自噬——脑缺血损伤中的双刃剑

近来研究表明,自噬可能成为卒中治疗过程中的新靶点,但是自噬在脑缺血过程中的激活是否提升神经元存活率一直存在争议,关于其对于脑缺血后神经元发挥的作用是保护还是加重损伤尚不明确。本文主要综述脑缺血后自噬在缺血/低氧损伤中可能发挥的双重作用及其可能的信号通路。     

A Double‐Edged Sword with Therapeutic Potential: An Updated Role of Autophagy in Ischemic Cerebral Injury

Cerebral ischemia is a severe outcome that could cause cognitive and motor dysfunction, neurodegenerative diseases and even acute death. Although the existence of autophagy in cerebral ischemia is undisputable, the consensus has not yet been reached regarding the exact functions and influence of autophagy in cerebral ischemia. Whether the activation of autophagy is beneficial or harmful in cerebral ischemia injury largely depends on the balance between the burden of intracellular substrate targeted for autophagy and the capacity of the cellular autophagic machinery. Furthermore, the mechanisms underlying the autophagy in cerebral ischemia are far from clear yet. This brief review focuses on not only the current understanding of biological effects of autophagy, but also the therapeutic potentials of autophagy in ischemic stroke. There are disputes over the exact role of autophagy in cerebral ischemia. Application of chemical autophagy inhibitor (e.g., 3‐methyladenine) or inducer (e.g., rapamycin) in vitro and in vivo was reported to protect or harm neuronal cell. Knockdown of autophagic protein, such as Beclin 1, was also reported to modulate the cerebral ischemia‐induced injury. Moreover, autophagy inhibitor abolished the neuroprotection of ischemic preconditioning, implying a neuroprotective effect of autophagy. To clarify these issues on autophagy in cerebral ischemia, future investigations are warranted.     

Exercise intensity influences cell injury in rat hippocampal slices exposed to oxygen and glucose deprivation

We evaluated the effects of two levels of daily forced exercise intensity (moderate and high) in the treadmill over cell susceptibility to oxygen and glucose deprivation (OGD) in hippocampal slices from Wistar rats. Moderate exercise decreased lactate dehydrogenase (LDH) release after OGD, while a significant increase in LDH release was observed in the high intensity group submitted to OGD. Our data corroborate the hypothesis that higher training intensity exacerbates brain damage, while a moderate intensity reduces the injury caused by in vitro ischemia.     

Enhanced SUMOylation and SENP-1 protein levels following oxygen and glucose deprivation in neurones

Here, we show that oxygen and glucose deprivation (OGD) causes increased small ubiquitin-like modifier (SUMO)-1 and SUMO-2/3 conjugation to substrate proteins in cultured hippocampal neurones. Surprisingly, the SUMO protease SENP-1, which removes SUMO from conjugated proteins, was also increased by OGD, suggesting that the neuronal response to OGD involves a complex interplay between SUMOylation and deSUMOylation. Importantly, decreasing global SUMOylation in cultured hippocampal neurones by overexpression of the catalytic domain of SENP-1 increased neuronal vulnerability to OGD-induced cell death. Taken together, these results suggest a neuroprotective role for neuronal SUMOylation after OGD.     

Autophagy and inflammation in ischemic stroke

Appropriate autophagy has protective effects on ischemic nerve tissue,while excessive autophagy may cause cell death.The inflammatory response plays an important role in the survival of nerve cells and the recovery of neural tissue after ischemia.Many studies have found an interaction between autophagy and inflammation in the pathogenesis of ischemic stroke.This study outlines recent advances regarding the role of autophagy in the post-stroke inflammatory response as follows.(1)Autophagy inhibits inflammatory responses caused by ischemic stimulation through mTOR,the AMPK pathway,and inhibition of inflammasome activation.(2)Activation of inflammation triggers the formation of autophagosomes,and the upregulation of autophagy levels is marked by a significant increase in the autophagy-forming markers LC3-II and Beclin-1.Lipopolysaccharide stimulates microglia and inhibits ULK1 activity by direct phosphorylation of p38 MAPK,reducing the flux and autophagy level,thereby inducing inflammatory activity.(3)By blocking the activation of autophagy,the activation of inflammasomes can alleviate cerebral ischemic injury.Autophagy can also regulate the phenotypic alternation of microglia through the nuclear factor-κB pathway,which is beneficial to the recovery of neural tissue after ischemia.Studies have shown that some drugs such as resveratrol can exert neuroprotective effects by regulating the autophagy-inflammatory pathway.These studies suggest that the autophagy-inflammatory pathway may provide a new direction for the treatment of ischemic stroke.     

Tissue kallikrein protects cortical neurons against in vitro ischemia-acidosis/reperfusion-induced injury through the ERK1/2 pathway

Human tissue kallikrein (hTK) gene transfer has been shown to protect neurons against cerebral ischemia/reperfusion (I/R) injury, and exogenous tissue kallikrein (TK) administration can enhance neurogenesis and angiogenesis following focal cortical infarction. Previous studies have reported that acidosis is a common feature of ischemia and plays a critical role in brain injury. However, little is known about the role of TK in ischemia-acidosis-induced injury, which is partially caused by the activation of acid-sensing ion channels (ASICs). Here we report that pretreatment of cultured cortical neurons with TK reduced cell death induced by either acidosis or oxygen and glucose deprivation-acidosis/reoxygenation (OGD-A/R). Immunocytochemical staining revealed that TK largely prevented OGD-A/R-induced neuronal morphological changes. We also observed that TK treatment protected cultured neurons from acidosis and OGD-A/R insults. TK exerted the neuroprotective effects by reducing production of reactive oxygen species (ROS), stabilizing the mitochondrial membrane potential (MMP) and inhibiting caspase-3 activation, and thereby attenuating oxidative stress and apoptosis. In addition, we found that activation of the extracellular signal-regulated kinase1/2 (ERK1/2) signaling cascade but not the PI3K/Akt signaling pathway was required for the survival-promoting effect of TK on neurons exposed to OGD-A/R. Moreover, blockade of ASICs had effects similar to TK administration, suggesting direct or indirect involvement of ASICs in TK protection. In conclusion, TK has antioxidant characteristics and is capable of alleviating ischemia-acidosis/reperfusion-induced injury, inhibiting apoptosis and promoting cell survival in vitro through activating the ERK1/2 signaling pathways. Therefore, TK represents a promising therapeutic strategy for ischemic stroke.     

大鼠全脑缺血再灌流后脑组织P53及P21蛋白的表达

目的　探讨大鼠全脑缺血再灌流后Ｐ５３、Ｐ２１蛋白的表达及其与迟发性神经元死亡（ＤＮＤ）的关系。方法　在４ 血管闭塞法全脑缺血模型上,采用ＨＥ及ＬＳＡＢ染色法,观察脑组织病理改变,检测脑组织Ｐ５３、Ｐ２１蛋白的表达,以及蛋白合成抑制剂放线菌酮对其的影响。结果　全脑缺血１５ ｍ ｉｎ 再灌流后,脑组织Ｐ５３、Ｐ２１蛋白表达增加,且两者分布接近。海马结构、丘脑、下丘脑等白质区（再灌流后６ ｈ）较皮层、海马的神经细胞核（２４ ｈ）先检测到Ｐ５３、Ｐ２１蛋白,７２ ｈ 表达达高峰。并且以缺血损伤最严重的海马ＣＡ１ 区Ｐ５３、Ｐ２１蛋白表达为强。另外,放线菌酮可抑制脑组织Ｐ５３、Ｐ２１蛋白的表达,并对ＤＮＤ具有一定的保护作用。结论　全脑缺血再灌流损伤后,脑组织Ｐ５３、Ｐ２１蛋白表达增加,放线菌酮可抑制其表达,并对ＤＮＤ起保护作用,提示Ｐ５３、Ｐ２１蛋白参与了全脑缺血后ＤＮＤ的凋亡机制,并对其起促进作用     

大鼠脑缺血再灌注时小胶质细胞或巨噬细胞的活化与增殖

目的 研究脑缺血再灌注时小胶质细胞/巨噬细胞的活化与增殖。方法 阻塞大鼠大脑中动脉2h再灌注0.5-48h制成脑缺血模型，以免疫组化与凝集素（GSI-B4）组化法观察小胶质细胞/巨噬细胞在脑组织的分布及形态特点。结果 0.5h时，呈现有大量形态各异的GSI-B4阳性小胶质细胞与巨噬细胞，随后下降；12-48h，数量再次且持续增高，并呈不同的形态，视前区，杏仁皮质核浅层及其脑膜有阳性巨噬细胞，3-48h，在缺血区有CD45阳性的活化小胶质细胞；缺血再灌注各组，室管膜及室管膜下层有PCNA强阳性细胞，杏仁核，杏仁皮质核，视前区及其软脑膜有PCNA强阳性细胞；有PCNA与CD45双标阳性的细胞。结论 缺血再灌注时，不同区域小胶质细胞形态呈明显的异形性，外源性巨噬细胞可能来自血单核细胞的浸润，脑膜巨噬细胞和室管膜及室管膜下层细胞的增生，浸润和迁移；CD45是小胶质细胞活化的标志。     

脑缺血再灌注脑内纹状体区细胞外液谷胱甘肽的动态变化及丹参的影响

采用脑内微透析技术，应用高压液相色谱电化学检测方法（ＨＰＬＣ－ＥＤ），活体动态观察沙土鼠全脑缺血３０分钟，再灌注１２０分钟的细胞外液中的谷胱甘肽（Ｇｌｕｔａｔｈｉｏｎｅ，ＧＳＨ）的变化及丹参对它的影响。结果显示：全脑缺血后，细胞外液ＧＳＨ水平迅速升高（Ｐ＜０．０１），缺血３０分钟达高峰为缺血前的５。８２倍。再灌注后ＧＳＨ水平明显降低，于３０分钟趋于正常。脑缺血前３０分钟给予丹参注射液不影响细胞外液ＧＳＨ水平。表明脑缺血及再灌注期，ＧＳＨ反应性增高。ＧＳＨ作为内源性抗氧化剂及ＮＭＤＡ受体拮抗剂在脑缺血损伤中起重要作用。     

半胱氨酸蛋白酶抑制剂减少大鼠脑缺血模型caspase-3的表达

目的 :研究半胱氨酸蛋白酶caspase 3及calpain抑制剂干预治疗对大鼠局灶性脑缺血 /再灌注模型caspase 3表达的影响。方法 :大鼠随机分为经左侧侧脑室注射caspase 3抑制剂Ⅲ组 (DEVD组 )、calpain抑制剂Ⅰ组 (ALLN组 )、联合治疗组 (DEVD +ALLN组 )、溶剂二甲基亚砜对照组 (DMSO组 )以及左侧大脑中动脉缺血 /再灌注对照组 (MCAO组 ) ,诱导左侧大脑中动脉缺血 2h ,再灌注2 4或 48h ;再灌注 2 4h进行TTC染色观察梗死灶的形成情况 ;用TdT介导的dUTP缺口末端标记技术 (TUNEL)检测鼠脑中的神经元凋亡情况 ;并分别通过原位杂交和免疫组化技术检测鼠脑中caspase 3mRNA及活性蛋白的表达。结果 :DMSO组的各项指标与MCAO组无明显差异 ;DEVD组或ALLN组缺血侧脑中细胞凋亡数、caspase 3mRNA及活性蛋白的表达均明显减少 ,联合治疗组作用更强。结论 :caspase 3与calpain抑制剂干预治疗可减少大鼠缺血再灌注脑区神经元凋亡和caspase 3的表达 ,从而产生神经保护作用 ,并具有潜在的临床应用价值。     

Neuroprotective effects of nimodipine and nifedipine in the NGF-differentiated PC12 cells exposed to oxygen-glucose deprivation or trophic withdrawal

The goal of this study was to compare the neuroprotective properties of the l-type Ca(2+) channel blockers, nimodipine and nifedipine, using nerve growth factor (NGF)-differentiated PC12 neuronal cultures exposed to oxygen-glucose deprivation (OGD) and trophic withdrawal-induced cell death. Nimodipine (1-100μM) conferred 65±13% neuroprotection upon exposure to OGD and 35±6% neuroprotection towards different trophic withdrawal-induced cell death measured by lactate dehydrogenase and caspase 3 activities. The time window of nimodipine conferred neuroprotection was detected during the first 5h but not at longer OGD exposures. Nifedipine (1-100μM), to a lower potency than nimodipine, conferred 30-55±8% neuroprotection towards OGD in PC12 cells and 29±5% in rat hypocampal slices, and 10±3% neuroprotection at 100μM towards trophic withdrawal-induced PC12 cell death. The ability to demonstrate that nimodipine conferred neuroprotection in a narrow therapeutic time-window indicates that the OGD PC12 model mimics the in vivo models and therefore suitable for neuroprotective drug discovery and development.     

去骨瓣减压术对大鼠局灶性脑缺血后脑水肿、血脑屏障、脑血流变化的影响

目的:观察去骨瓣减压术对局灶性脑缺血大鼠脑水肿、血脑屏障(BBB)及脑血流(CBF)的影响,探讨该手术对缺血性脑损害的保护机制。方法:改良Koizumi′s法制作脑缺血大鼠动物模型,脑缺血后6h行去骨瓣减压术,分别在术后3和7d观察脑水肿和血脑屏障的变化,另持续观察缺血1h 去骨瓣减压2h的CBF变化。结果:去骨瓣减压术后半暗区CBF增加更加持久和有效,在术后第3天时BBB破坏范围明显缩小,术后第7天时脑水含量明显降低。结论:去骨瓣减压术对局灶性脑缺血的保护作用可能与增加半暗区CBF、改善BBB破坏和减轻脑水肿有关。     

早期强制性使用运动疗法对局灶性脑梗死大鼠行为学评分及神经生长因子表达的影响

目的探讨卒中后早期强制性使用单侧肢体对局灶性脑梗死大鼠行为学评分及大脑皮层梗死周围神经生长因子(NGF)表达的影响。方法健康雄性Sprague-Dawley大鼠,用线栓法建立大脑中动脉永久性缺血(MCAO)模型,按照Zea-Longa方法对动物的行为缺陷进行评分,选取1～3分鼠60只,将60只大鼠随机分为2组,健侧前肢固定组30只,未固定组30只,7 d时解除固定,再采用随机区组法分为14 d时处死组和21 d处死组,各组动物在7 d时和处死前行神经功能评分(longa评分);采用免疫组织化学方法观察NGF蛋白的表达,采用原位杂交方法检测NGFmRNA表达。结果固定组与未固定组相比,早期强制性使用运动疗法可以改善患侧肢体14 d及21 d的行为学评分:在梗死周围皮质NGF蛋白和NGF mRNA的表达,固定组14 d时表达均明显高于非固定组．而在21 d时无统计学意义。结论早期强制性使用运动疗法可以改善神经功能的恢复,从基因水平来提高NGF的表达可能为其发挥作用的机制之一。     

CX3CL1/CX3CR1-mediated microglia activation plays a detrimental role in ischemic mice brain via p38MAPK/PKC pathway

The exact roles of activated microglia and fractalkine (CX3CL1)/fractalkine receptor (CX3CR1) signaling are not fully understood in brain ischemic injury and the findings reported are controversial. Here, we investigated the effects of CX3CR1 siRNA on the expression of CX3CR1, p38 mitogen-activated protein kinase (p38MAPK), Protein Kinase C (PKC) and inflammatory cytokines, microglia activation, white matter lesions, and cognitive function in mice treated with bilateral common carotid artery stenosis (BCAS) in vivo as well as effects of exogenous CX3CL1, CX3CR1 siRNA, and SB2035080 on expression of inflammatory cytokines in BV2 microglia treated with oxygen–glucose deprivation (OGD) in vitro. We showed that CX3CR1 siRNA significantly inhibited the increased expression of CX3CR1, p38MAPK, PKC as well as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6, and also attenuated microglia activation, white matter lesions, and cognitive deficits induced by BCAS in mice brain. We also showed that exogenous CX3CL1 could induce a further enhancement in TNF-α and IL-1β expression, which could be suppressed by CX3CR1 siRNA or by the p38MAPK inhibitor in OGD-treated BV2 microglial cells in vitro. Our findings indicated that CX3CL1/CX3CR1-mediated microglial activation plays a detrimental role in ischemic brain via p38MAPK/PKC signaling and also suggested that CX3CL1/CX3CR1 axis might be a putative therapeutic target to disrupt the cascade of deleterious events that lead to brain ischemic injury.