.

Cannabinoid CB(2) Receptor (CB(2)) activation has been shown to have immunomodulatory properties without psychotropic effects. The hypothesis of this study is that selective CB(2) agonist treatment can attenuate cerebral ischemia/reperfusion injury. Selective CB(2) agonists (O-3853, O-1966) were administered intravenously 1 h before transient middle cerebral artery occlusion (MCAO) or 10 mins after reperfusion in male mice. Leukocyte/endothelial interactions were evaluated before MCAO, 1 h after MCAO, and 24 h after MCAO via a closed cranial window. Cerebral infarct volume and motor function were determined 24 h after MCAO. Administration of the selective CB(2) agonists significantly decreased cerebral infarction (30%) and improved motor function (P<0.05) after 1 h MCAO followed by 23 h reperfusion in mice. Transient ischemia in untreated animals was associated with a significant increase in leukocyte rolling and adhesion on both venules and arterioles (P<0.05), whereas the enhanced rolling and adhesion were attenuated by both selective CB(2) agonists administered either at 1 h before or after MCAO (P<0.05). CB(2) activation is associated with a reduction in white blood cell rolling and adhesion along cerebral vascular endothelial cells, a reduction in infarct size, and improved motor function after transient focal ischemia.     

Protection against ischemic brain damage in rats by immunophilin ligand GPI-1046

To determine the effect of immunophilin ligand GPI-1046 on ischemic brain injury, 90 min of transient middle cerebral artery occlusion (MCAO) was carried out in rat brains. In contrast to cases treated with vehicle, the infarct volume was reduced greatly and rotamase activity was inhibited significantly at 24 hr of reperfusion by treatment with GPI-1046. Immunoreactivity and the number of cells stained positively for FKBP12, FKBP52, caspase-8, cytochrome c, and caspase-3 were also reduced markedly in the brain after GPI-1046 treatment. The present results suggest that GPI-1046 significantly decreased infarct volume and provided neuroprotective effect on rats after transient focal cerebral ischemia by inhibiting the increase of rotamase activity and of the number of FKBP12-, FKBP52-, caspase-8-, cytochrome c-, and caspase-3-positive cells in the ischemic area.     

Tumour expresion of tissue factor and tissue factor pathway inhibitor in ovarian cancer- relationship with venous thrombosis risk

Introduction

Ovarian cancer is known to display a particular association with venous thromboembolism (VTE) with reports up to 42% of patients developing thromboembolic complications. Tissue Factor (TF) and its inhibitor Tissue Factor Pathway Inhibitor (TFPI) have been implicated in VTE risk in cancer. The aim of this study was to measure tumour derived TF and TFPI and to investigate their potential role in VTE in ovarian cancer patients.

Methods

TF and TFPI mRNA expression was measured using TaqMan real time PCR in 99 ovarian tumour samples. Nineteen cases complicated by VTE were matched to 19 cases without VTE. TF and TFPI protein levels were measured using ELISA and immunohistochemistry was used to localize TF expression. The role of TF expression on overall survival was also determined.

Results

TF mRNA and protein expression was increased in tumours from patients with clear cell carcinoma (p < 0.001). TF protein expression was also increased in endometroid carcinoma (P < 0.01) compared with benign tumours. TFPI mRNA expression was increased in clear cell carcinoma (P < 0.01). TF mRNA and antigen level was increased in malignant tumours of patients who developed VTE compared with matched malignant õtumours of patients who remained thrombosis free (P < 0.01). There was no difference in TFPI expression between the two groups.

Conclusion

TF expression in ovarian cancer is significantly higher in patients who develop VTE. TF expression was increased in clear cell ovarian cancer and endometroid cancer and this may explain the higher risk of VTE in these subgroups. TF derived from these tumours may be the trigger for VTE in ovarian cancer.