自由基清除剂预处理对大鼠脑缺血再灌注损伤后脑组织细胞凋亡及其相关蛋白表达的影响

目的探讨自由基清除剂依达拉奉预处理对大鼠脑缺血再灌注损伤后神经细胞凋亡及其相关蛋白Bcl-2、Bax、热休克蛋白70(HSP70)表达的影响。方法将45只雄性SD大鼠随机分为假手术组、对照组、依达拉奉预处理组,每组15只。采用线栓法制作大鼠缺血2h再灌注24h模型。预处理组大鼠建模前12h腹腔注射依达拉奉(3mg/kg),对照组给予等容量生理盐水。再灌注24h后断头取脑,应用免疫组织化学法检测Bcl-2、Bax、HSP70蛋白表达,末端脱氧核糖核酸转移酶介导的原位缺口末端标记法检测凋亡细胞。结果依达拉奉预处理组和对照组大鼠缺血周围脑组织中凋亡细胞和Bcl-2、Bax及HSP70阳性细胞数比假手术组均明显增加(P<0.01);与对照组比较,其凋亡细胞和Bax阳性细胞数均明显减少(P<0.01),而Bcl-2和HSP70阳性细胞数明显增加(P<0.01)。结论细胞凋亡在缺血再灌注损伤中起着重要作用;依达拉奉可能通过上调Bcl-2、HSP70蛋白表达、下调Bax蛋白表达减轻大鼠脑缺血再灌注后的细胞凋亡,增加脑缺血再灌注损伤耐受性,从而起到神经保护作用。     

依达拉奉预处理对大鼠脑缺血-再灌注损伤的保护机制

目的探讨依达拉奉预处理对大鼠局灶性脑缺血-再灌注损伤的保护机制。方法将36只SD大鼠随机分为假手术组、缺血-再灌注组和依达拉奉组。依达拉奉组术前给予60 mg/(kg.d)的依达拉奉灌胃,共3 d。采用大脑中动脉线栓法制备大鼠缺血-再灌注损伤模型。比较各组神经功能缺损评分、脑梗死体积、血清神经元特异性烯醇化酶(NSE)含量及脑组织白介素-1β(IL-1β)和肿瘤坏死因子(TNF-α)含量。结果与缺血-再灌注组比较,依达拉奉组的神经功能缺损评分显著下降,脑梗死灶体积显著缩小,血清NSE含量明显降低(均P<0.01);脑组织IL-1β和TNF-α含量显著降低(P<0.05～0.01)。结论依达拉奉预处理可减少大鼠脑缺血-再灌注损伤后脑组织IL-1β和TNF-α表达,保护脑组织。     

依达拉奉对局灶性脑缺血再灌注损伤大鼠caspase-3、Bcl-2表达的影响

目的 对局灶性脑缺血再灌注损伤大鼠,给予自由基清除剂依达拉奉后,观察再灌注不同时间点脑组织caspase-3和Bcl-2蛋白表达情况,探讨依达拉奉对脑缺血再灌注损伤的保护作用.方法 制作局灶性脑缺血再灌注模型.随机分为正常组、假手术组、脑缺血组、依达拉奉组.假手术组于术后,脑缺血组和依达拉奉组于缺血1h后再灌注2h、6h、12h、24h、48h不同时间点,依达拉奉组于再灌注后30min腹腔内及皮下各注射依达拉奉1 次(3mg/kg.wt),30min 后重复1次.按时间点处死大鼠,灌注固定、取脑,行免疫组化染色,观察和计数不同脑区的caspase-3和Bcl-2蛋白表达阳性细胞数.结果 脑缺血组再灌注后2h在大脑额、顶叶皮质和海马均可见到caspase-3和Bcl-2阳性细胞,caspase-3表达高峰在12h;Bcl-2表达高峰在6h.依达拉奉组各时间点caspase-3阳性细胞较脑缺血组明显减少,而Bcl-2阳性细胞数明显增加 (均为P＜0.05).结论 依达拉奉可抑制caspase-3,提高Bcl-2的蛋白表达,对脑缺血再灌注损害有明显的保护作用.     

丁苯酞联合依达拉奉治疗急性缺血性脑卒中对细胞凋亡的影响

目的探讨丁苯酞注射液联合依达拉奉对急性缺血性脑卒中的临床疗效及对细胞凋亡的影响。方法选择急性脑梗死患者136例,随机分为联合用药组和依达拉奉组,每组68例。依达拉奉组给予依达拉奉注射液治疗,联合用药组在依达拉奉组治疗的基础上加用丁苯酞,共治疗14 d。比较治疗前后NIHSS、ADL评分,测定细胞脂质过氧化水平及抗氧化酶活性及血清Bax、Bcl-2含量。结果联合用药组治疗后NIHSS评分低于依达拉奉组,ADL评分高于依达拉奉组,丙二醛(MDA)、超氧化物歧化酶(SOD)水平低于依达拉奉组(P0.05);联合用药组治疗后血清Bax含量低于依达拉奉组,血清Bcl-2含量高于依达拉奉组(P0.05)。结论丁苯酞注射液联合依达拉奉治疗急性缺血性脑卒中,可减轻缺血再灌注过程中氧化应激反应及细胞凋亡,从而减轻脑缺血再灌注(I/R)所引起的脑损伤。     

升压联合亚低温治疗对局灶性脑缺血再灌注的脑保护作用

目的　观察升压联合亚低温治疗对大鼠局灶性脑缺血再灌注的脑保护作用。方法　 32只大鼠随机分为对照组、升压组、亚低温组、升压 亚低温组 ,采用大鼠局灶性脑缺血再灌注模型 ,观察各组神经功能缺损评分和脑梗死体积。结果　升压组、亚低温组及升压 亚低温组神经功能缺损评分 (P <0 .0 5 )、脑梗死体积 (P <0 .0 1)均明显低于对照组 ;升压 亚低温组脑梗死体积明显低于升压组和亚低温组 (P <0 .0 5 )。结论　升压、亚低温对局灶性脑缺血再灌注损伤有明显脑保护作用 ,升压联合亚低温应用效果更佳     

亚低温联合黄芪对大鼠局灶脑缺血再灌注损伤的神经保护作用

目的探讨亚低温(32±1℃)联合黄芪对大鼠局灶脑缺血再灌注损伤的协同保护作用。方法采用改良线栓法制备大鼠大脑中动脉闭塞再灌注模型,将24只SD大鼠随机分为四组(每组6只)常温组;亚低温组,再灌注后立即诱导亚低温并持续12小时;黄芪组,脑缺血后立即按1g/kg剂量腹腔注射黄芪;黄芪加亚低温组,脑缺血后立即按1g/kg剂量腹腔注射黄芪,再灌注后立即诱导亚低温并持续12小时。每组均脑缺血6小时后再灌注。观察脑梗死体积、神经功能缺损评分和病理改变。结果黄芪加亚低温组脑梗死体积为112.41±1664mm3,亚低温组为14668±14.88mm3,经两因素方差分析有显著性意义(P＜0.05)。结论亚低温联合黄芪对脑缺血再灌注损伤具有协同保护作用。     

亚低温对大鼠全脑缺血-再灌注损伤后神经细胞凋亡的治疗时间窗研究

目的本研究通过观察大鼠全脑缺血-再灌注损伤后不同时间段亚低温对Bcl-2、Bax表达及二者比值的影响,探讨亚低温的保护作用及最佳治疗时间窗。方法 Wistar雄性大鼠随机分为对照组及亚低温组。亚低温组根据亚低温时点不同分为缺血后、再灌注0、6、12、24h 5个亚组。采用四条血管阻断方法(Puisinelli-4VO法)制备大鼠全脑缺血-再灌注模型,流式细胞仪对海马组织的Bcl-2、Bax蛋白的表达进行测定。结果亚低温可使大鼠海马细胞的Bcl-2表达增高、Bax表达降低,以再灌注后0 h亚低温效果最佳,随再灌注时间延长,效果下降。结论亚低温可有效干预大鼠全脑缺血-再灌注损伤后神经细胞的凋亡,且最佳治疗时间窗为再灌注后0h,但再灌注24h也有一定疗效。     

依达拉奉对大鼠脑梗死溶栓治疗后MMP-9表达的影响

目的研究依达拉奉对大鼠脑梗死溶栓治疗后脑组织中MMP-9表达及血脑屏障的影响,探讨依达拉奉对脑梗死溶栓治疗后再灌注损伤的保护机制。方法采用SD大鼠自体血栓栓塞法制备大脑中动脉闭塞模型,并将SD大鼠随机分为假手术组、尿激酶溶栓治疗组(UK)、尿激酶+依达拉奉治疗组(UK+ED),12h后分别以免疫组织化学法和比色法对SD大鼠脑组织中MMP-9表达水平和伊文思蓝含量进行测定。结果与尿激酶溶栓治疗组相比,尿激酶+依达拉奉组SD大鼠缺血侧脑组织MMP-9表达水平和EB含量均显著降低,差异具有统计学意义(P值均<0.01)。结论依达拉奉可能通过下调MMP-9表达,减轻血脑屏障的破坏,减轻溶栓后脑缺血再灌注损伤。     

依达拉奉对大鼠局灶性脑缺血再灌注损伤后细胞凋亡及caspase-3蛋白表达的影响

目的 探讨依达拉奉对大鼠局灶性脑缺血再灌注损伤后神经功能损伤、细胞凋亡及caspasc-3蛋白表达的影响.方法 雄性SD大鼠24只采用随机数字表法分为假手术组、脑缺血再灌注组、生理盐水治疗组、依达拉奉治疗组,每组6只.除假手术组外,其余3组均采用大脑中动脉线栓法制作大鼠局灶性脑缺血再灌注损伤模型.依达拉奉治疗组于脑缺血开始时及再灌注后12 h分别腹腔注射依达拉奉3 mg/kg,生理盐水治疗组同时间注射等量生理盐水;假手术组同样过程造模,但不插入尼龙线造成缺血.造模后24 h后进行大鼠神经行为学评分;应用免疫组织化学及Western blot检测caspase-3蛋白表达水平的变化;利用原位缺口末端标记法(TUNEL法)研究神经细胞凋亡的变化.结果 与脑缺血再灌注组及生理盐水治疗组相比,依达拉奉治疗组大鼠神经行为学评分明显减少,caspase-3免疫阳性细胞及蛋白表达明显减少,凋亡细胞也减少,差异均有统计学意义(P<0.05).结论 依达拉奉能有效减轻脑缺血灌注损伤后神经细胞凋亡.改善神经功能缺损症状,推测其机制与抑制caspase-3蛋白表达有关.     

依达拉奉对脑缺血再灌注损伤保护作用机制的研究

目的 探讨依达拉奉对脑缺血再灌注损伤的保护机制.方法 将45只健康雄性Wistar大鼠随机分为假手术组、生理盐水对照组、依达拉奉干预组各15只,采用线栓法制作大鼠大脑中动脉缺血模型,脑缺血2h再灌注即刻及12h干预组给予依达拉奉3mr/kg,对照组给予等量生理盐水分别腹腔注射.于24h后断头取脑,免疫组化法测细胞色素C(Cyt C)、半胱氨酸蛋白酶-3(Caspase-3),TUNEL法检测神经细胞凋亡,化学比色法测MDA、SOD,TTC染色测梗死体积.结果 假手术组无梗死现象,免疫反应阳性细胞及凋亡细胞亦少见.依达拉奉十预组与生理盐水对照组相比,Cyt C阳性细胞数、Caspase-3阳性细胞数及凋亡细胞数均明显减少,MDA含量减少,SOD活性有所恢复,差异均有统计学意义.生理盐水对照组可见明显大脑中动脉供血区梗死灶,依达拉奉干预组亦可见到梗死灶,与对照组比梗死体积占全脑体积的百分比明显缩小(t=6.576,P＜0.01).结论 依达拉奉有自由基清除作用,减少了Cyt C的释放,抑制了细胞凋亡,并缩小了梗死体积;依达拉奉可能通过线粒体途径抑制细胞凋亡.对脑缺血再灌注损伤有保护作用.     

病变侧亚低温对局部脑缺血再灌流损伤有关因素的影响

目的　研究病变侧脑亚低温对脑缺血再灌流损伤梗塞体积、 Ｎ Ｏ 的影响确定病变侧亚低温的疗效, 探讨机理。方法　应用可反馈控温半导体致冷块对大鼠局灶脑缺血模型病变侧降温至３２ ～３３ ℃研究持续缺血及再灌流损伤的保护作用及有关因素的影响。结果　持续缺血１０ 分钟低温组及缺血４０ 分钟再灌流并低温组梗塞体积均小于常温对照组。亚低温组 Ｎ Ｏ 含量明显低于常温对照组。结论　病变侧亚低温对脑缺血再灌流损伤在一定时间窗内有明显保护作用, 而亚低温使 Ｎ Ｏ 产生减少可能是其脑保护作用的部分机制。     

Pretreatment with scutellaria baicalensis stem-leaf total flavonoid prevents cerebral ischemia- reperfusion injury

Pretreatment with scutellaria baicalensis stem-leaf total flavonoid has protective effects against ischemia and attenuates myocardial ischemia-reperfusion injury. In this study, rats were given scutellaria baicalensis stem-leaf total flavonoid intragastrically at 50, 100, and 200 mg/kg per day for 7 days before focal cerebral ischemia-reperfusion injury models were established using the suture method. We then determined the protective effects of scutellaria baicalensis stem-leaf total flavon- oid pretreatment on focal cerebral ischemia-reperfusion injury. Results showed that neurological deficit scores increased, infarct volumes enlarged, apoptosis increased and Bcl-2 and Bax protein expression were upregulated at 24 hours after reperfusion. Pretreatment with scutellaria baicalensis stem-leaf total flavonoid at any dose lowered the neurological deficit scores, reduced the infarct volume, prevented apoptosis in hippocampal cells, attenuated neuronal and blood-brain barrier damage and upregulated Bcl-2 protein expression but inhibited Bax protein expression. Doses of 100 and 200 mg/kg were the most efficacious. Our findings indicate that pretreatment with scutel- laria baicalensis stem-leaf total flavonoid at 100 and 200 mg/kg can improve the neurological func- tions and have preventive and protective roles after focal cerebral ischemia-reperfusion injury.     

Neuroprotective effects of total saponins from Rubus parvifolius L. on cerebral ischemia/ reperfusion injury in rats

This study examines the neuroprotective effects and mechanisms of action of total saponins from Rubus parvifolius L. (TSRP) on focal cerebral ischemia and reperfusion injury in rats. Focal cerebral ischemia and reperfusion injury was performed in rats using the suture method. The results indicate that intragastric injection of TSRP, at 5, 10 and 20 mg/kg, could decrease neurological impairment, reduce cerebral infarct volume, diminish pathological changes, and significantly inhibit the apoptosis of neurons surrounding the ischemic area. In addition, TSRP upregulated the expression of the anti-apoptotic factor Bcl-2, at the protein and mRNA levels, and it downregulated the expression of the pro-apoptotic factor Bax, at the protein and mRNA levels. These findings indicate that TSRP protects against cerebral ischemia/reperfusion injury, and that it may do so by regulating the expression of Bcl-2 and Bax.     

局部亚低温对脑缺血再灌注治疗时间的影响

目的观察有效再灌注时间窗内实施局部亚低温对不同时间点血管再通(再灌注)的影响,并探讨其机制。方法将150只雄性大鼠随机分为大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)24h组10只;常温再灌注组50只;MCAO 2h进行亚低温再灌注组50只;MCAO 3h进行亚低温再灌注组40只。动物均于再灌注24h后处死,但MCAO 24h组大脑中动脉闭塞24h后直接处死。采用TTC染色观察梗死体积,用干-湿质量法测定脑含水量,用原位末端标记(TUNEL)观察神经细胞凋亡的变化,采用免疫组化法检测大鼠模型的Bcl-2、Bax及水通道蛋白4(aquaporin4,AQP4)的表达。结果 MCAO 24h组的梗死体积、脑含水量、TUNEL阳性细胞数、及Bcl-2、Bax、AQP4的表达相比较,常温组2～3h有统计学差异(P<0.01),4～6h没有统计学差异(P>0.05);MCAO 2h亚低温再灌注组2～5h有统计学差异(P<0.01),6h没有统计学差异(P>0.05);MCAO 3h亚低温再灌注组3～4h有统计学差异(P<0.01),5～6h没有统计学差异(P>0.05)。亚低温再灌注组各时相点的梗死体积、脑含水量、TUNEL阳性细胞数、Bax、AQP4的表达均显著低于相应常温组,Bcl-2的表达显著高于常温组(P<0.01,P<0.05)。结论实施局部亚低温可延长再灌注治疗时间窗,而且亚低温开始时间越早,其延长时间窗的效果就越显著。这为解决在时间窗内就诊的患者因检查等错过再灌注治疗这一临床难题的解决提供了重要的实验室依据。其机制可能与抑制半暗带细胞的凋亡和改善微循环有关。     

Mild hypothermia reduces infarct size resulting from temporary but not permanent focal ischemia in rats.

BACKGROUND AND PURPOSE: Mild hypothermia (32-35 degrees C) has been repeatedly shown in laboratory models to reduce damage resulting from global cerebral ischemic insults. Little information is available, however, regarding the protective potential of mild hypothermia against focal ischemia. We designed the present study to determine whether mild hypothermia influences outcome from either temporary or permanent middle cerebral artery occlusion in the rat. METHODS: In experiment 1 (permanent occlusion), mechanically ventilated, halothane-anesthetized spontaneously hypertensive rats underwent permanent ligation of the middle cerebral artery. Pericranial temperature was maintained at either 37 degrees C (n = 11) or 33 degrees C (n = 11) during the first 2 hours of occlusion. In experiment 2 (temporary occlusion), the vessel was occluded for 1 hour only. Pericranial temperature was controlled at either 37 degrees C (n = 12) or 33 degrees C (n = 14) during ischemia and for 1 hour after reperfusion. In both experiments, the rats were allowed to recover, with neurological function scored at 24 and 96 hours after onset of ischemia. Cerebral infarct volume (as determined by nitro blue tetrazolium staining) was planimetrically evaluated 96 hours after onset of ischemia. RESULTS: No difference in infarct volume was observed between groups undergoing permanent occlusion (177 +/- 53 mm3 for 37 degrees C rats, 167 +/- 71 mm3 for 33 degrees C rats [mean +/- SD]). Although neurologic function correlated with infarct volume at 96 hours (all animals in experiment 1 combined; p less than 0.01), we were unable to demonstrate an intergroup difference in function. In animals undergoing temporary occlusion, mean +/- SD infarct volume was 48% less in the hypothermic group (89 +/- 54 mm3 for 37 degrees C, 46 +/- 31 mm3 for 33 degrees C; p less than 0.03). Neurological function again correlated with infarct size (p less than 0.02), but improvement in function approached significance for the hypothermic group (p less than 0.06) at 24 hours after reperfusion only. CONCLUSIONS: Benefits from mild hypothermia may be obtained under conditions of temporary but not permanent middle cerebral artery occlusion in the rat.     

Comparison of the anti-apoptotic effects of 15-and 35-minute suspended moxibustion after focal cerebral ischemia/reperfusion injury

Heat-sensitive suspended moxibustion has a neuroprotective effect against focal cerebral ischemia/reperfusion injury, but the underlying mechanisms remain unclear. The duration of heat-sensitive suspended moxibustion(usually from 30 minutes to 1 hour) is longer than traditional suspended moxibustion(usually 15 minutes). However, the effects of 15-and 35-minute suspended moxibustion in rats with cerebral ischemia/reperfusion injury are poorly understood. In this study, we performed 15-or 35-minute suspended moxibustion at acupoint Dazhui(GV14) in an adult rat model of focal cerebral ischemia/reperfusion injury. Infarct volume was evaluated with the 2,3,5-triphenyltetrazolium chloride assay. Histopathological changes and neuronal apoptosis at the injury site were assessed by hematoxylin-eosin staining and terminal deoxynucleotidyl transferase d UTP nick end labeling assay. Caspase-9 and caspase-3 expression at the injury site was detected using immunofluorescent staining. Bax and Bcl-2 expression at the injury site was assessed using western blot assay. In the 35-minute moxibustion group, infarct volume was decreased, neuronal apoptosis was reduced, caspase-9, caspase-3 and Bax expression was lower, and Bcl-2 expression was increased, compared with the 15-minute moxibustion group. Our findings show that 35-minute moxibustion has a greater anti-apoptotic effect than 15-minute moxibustion after focal cerebral ischemia/reperfusion injury.     

肢体缺血预处理对脑缺血再灌注损伤大鼠自噬的影响

目的探讨肢体缺血预处理对脑缺血再灌注损伤大鼠自噬的影响。方法将60只Wistar大鼠随机分为假手术组(Sham组)、缺血再灌注组(I/R组)、肢体缺血预处理组(LIPC组)、3-甲基嘌呤组(3-MA组),每组15只。制作脑缺血再灌注、肢体缺血预处理及3-MA干预大鼠模型,在脑缺血2 h再灌注24 h后进行神经功能缺陷评分和脑梗死体积测定,HE染色观察细胞形态学改变,Western Bloting法检测自噬相关蛋白Beclin-1、Cathepsin B的表达。结果与I/R组比较,LIPC组神经功能缺陷评分降低(P<0.05),脑梗体积明显减小(P<0.05),细胞损伤、坏死减轻(P<0.05),Beclin-1、Cathepsin B的蛋白表达明显减弱(P<0.05)。结论 LIPC对缺血再灌注损伤大脑具有保护作用,其机制可能与减弱自噬水平有关。     

自由基清除剂联合钙离子拮抗剂对脑缺血神经元凋亡的影响

【摘要】 目的 观察自由基清除剂联合钙离子拮抗剂对大鼠脑缺血半暗带神经元凋亡及B细胞淋巴瘤/白血病-2（B cell lymphoma/leukemia-2,Bcl-2）和Bcl-2相关X蛋白（Bcl-2 associated X protein,Bax）表达的影响。 方法 线栓法制备大鼠大脑中动脉缺血再灌注模型,随机分为假手术组、对照组和干预组,干预组又分为自由基组、钙离子组及联合干预组。对照组及各干预组根据缺血再灌注时间分为再灌注6 h、24 h、48 h、72 h及5 d组,每组各16只大鼠。对照组大鼠每日腹腔注射3 mg/kg生理盐水及灌胃给服12 mg/kg淀粉,自由基组大鼠每日腹腔注射3 mg/kg依达拉奉,钙离子组大鼠每日灌胃给服12 mg/kg尼莫地平,联合干预组大鼠每日腹腔注射3 mg/kg依达拉奉及灌胃给服12 mg/kg尼莫地平。观察各组大鼠缺血侧脑梗死面积、缺血半暗带Bcl-2、Bax蛋白表达及神经元凋亡的变化。 结果 缺血再灌注后6 h,各干预组与对照组比较,大鼠缺血侧脑梗死面积、脑缺血半暗带神经元凋亡及凋亡相关Bcl-2、Bax蛋白表达差异无显著性（P均>0.05）;缺血再灌注后48 h、72 h和5 d,与同时间段对照组比较,各干预组大鼠脑缺血半暗带区Bcl-2蛋白表达增强,Bax蛋白表达降低,凋亡神经元数减少,梗死区面积缩小,差异有显著性（P均＜0.05）;与自由基组及钙离子组比较,联合干预组大鼠上述指标改变更显著,差异有显著性（P均＜0.001）;自由基组与钙离子组组间比较,上述各指标差异无显著性（P均>0.05）。 结论 在脑缺血再灌注损伤中,联合应用自由基清除剂和钙离子拮抗剂对缺血半暗带神经元具有较好的协同保护作用。