抗白细胞药物在保护大鼠局灶性脑缺血中的时间窗研究

目的 研究抗白细胞药物，环磷酰胺，氯化奎宁，秋水仙碱在保护大鼠局灶性脑缺血中的时间窗问题。方法 用线栓法建立大鼠大脑中动脉区缺血再灌注模型，检测于栓塞后１ｈ，６ｈ，１２ｈ，２４ｈ四个不同的时间窗应用抗白细胞药物对大鼠局灶性脑缺血后梗塞体积及梗塞区小胶质细胞数的影响。结果 抗白细胞药物于１ｈ６ｈ，１２ｈ三个时间窗给药，其疗效相似（Ｐ〉０．０５），显著优于２４ｈ给药组和对照组（Ｐ〈０．０１），而２４ｈ     

头孢曲松钠对大鼠脑缺血损伤的保护作用及其机制

目的 探讨在大鼠局灶性脑缺血模型中应用头孢曲松钠对脑缺血损伤的保护作用及其相关机制.方法 制备Wistar大鼠局灶性脑缺血模型,并按随机数字表法分为单纯缺血组(MCAO组)、头孢曲松钠治疗组(MCAO+CTX组)和盐水对照组,其中MCAO+CTX组为缺血90min时给予头孢曲松钠200 mg/kg.缺血后24 h、48 h、7 d时对各组大鼠进行神经行为学评分和脑水肿程度测定,同时比较各组大鼠皮层和海马谷氨酸转运体功能的差异.结果 随着缺血时间延长,各组大鼠神经行为学评分逐渐提高;脑水肿在缺血后24 h、48 h时逐渐加重,至7 d时已逐渐消退.与MCAO组比较,各时间点MCAO+CTX组大鼠神经行为学评分明显提高,脑水肿程度明显减轻,伤侧皮层及海马谷氨酸转运体功能明显增强,差异均有统计学意义(P＜0.05).结论 头孢曲松钠对大鼠局灶性脑缺血损伤具有保护作用,其机制可能与增强谷氨酸转运体功能从而减轻谷氨酸神经毒性作用有关.

Abstract：

Objective To explore the neuroprotective effect of ceftriaxone on cerebral ischemia injury in rats with focal cerebral ischemia and its possible mechanism. Methods Focal cerebral ischemic models were established in Wistar rats and randomly divided into ischemic group (performed middle cerebral artery occlusion [MCAO]), ceftriaxone (CTX) therapy group (given CTX at a dosage of 200 mg/kg 90 min after MCAO) and control group (given physiological saline only). Twenty-four and 48 h, and 7 d after MCAO, neurological behaviors and cerebral edema level were evaluated in these 3 groups;glutamate transporter function in the cortex and hippocampus of rats was compared between each 2 groups. Results With time extended, neurological behaviors scores were obviously elevated in every group;and cerebral edema became worse at 24 and 48 h and decreased 7 d after MCAO. As compared with that in the ischemic group, glutamate transporter function, level of edema and neurological behaviors scores in cortex and hippocampus of rats in the CTX therapy group were statistically increased at different ischemic time points (P<0.05). Conclusion Ceftriaxone has a neuroprotective effect against focal cerebral ischemia in rats, which may relate to increased glutamate transporter function and reduced glutamate neurotoxicity.     

Neurovascular and neuronal protection by E64d after focal cerebral ischemia in rats

Calpains and cathepsins are two families of proteases that play an important role in ischemic cell death. In this study, we investigated the effect of E64d, a mu-calpain and cathepsin B inhibitor, in the prevention of neuronal and endothelial apoptotic cell death after focal cerebral ischemia in rats. Rats underwent 2 hr of transient focal ischemia from middle cerebral artery occlusion (MCAO) and were sacrificed 24 hr later. E64d (5 mg/ kg intraperitoneally) was administered 30 min before MCAO. Assessment included neurological function, infarction volume, brain water content, blood-brain barrier permeability, histology, and immunohistochemistry. The E64d-treated rats had significant brain protection against ischemic damage. We observed a reduction of infarction volume, brain edema, and improved neurological scores in E64d-treated rats compared with the nontreated control. Furthermore, there was a remarkable reduction in both proteases and caspase-3 activation and apoptotic changes in both neurons and endothelial cells in E64d-treated rats. These results suggest that E64d protects the brain against ischemic/reperfusion injury by attenuating neuronal and endothelial apoptosis.     

抗白细胞药物在大鼠局灶性脑缺血中的保护作用

目的研究抗白细胞药物：环磷酰胺、氯化奎宁、秋水仙碱在大鼠局灶性脑缺血中的作用。方法用线栓法建立大鼠大脑中动脉区缺血再灌注模型，检测抗白细胞药物对大鼠局灶性脑缺血后外周血白细胞总数，梗塞区小胶质细胞数及对梗塞体积的影响。结果三种抗白细胞药物均能抑制外周血白细胞活化和梗塞区小胶质细胞的激活，缩小梗塞体积。结论抗白细胞药物对缺血性脑梗塞有保护作用，且联合用药优于单一用药     

高压氧预处理可对大鼠局灶性脑缺血再灌注损伤的保护作用

目的研究短期高压氧预处理后是否可减轻大鼠局灶性脑缺血再灌注损伤。方法选取成年雄性Wister大鼠,采用连续5d,每天1次,3.0ATA,100%O2高压氧(HBO)预处理,每次60min,末次预处理后24h,运用改良的经典线栓法制作MCAO模型,再灌注2h。将实验大鼠分为假手术组、MCAO组、HBO+MCAO组(n=5)。造模后24h观察各组动物的一般精神状态及体重变化情况、用Rogers DC and Hunter AJ描述的神经功能7分评分法对神经功能损伤进行评估,TTC染色测梗死面积,并对脑组织进行石蜡切片,行Nissl、TUNEL染色,利用显微镜对神经细胞进行计数。结果假手术组无神经功能缺失,TTC染色未见梗死灶,镜下观察未见坏死细胞。MCAO组和HBO+MCAO组均有不同程度的神经功能缺损,且HBO+MCAO组神经功能缺失较MCAO组轻;TTC染色MCAO组的梗死面积明显大于HBO+MCAO组;镜下观察,MCAO组梗死区内尼氏小体明显少于HBO+MCAO组。结论短期高压氧预处理后可减轻大鼠局灶性脑缺血再灌注损伤。     

N-甲基-D-天冬氨酸受体1反义抑制剂治疗局灶性脑缺血作用机制的实验研究

目的 探讨N-甲基-D-天冬氨酸受体1（NMDAR1）反义抑制剂（ASODN）治疗急性脑梗死的作用机制。方法 采用Koizumi‘s线栓法制作可复流MCAO大鼠模型。40只8-12月龄健康，雄性Wistar大鼠随机分为4组；（1）MCAO组；（2）MCAO PBS组；（3）MCAO MSODN组；（4）MCAO ASODN组。实验各组分别于MCAO后2h,24h经侧脑室注射给药，MCAO后第5天处死动物。取脑组织进行NMDAR1免疫组织化学及NMDAR1mRNA原位杂交检测。检测结果经CMIAS图像分析系统进行定量分析。结果 （1）ASODN治疗组NMDAR1阳性细胞在海马CA1区，CA3区及DG区的数密度，光密度显著低于MCAO组；（2）ASODN治疗组NMDAR1mRNA阳性细胞在海马各区及大脑皮层的数密度，光密度与MCAO组无显著差异。结论 NMDAR1反义抑制剂可能主要作用于NMDAR1基因的蛋白翻译过程（胞浆机制），抑制缺血后神经元NMDAR1的产生。下调NMDAR1的数量及功能，减轻缺血后迟发性神经元坏死。达到脑保护作用。     

硫酸镁在大鼠局灶脑缺血中的保护作用

目的 研究非竞争性谷氨酸受体拮抗剂－－硫酸镁在大鼠局灶脑缺血中的作用。方法 采用线栓法建立大鼠右侧大脑中动脉区永久脑缺血模型,分别于缺血前半小时,、缺血后第１、３、６、１２小时静滴１０％硫酸镁溶液,滴速１．５ｍｌ／ｈ,通过神经功能评分、梗死体积及含水量的改变、病理学检查,探讨硫酸镁对脑缺血的保护作用及治疗时间窗。结果 缺血６小时内应用硫酸镁能改善运动功能,减轻脑水肿,缩经体积。结论 硫酸镁具有明显     

Progesterone administration during reperfusion, but not preischemia alone, reduces injury in ovariectomized rats.

Although progesterone is neuroprotective in traumatic brain injury, its efficacy in stroke is unclear. The authors determined whether there are infarction differences after middle cerebral artery occlusion (MCAO) in ovariectomized rats treated acutely with progesterone before MCAO or both pre- and postischemia. Rats received vehicle, 5 (P5), 10 (P10), or 20 (P20) mg/kg progesterone intraperitoneally 30 minutes before MCAO. In another cohort, animals received vehicle or 5 (P5R) mg/kg progesterone intraperitoneally 30 minutes before MCAO, at reperfusion initiation, and at 6-hour reperfusion. Animals underwent 2-hour MCAO by the intraluminal filament technique, followed by 22-hour reperfusion. Cortical (CTX) and caudate-putamen (CP) infarctions were determined by 2,3,5-triphenyltetrazolium chloride staining and digital image analysis. End-ischemic and early reperfusion regional cerebral blood flow (CBF) was measured by [ C]-iodoantipyrine quantitative autoradiography in vehicle- or progesterone (5 mg/kg)-treated rats. Cortical infarction (% contralateral CTX) was 31 +/- 30% (vehicle), 39 +/- 23% (P5), 41 +/- 14% (P10), and 28 +/- 20% (P20). Caudate-putamen infarction (% contralateral CP) was 45 +/- 37% (vehicle), 62 +/- 34% (P5), 75 +/- 17% (P10), and 52 +/- 30% (P20). In vehicle and P5R groups, CTX infarction was 37 +/- 20% and *20 +/- 17%, respectively (* < 0.05 from vehicle). In vehicle and P5R groups, CP infarction was 63 +/- 26% and 43 +/- 29%, respectively. End-ischemic regional CBF and CBF recovery during initial reperfusion was unaffected by progesterone treatment. These data suggest that progesterone administration both before MCAO and during reperfusion decreases ischemic brain injury.     

人参皂甙单体Rb1对大鼠脑缺血损伤保护作用的实验研究

目的 观察人参皂甙单体Rb1 对大鼠实验性脑缺血的保护作用。方法 健康成年雄性清洁级SD大鼠随机分为假手术组(Sham)、缺血对照组(Con)、干预组(Tre),干预组又分为Rb130 mg/kg、60 mg/kg及90 mg/kg三个不同剂量组。缺血对照组采用线栓法建立大脑中动脉闭塞模型,缺血2 h后拨出线栓再灌注; 各干预组用相应剂量Rb1 ip qd×7 d,末次给药后3 h内用同样方法建立大脑中动脉闭塞模型及再灌注; 假手术组不插入线栓,余操作相同。术后48 h取标本TTC染色测梗死体积、干湿重法脑组织含水量测定、Tunnel法测定调亡细胞数及NgR表达的免疫组化测定。结果 各干预组的梗死体积分别为30 mg/kg组(27.629±1.401)%,60 mg/kg组(24.164±1.710)%,90mg/kg组(21.955±2.556)%,缺血对照组为(29.846±1.153)%; 脑组织含水量为30 mg/kg组(80.079±0.726)%,60 mg/kg组(78.984±0.902)%,90 mg/kg组(77.855±0.258)%,假手术组与缺血对照组分别为(76.517±0.37)%、(81.799±1.065)%; 调亡细胞数分别为30 mg/kg组(89.000±10.296),60 mg/kg组(59.000±12.522),90 mg/kg组(36.667±19.054),假手术组与缺血对照组分别为(1.600±1.517)、(132.667±22.223); NgR表达阳性面积分别为30 mg/kg组(84.827±3.870),90 mg/kg组(66.040±5.541),60 mg/kg组(75.577±7.150),假手术组与缺血对照组分别为(48.355±9.720)、(91.485±5.822)。结论 人参皂甙单体Rb1对大鼠实验性脑缺血有保护作用,能减轻缺血再灌注损伤所致的脑水肿及梗塞面积,减少细胞调亡,并且该保护作用呈剂量依赖性。对NgR表达的干预提示Rb1可能在脑缺血死后神经可塑性中起促进作用。     

Dynorphin A (1–8) inhibits oxidative stress and apoptosis in MCAO rats,affording neuroprotection through NMDA receptor and κ-opioid receptor channels

The contents of Dynorphin A(1–8) decreased gradually in ischemic cortices in rats and an intracerebroventricular administration of synthetic Dynorphin A(1–8) reduced the volume of cerebral infarction in our previous research. However, the specific protective mechanism is unclear and Dynorphin A(1–8) is unlikely to cross the blood-brain barrier (BBB) by noninvasive oral or intravenous administration as a macromolecule neuropeptide. In this study, intranasal administration was used to middle cerebral artery occlusion(MCAO) rats to assessed the therapeutic effects of Dynorphin A(1–8) by evaluating behavior, volume of cerebral infarct, cerebral edema ratio, histological observation. Then apoptosis neuron rate was detected by TUNEL staining. Immunohistochemical staining was carried out to explore the alteration of Bcl-2, Bax and Caspase-3. Finally, κ-opioid receptor antagonist and N-methyl-d-aspartate(NMDA) receptor antagonist were used to explore its possible mechanism. We found that MCAO rats under intranasal administration of Dynorphin A(1–8) showed better behavioral improvement, higher extent of Bcl-2, activity of SOD along with much lower level of infarction volume, brain water content, number of cell apoptosis, extent of Bax and Caspase-3, and concentration of MDA compared with those in MCAO model group and intravenous Dynorphin A(1–8) group. Administration of nor-BNI or MK-801 reversed these neuroprotective effects of intranasal Dynorphin A(1–8). In summary, Dynorphin A(1–8), with advantages of intranasal administration, could be effectively delivered to central nervous system(CNS). Dynorphin A(1–8) inhibited oxidative stress and apoptosis against cerebral ischemia/reperfusion injury, affording neuroprotection through NMDA receptor and κ-opioid receptor channels.     

NR_1反义抑制剂对局灶性脑缺血作用的初步研究

目的　探讨NR1反义寡核苷酸对局灶性脑缺血的治疗作用。方法　于大鼠大脑中动脉闭塞后2小时、2 4小时分别经侧脑室注射磷酸缓冲液 (PBS)、错义寡核苷酸 (MSODN)及反义寡核苷酸 (ASODN) ,然后在不同时间点进行神经功能缺损评分 ,术后第 5天进行Nissl染色、TTC染色及梗死体积比测定。结果　各组局灶性脑缺血的神经功能缺损评分无显著性差异 ;反义寡核苷酸治疗组的梗死体积比显著低于单纯缺血组 ;反义寡核苷酸治疗组海马各区神经元损伤轻 ,神经元丢失相对较少。结论　局灶性脑缺血后侧脑室注入NR1反义寡核苷酸 ,可以减轻缺血脑组织病理学损害 ,具有脑保护作用。     

Deficiency of PAR4 attenuates cerebral ischemia/reperfusion injury in mice

Stroke is the third leading cause of death in the USA. Antithrombotic therapy targeting platelet activation is one of the treatments for ischemic stroke. Here we investigate the role of one of the thrombin receptors, protease-activated receptor 4 (PAR4), in a mouse transient middle cerebral artery occlusion (MCAO) model. After a 60 min MCAO and 23 h reperfusion, leukocyte and platelet rolling and adhesion on cerebral venules, blood–brain barrier (BBB) permeability, and cerebral edema were compared in PAR4-deficient mice and wild-type mice. Cerebral infarction volume and neuronal death were also measured. PAR4−/− mice had more than an 80% reduction of infarct volume and significantly improved neurologic and motor function compared with wild-type mice after MCAO. Furthermore, deficiency of PAR4 significantly inhibits the rolling and adhesion of both platelets and leukocytes after MCAO. BBB disruption and cerebral edema were also attenuated in PAR4−/− mice compared with wild-type animals. The results of this investigation indicate that deficiency of PAR4 protects mice from cerebral ischemia/reperfusion (I/R) injury, partially through inhibition of platelet activation and attenuation of microvascular inflammation.     

NO对兔局灶脑缺血脑水肿和脑梗塞灶的影响

本文在兔MCAO局灶脑缺血模型基础上,利用外源性一氧化氮(NO)前体物质L-精氨酸和NOS抑制剂L-NNA研究NO对脑缺血后脑水肿和脑梗塞的影响。NZW兔42只随机分为7组,每组6只,计对照组、假手术组、MCAO组、MCAO+NS组、MCAO+L-Arg组、MCAO+D-Arg组及MCAO+L-Arg+L-NNA组。结果提示:与单纯MCAO组比较,L-Arg组的脑组织H_2O、Na~+、Ca~(2+)含量明显下降(P<0.01),脑梗塞灶亦明显缩小(P<0.01)。从而显示L-Arg系通过产生NO促使血管舒张而减轻脑水肿和缩小梗塞灶,为临床改进脑缺血的治疗提供依据。     

胰岛素治疗肾血管性高血压大鼠脑梗死的实验研究

目的 研究葡萄糖、胰岛素对肾血管高血压大鼠脑梗死的不同影响。方法 采用易卒中型肾血管性高血压大鼠,在单侧大脑中动脉闭塞术后３０分钟,分别腹腔注射葡萄糖、胰岛素和生理盐水,２４小时内记录动物神经症状并处死,光镜、电镜观察脑组织病理及超微结构的改变。结果 葡萄糖组脑缺血和脑水肿较对照组严重;神经元线粒体高度肿胀,数目减少;血脑屏障内皮细胞较对照组明显肿胀、变形,线粒体肿胀。胰岛素组上述组织病理及超微结     

Cerebroprotective action of a Na+/Ca2+ channel blocker NS-7. I. Effect on the cerebral infarction and edema at the acute stage of permanent middle cerebral artery occlusion in rats

The effect of a novel Na+/Ca2+ channel blocker NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy)pyrimidine hydrochloride] on the cerebral infarction, edema and brain energy metabolism was investigated in rats after permanent middle cerebral artery occlusion (MCAO). The infarction and brain water content were evaluated at 48 h and 24 h after MCAO, respectively. A single bolus injection of NS-7 (0.03125-0.25 mg/kg) immediately after MCAO produced a dose-dependent reduction in the infarct volume as well as edema both in the cerebral cortex and striatum. Glycerol (4 g/kg) also decreased water content both in the occluded and non-occluded brain, but it did not reduce the size of cerebral infarction. Unlike glycerol, NS-7 did not change the water content in non-occluded brain. Moreover, a significant protective action was still observed even when NS-7 was injected once at 12 h after occlusion. In addition, NS-7 significantly reversed the decrease in tissue ATP content observed at 3 h but not at 0.5 h after MCAO. These findings suggest that a Na+/Ca2+ channel blocker NS-7 protects cerebral tissues against ischemic insults by improving the disturbance of cerebral energy metabolism and suppressing the cerebral edema.     

Collateral augmentation treatment with a combination of acetazolamide and head-down tilt in a rat ischemic stroke model

Cerebral collaterals is crucially important in the pathophysiology of acute ischemic stroke and associated with outcome after reperfusion therapy. We explored the effectiveness of collateral augmentation treatment with a combination of acetazolamide (ACZ) and head-down tilt (HDT) in the transient middle cerebral artery occlusion (MCAO) rat model. Transient MCAO was induced in all animals for 1.5 h, followed by reperfusion for 22.5 h. Seventy-two male Wistar rats were divided into four treatment groups: control, ACZ, HDT, and combination. Twenty sham rats, which underwent surgery, were randomly allocated to these groups. Twenty-four hours after MCAO or sham surgery, we measured the infarction volume, brain edema (aquaporin-4 [AQP4], and brain water content), and neurological deficits (Garcia and Longa tests). Collateral augmentation treatments were associated with reduced infarction volume, less brain edema, and better neurological outcomes compared with untreated animals. More specifically, ACZ and HDT treatments resulted in small infarction volumes, and HDT was associated with a low AQP4 expression and improved neurological score, while the combination of ACZ and HDT improved neurological scores and reduced brain water content. This study shows that collateral augmentation treatments are associated with a better stroke prognosis compared with untreated animals after transient MCAO. The combination of ACZ and HDT seems to have some synergistic effect, but was not proven to be superior to HDT treatment alone.     

Atorvastatin Pretreatment Attenuates Ischemic Brain Edema by Suppressing Aquaporin 4

Background: Cerebral edema, a serious complication of acute cerebral infarction, has a crucial impact on morbidity and mortality in the early stage of cerebral infarction. And aquaporin 4 (AQP4), a bidirectional water transporting protein, plays a pivotal role in edema formation. At experimental model, it has proven that atorvastatin could exert pleiotropic neuroprotection on acute cerebral infarction independent of its cholesterol-lowering action. It was a common protective manifestation that atorvastatin can reduce the infarct volume and cerebral edema. However, little is known about atorvastatin improving ischemic brain edema by regulating AQP4 expression. This study intended to investigate the neuroprotection effects of atorvastatin pretreatment in rats with cerebral ischemia and further explore the potential relationship between atorvastatin and AQP4 expression. Methods: Fifty-one adult male Sprague Dawley rats were randomly divided into 3 groups: sham, middle cerebral artery occlusion (MCAO), and atorvastatin pretreatment (Ator) group. For Ator group, 20 mg/kg of atorvastatin injectable suspension was administered once for 7days by gavage before operation, whereas the others were administered the same volume of saline matching. Except for sham group, MCAO and Ator groups were subjected to permanent MCAO by modified intraluminal suture method. Infarct volume, neurological deficit, brain water content (BWC), immunohistochemistry, western blot, and polymerase chain reaction (PCR) were measured at 24 hours after MCAO. Results: Compared with sham group, the mNSS, infarct volume, and BWC of ischemic hemisphere were significantly increased (P < 0.001) in MCAO group. Positive cells and protein levels of p-p38MAPK and AQP4 in peri-infarction were significantly increased (P < 0.01). The mRNA levels of p38MAPK and AQP4 were also prominently upregulated (P < 0.01). Interestingly, preadministration of atorvastatin dramatically decreased infarct volume and the BWC of ischemic hemisphere compared with MCAO group (P < 0.05). The overexpressions of p-p38MAPK and AQP4 in peri-infarction were significantly decreased (P < 0.05) and their mRNA levels were downregulated by atorvastatin pretreatment (P < 0.05). Neurological deficits were also dramatically improved (P < 0.001). Conclusion: To the best of our knowledge, this is the first study that demonstrates an effect of atorvastatin on expression of AQP4, and we propose that decreased AQP4 expression through a p38MAPK-suppression pathway may be the mechanism of atorvastatin alleviating ischemic cerebral edema.     

大鼠局灶性脑缺血后神经营养因子表达的自然变化

目的 观察大鼠局灶性脑缺血后大鼠神经行为、梗死体积、组织形态及缺血半暗带神经生长因子(NGF)、脑源性神经营养因子(BDNF)的表达水平变化。方法 将健康雄性SD大鼠24只随机分为2组,Ⅰ组(假手术组); Ⅱ组(脑缺血组)。用线栓法建立动物模型,不给予再灌注,各组在术后48h断头取脑,处死前行神经功能评分,用氯化三苯基四氮唑(TTC)染色计算脑梗死体积,用HE染色观察组织学形态,用免疫组织化学染色观察NGF、BDNF的表达水平。结果 Ⅰ组在神经功能评分、梗死体积、组织形态及大脑皮层相应部位NGF、BDNF阳性细胞数均正常。与Ⅰ组比较,Ⅱ组的神经功能评分和梗死体积均严重受损; 光镜下Ⅱ组缺血性病理改变较重,与Ⅰ组比较,Ⅱ组缺血半暗带NGF、BDNF阳性神经元数增加(P<0.05)。结论 脑缺血本身可上调缺血半暗带NGF、BDNF的表达水平。     

灵芝甾醇对大鼠局灶性脑缺血再灌注损伤的保护作用

目的观察灵芝甾醇(GS)对局灶性脑缺血再灌注损伤(I／R)大鼠的保护作用，并对其作用原理进行初步探讨。方法复制大鼠大脑中动脉阻断再灌注模型(MCAO／R)，分别采用TTC染色法、神经功能评分法观察GS对大鼠大脑梗死体积和行为学评分的影响，同时观察GS对脑组织形态学和MDA水平、SOD活性的影响。结果GS能够降低I／R大鼠脑梗死体积和行为学评分。减轻受损大鼠皮层脑组织的病理改变，抑制脑组织中MDA的生成，提高Mn-SOD的活性。结论GS对大鼠缺血再灌注损伤有一定保护作用，其作用机制与增强Mn-SOD活性，减轻I／R中氧化损伤有关。