脑缺血大鼠大脑皮质NMDA受体的早期表达

目的　探讨脑缺血和缺血再灌注早期大脑皮质NMDA受体的变化规律。方法　健康雄性SD大鼠 4 8只 ,随机分为 6组 :假手术对照组、单纯脑缺血 1 5min、2 0min和 30min组及脑缺血 1 5min再灌流 30min和 2 4h组 ;4血管脑缺血动物模型 ;连续冰冻冠状切片观察 ,NR1 免疫组化染色特异性地显著NMDA受体的变化 ,图象分析及计算阳性单位PU值。结果　①单纯脑缺血各组PU值分别为 7 5 5± 1 81、6 91± 2 5 3和 6 0 9± 1 5 0 ,与对照组PU值 9 0 4± 1 5 7相比呈下降趋势 (P≤ 0 0 5 ) ;②再灌流 30min和 2 4h组PU值分别为 5 76± 2 2 4和 4 73± 1 34,与对照组PU值相比明显减少 36 2 9%～ 4 7 6 8% ,有统计学意义(P <0 0 5 ) ;③再灌流 0min、30min和 2 4h组两两比较 ,除 2 4h和 0min组相比NR1 阳性反应物减少有统计学意义 (P <0 0 5 )外 ,其它组别间NMDA受体下降的变化无显著性差异 (P >0 0 5 )。结论　脑缺血和缺血再灌流早期NMDA受体都存在下行调节 ,这种调节可能是一种保护性作用     

Influence of duration of focal cerebral ischemia and neuronal nitric oxide synthase on translocation of apoptosis-inducing factor to the nucleus

Translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus can play a major role in neuronal death elicited by oxidant stress. The time course of nuclear translocation of AIF after experimental stroke may vary with the severity of injury and may be accelerated by oxidant stress associated with reperfusion and nitric oxide (NO) production. Western immunoblots of AIF on nuclear fractions of ischemic hemisphere of male mice showed no significant increase with 1 h of middle cerebral artery occlusion and no reperfusion, whereas increases were detectable after 6 and 24 h of permanent ischemia. However, as little as 20 min of reperfusion after 1 h of middle cerebral artery occlusion resulted in an increase in nuclear AIF coincident with an increase in poly(ADP-ribose) polymer (PAR) formation. Further nuclear AIF accumulation was seen at 6 and 24 h of reperfusion. In contrast, 20 min of reperfusion after 2 h of occlusion did not increase nuclear AIF. In this case, nuclear AIF became detectable at 6 and 24 h of reperfusion. With brief occlusion of 30 min duration, nuclear AIF remained undetectable at both 20 min and 6 h and became evident only after 24 h of reperfusion. Inhibition of neuronal NO synthase attenuated formation of PAR and nuclear AIF accumulation. Gene deletion of neuronal NO synthase also attenuated nuclear AIF accumulation. Therefore, reperfusion accelerates AIF translocation to the nucleus when focal ischemia is of moderate duration (1 h), but is markedly delayed after brief ischemia (30 min). Nuclear translocation of AIF eventually occurs with prolonged focal ischemia with or without reperfusion. Neuronally-derived NO is a major factor contributing to nuclear AIF accumulation after stroke.     

Effects of bis(7)-tacrine on spontaneous synaptic activity and on the nicotinic ACh receptor of Torpedo electric organ.

Bis(7)-tacrine is a potent acetylcholinesterase inhibitor in which two tacrine molecules are linked by a heptylene chain. We tested the effects of bis(7)-tacrine on the spontaneous synaptic activity. Miniature endplate potentials (MEPPs) were recorded extracellularly on slices of electric organ of Torpedo marmorata. Bis(7)-tacrine, at a concentration of 100 nM, increased the magnitudes that describe MEPPs: amplitude, area, rise time, rate of rise, and half-width. We also tested the effect of bis(7)-tacrine on nicotinic acetylcholine receptors by analyzing the currents elicited by acetylcholine (100 microM) in Torpedo electric organ membranes transplanted in Xenopus laevis oocytes. Bis(7)-tacrine inhibited the acetylcholine-induced currents in a reversible manner (IC(50) = 162 nM). The inhibition of nicotinic acetylcholine receptors was not voltage dependent, and bis(7)-tacrine increased the desensitization of nicotinic acetylcholine receptors. The Hill coefficient for bis(7)-tacrine was -0.72 +/- 0.02, indicating that bis(7)-tacrine binds to the nicotinic acetylcholine receptor in a molecular ratio of 1:1, but does not affect the binding of alpha-bungarotoxin with the nicotinic acetylcholine receptor. In conclusion, bis(7)-tacrine greatly increases the spontaneous quantal release from peripheral cholinergic terminals at a much lower concentration than tacrine. Bis(7)-tacrine also blocks acetylcholine-induced currents of Torpedo electric organ, although the mechanism is different from that of tacrine: bis(7)-tacrine enhances desensitization, whereas tacrine reduces it.     

大鼠局灶性脑缺血再灌注中nNOS源性NO对Bcl-2、Bax表达的影响

为研究大鼠局灶性脑缺血再灌注早期神经元型一氧化氮合酶(nNOS)来源的一氧化氮(NO)和过氧亚硝基阴离子(ONOO-)对Bcl-2和Bax蛋白表达的影响,本实验闭塞大鼠左侧大脑中动脉造成局灶性脑缺血模型,给予选择性nNOS抑制剂-7硝基吲唑,应用免疫组化法检测Bcl-2和Bax蛋白表达的变化。结果表明:50mg/kg、25mg/kg剂量的7-硝基吲唑可使Bcl-2蛋白表达升高,Bax表达降低。提示:局灶性脑缺血再灌注早期,nNOS来源的NO可能通过下调Bcl-2、上调Bax促进细胞凋亡的发生。     

大鼠脑缺血再灌流后基质金属蛋白酶-2和9的表达

目的：探讨大鼠局灶性脑缺血再灌流后基质金属蛋白酶(MMP)-2和-9(MMP-9)表达的变化规律与脑水肿的关系。方法：用线栓法制作大鼠左侧大脑中动脉阻塞再灌流模型(MCAO),用免疫组化技术分别观察脑缺血再灌流不同时间点MMP-2和MMP-9的表达。结果：(1)脑缺血再灌流后24h可见MMP-2阳性细胞开始出现,3～7d时阳性细胞数达高峰,显色最深,至14d时仍有表达,略高于假手术组。(2)脑缺血再灌流后6h缺血区内MMP-9阳性细胞开始出现,12h明显增高,至2d达高峰,3d后阳性细胞数开始减少,至14d时恢复到基础水平,各相邻时间点比较差异显著。结论：脑缺血再灌流后,病变区MMP-2和MMP-9表达增加,二者在脑缺血再灌流后脑水肿方面起重要作用。     

Anti-oxidative effects of d-allose, a rare sugar, on ischemia-reperfusion damage following focal cerebral ischemia in rat

The present study investigates the anti-oxidative effects of D-allose on ischemic damage. Rats were subjected to transient middle cerebral artery occlusion (MCAO) for 1 h under pentobarbital anesthesia. D-allose was intravenously infused during occlusion and a further 1 h after reperfusion (400 mg/kg). The effects of D-allose on focal cerebral ischemia were examined by measuring brain damage (infarction and atrophy volume) and behavioral deficits 7 days after MCAO. In another set of rats, apurnic/apyrimidic abasic sites (AP-sites) and 8-hydroxy-2'-deoxyguanosine (8-OHdG), oxidative stress markers, were investigated 24 h after MCAO to examine the anti-oxidative effects of D-allose. Brain damage and behavioral deficits were significantly decreased by D-allose administration compared to vehicle. The number of AP-sites and 8-OHdG levels were also reduced by D-allose. Thus, the present study suggests that D-allose has anti-oxidative effects and induces neuroprotection in focal cerebral ischemia.     

大鼠局灶性脑缺血/再灌注后缺血脑区缓激肽含量的变化

目的研究大鼠局灶性脑缺血/再灌注后缺血脑区缓激肽含量的变化。方法用线栓法制作SD大鼠大脑中动脉阻塞的脑缺血/再灌注动物模型,用免疫细胞化学法结合图像分析技术,检测缺血脑区缓激肽样免疫反应阳性物的平均光密度(A)值作为缓激肽的相对含量,比较局灶性脑缺血3h组、假手术对照组、正常对照组和再灌注30min、2h、4h、16h组缓激肽的相对含量。结果缺血脑区缓激肽样免疫反应阳性物的A值于脑缺血3h/再灌注2h后明显增高(P<0.05),随后下降至增高前水平。结论大鼠局灶性脑缺血/再灌注后,缺血脑区缓激肽含量于再灌注2h后明显增高,其可能在脑缺血后脑水肿的发生中起着重要作用。     

NGF对局灶性脑缺血再灌注大鼠海马和顶叶皮质内CREB mRNA表达的影响

探讨外源性神经生长因子(nervegrowthfactor,NGF)对局灶性脑缺血再灌注大鼠海马和顶叶皮质内的cAMP反应元件结合蛋白(cyclicAMPresponseelementbindingprotein,CREB)mRNA表达的影响。用线栓法制作大鼠局灶性脑缺血再灌注模型,运用原位杂交和图像分析技术检测大鼠缺血侧海马CA1区和顶叶皮质内CREBmRNA的表达。结果显示:缺血再灌注组缺血侧海马CA1区和顶叶皮质CREBmRNA阳性反应产物平均光密度(OD)比假手术组减少(P<0.05),NGF组CA1区和顶叶皮质内的CREBmRNA阳性产物平均光密度高于缺血再灌注组(P<0.05)。本研究结果提示NGF可以上调局灶性脑缺血再灌注大鼠海马和顶叶皮质缺血神经元CREBmRNA的表达,NGF对缺血神经元的保护作用可能通过激活CREB的转录与翻译,从而启动一系列信号通路来实现。     

Effect of bis(7)-tacrine on cognition in rats with chronic cerebral ischemia

Bis(7)-tacrine (B7T), a novel dimeric acetyl cholinesterase (AChE) inhibitor, has multiple neuroprotective activities against neuronal damage. However, its therapeutic effects in chronic cerebral ischemia remain unknown. In the present study, adult male Sprague-Dawley rats were subjected with permanent ligation of the bilateral common carotid arteries to investigate the roles of B7T on cognitive function, neuronal apoptosis and neurogenesis in the hippocampus. Results from spatial navigation test showed that chronic cerebral ischemia impaired spatial learning, B7T treatment shorten escape latency of ischemia rats as compared with saline-treated rats. Probe trial test indicated that spatial memory deficit of chronic cerebral ischemic animals was reversed by B7T treatment. Immunohistochemical results showed that B7T reduced neuronal apoptosis in the hippocampal CA1 region as compared with ischemia rats, and B7T treatment increased neurogenesis in the hippocampus. These findings suggest that B7T may exert its neuroprotective effects by inhibiting apoptosis and promoting neurogenesis in 2VO rats.     

Spatio-temporal expression of cysteinyl leukotriene receptor-2 mRNA in rat brain after focal cerebral ischemia

Cysteinyl leukotrienes (CysLTs) induce inflammatory responses mediated by activating CysLT(1) and CysLT(2) receptors. We have recently reported that CysLT(1) receptor expression is increased in rat brain after focal cerebral ischemia and the increased expression is spatio-temporally related to acute neuronal injury and late astrocyte proliferation. Here we report spatio-temporal expression of CysLT(2) receptor mRNA in rat brain after focal cerebral ischemia induced by 30min of middle cerebral artery occlusion. We found that the neuron density was gradually decreased or disappeared in the ischemic core and boundary zone during 14 days after reperfusion, and the astrocyte population in the boundary zone was increased 3-14 days after reperfusion. In the ischemic core, the expression of CysLT(2) receptor mRNA was increased at 6, 12 and 24h and then recovered at 3, 7 and 14 days after reperfusion. In the boundary zone, the expression was significantly increased 3, 7 and 14 days after reperfusion. The results suggest that CysLT(2) receptor may be related to the acute neuronal injury and late astrocyte proliferation in the ischemic brain.     

Tacrolimus (FK506) attenuates biphasic cytochrome c release and Bad phosphorylation following transient cerebral ischemia in mice

Tacrolimus (FK506) has a neuroprotective action on cerebral infarction produced by cerebral ischemia, however, detailed mechanisms underlying this action have not been fully elucidated. We examined temporal profiles of survival-and death-related signals, Bad phosphorylation, release of cytochrome c (cyt.c), activation of caspase 3 and DNA fragmentation in the brain during and after middle cerebral artery occlusion (MCAo) in mice, and then examined the effect of tacrolimus on these signals. C57BL/6J mice were subjected to transient MCAo by intraluminal suture insertion for 60 min. Tacrolimus (1 mg/kg, i.p.) was administered immediately after MCAo. There were biphasic increases in the release of cyt.c in the ischemic core and penumbra; with the first increase toward the end of the occlusion period and the second increase 3-12 h after reperfusion. Tacrolimus significantly inhibited the increase of cytosolic cyt.c during ischemia and reperfusion. Phosphorylated Bad, Ser-136 (P-Bad(136)) and Ser-155 (P-Bad(155)) were detected 30 min after MCAo and after reperfusion in the ischemic cortex, respectively. Tacrolimus increased P-Bad(136) during ischemia and prolonged P-Bad(155) expression after reperfusion. Tacrolimus also decreased caspase-3 and terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling-positive cells, and reduced the size of infarct 24 h after reperfusion. Our study provided the first evidence that the neuroprotective action of tacrolimus involved inhibition of biphasic cyt.c release from mitochondria, possibly via up-regulation of Bad phosphorylation at different sites after focal cerebral ischemia and reperfusion.     

尼古丁对大鼠局灶性脑缺血再灌注损伤的神经保护作用

目的:研究尼古丁对大鼠局灶性脑缺血再灌注损伤是否具有神经保护作用。方法:在大鼠大脑中动脉堵塞(MCAO)前30min给予腹腔注射尼古丁酒石酸盐溶液,观察局灶性脑缺血2h再灌注24h后,大鼠神经行为学评分及脑梗死容积的变化。结果:再灌注24h后,与单纯缺血再灌注组相比,给予尼古丁酒石酸盐溶液注射可以改善动物的神经行为学评分、减少脑梗死容积百分比(P0.05)。结论:尼古丁对大鼠局灶性脑缺血再灌注损伤具有神经保护作用。     

Intracerebroventricular injection of human prostatic acid phosphatase has potent neuroprotective effects against transient focal cerebral ischemia in rats

Though the potential use of adenosine as a neuroprotective agent has long been realized, there are currently no adenosine-based therapies for the prevention or treatment of cerebral ischemia and reperfusion injury. Prostatic acid phosphatase (PAP), an enzyme that has long served as a diagnostic marker for prostate cancer, has been recently demonstrated to exhibit ecto-5′-nucleotidase activity, and dephosphorylate endogenous extracellular AMP to adenosine. We therefore tested the hypothesis that PAP has sustained and potent neuroprotective effects against cerebral ischemia in the rat model of middle cerebral artery occlusion. We found that hPAP produced significant neuroprotection against focal cerebral ischemia, as evident from significant reduction in cerebral infarction and neurological deficits. The therapeutic time window for hPAP in rat focal cerebral ischemia model was limited from 6 h before ischemia to 1.5 h after reperfusion. The present study suggested that PAP is a potential candidate for the prevention and treatment of cerebral ischemic injury, especially during perioperative period.     

局灶性脑缺血后鼠脑组织tPA基因表达及其与细胞凋亡

目的 探讨局灶性脑缺血后组织型纤溶酶原激活物（tＰＡ）基因的表达及其与细胞凋亡的 关系。方法 用原位杂交和免疫组织化学方法检测tＰＡ基因在大鼠局灶性脑缺血（大脑中 动脉阻塞）后的表达,用ＴＵＮＥＬ法检测缺血不同时间的细胞凋亡状况。结果 脑缺血６h缺血中心部位的胶质细胞和梗死灶周边缺血半影区的神经元均观察到tＰＡ免疫反应性信号,在大脑皮层后肢体区、顶皮层区、海马区和齿状回可见凋亡细胞,但缺血１８h缺血侧神     

Rapid tolerance to focal cerebral ischemia in rats is induced by preconditioning with electroacupuncture: window of protection and the role of adenosine

The aim of the present study was to investigate the first protective window of preconditioning with electroacupuncture (EA) against focal cerebral ischemia, and to explore whether adenosine is involved in the rapid tolerance phenomenon. Sixty-four male Sprague-Dawley rats were randomly assigned to eight groups (n=8 in each). Animals in the control group received no treatment, and animals in EA1-EA4 groups received EA at 0.5, 1, 2 and 3 h before induction of focal cerebral ischemia, respectively. Rats in DPCPX group were intraperitoneally injected with 1 mg kg-1 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), 3 h before induction of focal cerebral ischemia. Animals in vehicle group and EA+DPCPX group were pretreated with 1 ml kg-1 dimethyl sulfoxide (DMSO, the solvent of DPCPX) and 1 mg kg-1 DPCPX 30 min before preconditioning with EA, respectively. All rats were anesthetized with 40 mg kg-1 pentobarbital sodium intraperitoneally. Animals that required EA preconditioning, received EA with intensity of 1 mA and frequency of 15 Hz at the Baihui acupoint (GV 20) for 30 min. The focal cerebral ischemia was produced by the right middle cerebral artery occlusion (MCAO) for 120 min. The neurologic deficit scores (NDS) and brain infarct volumes were evaluated at 24 h after reperfusion. All rats survived until 24 h after reperfusion. Preconditioning with EA at 2 h before induction of focal cerebral ischemia improved neurologic outcome (P<0.05 versus control) and reduced the infarct volume (P<0.01 versus control) at 24 h after reperfusion. These beneficial effects were reversed by pretreatment with 1 mg kg-1 DPCPX, whereas this agent itself did not affect the NDS and volume in drug-ischemic controls after ischemia. The results show that preconditioning with single EA session induces rapid tolerance to focal cerebral ischemia. The rapid ischemic tolerance appears at 2 h (but not at 0.5, 1, or 3 h) after preconditioning, and is possibly mediated through an adenosine A1 receptor-related mechanism.     

脑缺血及再灌流早期大鼠海马CA_1区NMDA受体的变化

探讨脑缺血和缺血再灌流早期海马CA1 区NMDA受体的变化规律,选用雄性SD大鼠 48只,随机分为:假手术对照组、单纯脑缺血 15min、20min和 30min组及脑缺血 15min再灌流 0min、30min和 24h组,参照Pulsinelli Brierley(4VO)法制作脑缺血模型,取脑,连续冰冻冠状切片,NR1免疫组化染色并进行图像分析及计算阳性单位PU值。结果显示: (1 )单纯脑缺血 15min、20min和 30min组PU值分别为 5. 89±0. 92、5. 18±1. 63和 4. 89±2. 69,与对照组PU值 7. 56±2. 35相比,下降 22. 09% ~35. 32% (P≤0.05); (2)再灌流 30min和 24h组PU值分别为 4. 20±1. 37和 2. 99±1. 28,与对照组PU值相比,减少 44. 44% ~60. 45%,有统计学意义(P<0.05),均明显降低; (3)再灌流 0min、30min和 24h组两两比较, 24h和 0min组的NMDA受体减少有统计学意义(P<0.05)。上述结果提示,脑缺血和缺血再灌流早期海马CA1 区NMDA受体存在下行调节,这可能是脑缺血后自身的一种保护性调节。     

阿托伐他汀对大鼠缺血再灌注脑组织中NADPH氧化酶源性超氧阴离子的抑制作用

目的：脑组织在缺血再灌注的早期，超氧阴离子的大量生成加重了脑组织损伤，本实验研究阿托伐他汀对缺血再灌注脑组织保护作用的可能机制。方法:成年雄性Sprague-Dawley大鼠经线栓法阻断大脑中动脉建立脑缺血再灌注模型，再灌注前经腹腔给予阿托伐他汀（立普妥）治疗。脑梗死灶体积用四唑氮蓝染色后测量；NADPH氧化酶酶活性和超氧阴离子水平使用光泽精增强化学发光法定量测定；NADPH氧化酶膜亚基gp91phox、膜易位亚基p47phox和小GTP酶Rac-1蛋白的表达用蛋白质印迹分析。结果:缺血半暗区的NADPH氧化酶活性和超氧阴离子水平增高，于再灌注2 h达到高峰，但缺血中心区的NADPH氧化酶活性和超氧阴离子水平无明显增高。阿托伐他汀预治疗能抑制再灌注2 h后缺血半暗区的NADPH氧化酶活性和超氧阴离子增高，减少膜亚基gp91phox蛋白的表达和预防细胞质亚基p47phox蛋白易位至细胞膜。结论:阿托伐他汀对缺血再灌注脑组织NADPH氧化酶源性超氧阴离子的抑制作用，是其脑保护作用机制之一。     

小鼠脑缺血时自噬相关基因5的抗损伤作用

 目的: 观察自噬相关基因5(Atg5)在小鼠脑缺血再灌注损伤中的抗损伤作用。方法: 将雄性BALB/c小鼠随机分为假手术(sham)组、缺血再灌注(I/R)组、Atg5 siRNA组和control siRNA组。I/R组采用大脑中动脉阻塞(MCAO)60 min后再灌注24 h。Atg5 siRNA组和control siRNA组将5 μL Atg5 siRNA或scrambled siRNA在MCAO前24 h侧脑室注射。实时荧光定量PCR和Western blot检测Atg5的表达;2,3,5-氯化三苯基四氮唑(TTC)染色法检测抑制Atg5对缺血再灌注损伤后脑梗死面积和水肿率的影响;神经行为学评分法检测抑制Atg5对缺血再灌注损伤后神经症状的影响。结果: MCAO后再灌24 h,缺血半影区Atg5 mRNA和蛋白水平显著增高(P<0.05);Atg5 siRNA明显降低缺血再灌后Atg5 mRNA和蛋白的表达(P<0.05);侧脑室给予Atg5 siRNA能显著增加脑梗死面积和水肿率,并加重神经行为学损伤(P<0.05)。结论: 沉默Atg5加重小鼠脑缺血再灌损伤,提示MCAO后诱导的 Atg5 可减轻小鼠局灶性脑缺血再灌注损伤。     

降钙素基因相关肽对局灶性脑缺血再灌注大鼠海马Fas mRNA表达的调节作用

目的研究外源性降钙素基因相关肽(CGRP)对局灶性脑缺血再灌注大鼠海马Fas mRNA表达的影响,探讨降钙素基因相关肽对缺血再灌注脑神经组织的作用。方法用线栓法制备大鼠大脑中动脉阻塞(MCAO)模型,应用原位杂交和显微图象分析方法检测局灶性脑缺血再灌注大鼠海马Fas mRNA的表达。结果假手术组大鼠海马未见Fas mRNA阳性表达;缺血再灌注组(缺血再灌注不同时间段6 h,12 h,24 h,48h,72 h)海马Fas mRNA明显过表达;注射CGRP后海马区Fas mRNA阳性表达细胞平均光密度值明显低于缺血再灌注组(P<0.01)。结论经颈动脉注入外源性CGRP下调缺血神经元Fas mRNA的表达,可能是外源性CGRP对缺血神经元保护作用的机制之一。     

缺血后适应对大鼠脑缺血/再灌注损伤的影响

目的：探讨缺血后适应对大鼠脑缺血/再灌注损伤的影响。方法:应用线栓法制作大鼠脑缺血/再灌注损伤模型；21只雄性SD大鼠随机分为缺血/再灌注组、夹闭单侧颈总动脉后处理组和夹闭双侧颈总动脉后处理组，每组7只。再灌注48 h，测定脑梗死体积；拔栓后1 h及处死大鼠前进行神经功能测定；梗死即刻、梗死后10 min、术中1 h、拔栓后即刻、每次夹/松颈总动脉时、干预后30 min等15个时点监测脑血流。结果:夹闭单侧、双侧颈总动脉后处理组大鼠脑组织梗死体积与缺血/再灌注组相比明显减小，有显著差异；3组脑血流各个时点方差分析差异无显著，但是夹闭双侧颈总动脉后处理组干预30 min后脑血流百分比较缺血/再灌注组、夹闭单侧颈总动脉后处理组降低9％。手术后1 h 3组神经功能评分P<0.05，差异显著，夹闭单侧、双侧颈总动脉后处理组神经功能缺损均比缺血/再灌注组减轻。结论:缺血后适应能够明显减小梗塞体积，改善大鼠术后1h神经功能评分，可能与缺血后适应调节早期再灌注时血流动力学状态有关。