3-n-butylphthalide (NBP) attenuated neuronal autophagy and amyloid-beta expression in diabetic mice subjected to brain ischemia

Abstract

Objective: The aim of this study was to investigate the protective effect of dl-3-n-butylphthalide (NBP) on brain damage in streptozotocin (STZ)-induced diabetic mice subjected to cerebral ischemia.

Methods: we pretreated diabetic mice with NBP orally for 4 weeks prior and 2 days after transient common carotid artery occlusion (CCAO) operation. Immunohistochemistry and transmission electron microscopy were performed to investigate the neuronal loss, astrocytes activation, amyloid-beta (Abeta) protein expression, and autophagy activation.

Results: The results showed that diabetes increased stroke-induced neuronal loss, astrocytes activation, Abeta generation, and autophagy activity, while NBP administration attenuated these changes. Immunofluorescence double staining of Abeta with autophagosome-specific antibody LC3 showed that most elevated Abeta⁺ signal was co-localized with LC3⁺ signal.

Conclusion: Our finding suggests that NBP attenuates Abeta generation promoted by diabetes in ischemia might act through inhibiting abnormally activated neuronal autophagy. Therefore, treatment with NBP to modulate autophagy might provide a novel therapeutic strategy for diabetes by preventing ischemic brain damage and depressing the risk of post-stroke dementia.     

Severe global cerebral ischemia-induced programmed necrosis of hippocampal CA1 neurons in rat is prevented by 3-methyladenine: a widely used inhibitor of autophagy

The true programmed mechanisms of delayed neuronal death induced by global cerebral ischemia/reperfusion injury remain incompletely characterized. Autophagic cell death and programmed necrosis are 2 kinds of programmed cell death distinct from apoptosis. Here, we studied the death mechanisms of hippocampal cornu ammonis 1 neuronal death after a 20-minute severe global ischemia/reperfusion injury in young adult rats and the effects of 3-methyladenine (3-MA), a widely used inhibitor of autophagy. The morphological changes detected by electron microscopy, together with the activation of autophagy, transferase-mediated UTP nick end-labeling-positive neurons, and delayed death, demonstrated that cornu ammonis 1 neuronal death induced in this paradigm was programmed necrosis. No significant activation of caspase-3 after injury was detected by Western blot and immunohistochemistry. Treatment with 3-MA provided time-dependent protection against cornu ammonis 1 neuronal death at 7 days of reperfusion when it was administered before ischemia; administration 60 minutes after reperfusion was not beneficial. The redistribution of the lysosomal enzyme cathepsin B after injury was inhibited by 3-MA administered before ischemia, suggesting that this might be another important mechanism for the protective effect of 3-MA in ischemic neuronal injury.     

15d-PGJ_2对神经元氧糖剥夺/复氧损伤的影响

目的观察过氧化物酶体增殖物激活受体γ(PPAR-γ)激动剂15d-PGJ_2对神经元氧糖剥夺/复氧(OGD/R)损伤的影响,并探讨其可能的作用机制。方法采用原代培养的小鼠胎鼠大脑皮质神经元,建立OGD/R损伤模型。将培养的神经元细胞进行分组,Western blot检测各组神经元内LC3-1、LC3-Ⅱ、Bcl-2、Beclin1、AMPK、m TOR及p70S6K等蛋白的表达;MTT法检测细胞活性,乳酸脱氢酶(LDH)漏出率测定细胞毒性。结果 OGD/R损伤6 h、12 h及24 h后神经元LC3-Ⅱ、Beclin 1表达明显增高,而p62表达持续下降,神经元的生存率明显下降,LDH漏出率增加。15d-PGJ_2可显著降低LC3-Ⅱ和Beclin 1表达水平,改善神经元的生存率及降低LDH的漏出率。OGD/R后Bcl-2的蛋白表达水平均明显减少,而Beclin 1表达则显着增加。15d-PGJ_2预处理显著增加OGD/R 24 h的Bcl-2表达量。利用Bcl-2 siRNA或scRNA转染神经元细胞发现,Bcl-2 siRNA可消除15d-PGJ_2对OGD/R后各时间点的抑制效应。结论 15d-PGJ_2能够有效地对神经元OGD/R损伤起到保护作用,其机制可能是通过上调Bcl-2的表达进而抑制自噬的发生实现的。     

Preservation of neuronal functions by exosomes derived from different human neural cell types under ischemic conditions

Stem cell‐based therapies have been reported in protecting cerebral infarction‐induced neuronal dysfunction and death. However, most studies used rat/mouse neuron as model cell when treated with stem cell or exosomes. Whether these findings can be translated from rodent to humans has been in doubt. Here, we used human embryonic stem cell‐derived neurons to detect the protective potential of exosomes against ischemia. Neurons were treated with in vitro oxygen–glucose deprivation (OGD) for 1 h. For treatment group, different exosomes were derived from neuron, embryonic stem cell, neural progenitor cell and astrocyte differentiated from H9 human embryonic stem cell and added to culture medium 30 min after OGD (100 μg/mL). Western blotting was performed 12 h after OGD, while cell counting and electrophysiological recording were performed 48 h after OGD. We found that these exosomes attenuated OGD‐induced neuronal death, Mammalian target of rapamycin (mTOR), pro‐inflammatory and apoptotic signaling pathway changes, as well as basal spontaneous synaptic transmission inhibition in varying degrees. The results implicate the protective effect of exosomes on OGD‐induced neuronal death and dysfunction in human embryonic stem cell‐derived neurons, potentially through their modulation on mTOR, pro‐inflammatory and apoptotic signaling pathways.     

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.     

Necroptosis,a novel form of caspase‐independent cell death,contributes to neuronal damage in a retinal ischemia‐reperfusion injury model

Necroptosis is programmed necrosis triggered by death receptor signaling. We investigated whether necroptosis contributes to neuronal damage and functional impairment in a model of retinal ischemia. Methods: Sprague‐Dawley rats were subjected to raised intra‐ocular pressure for 45 min and received intravitreal injections of the specific necroptosis inhibitor, Nec‐1, its inactive analogue (Nec‐1i) or vehicle. Seven days after ischemia, ERGs were performed and then the eyes were enucleated for histological analysis. In other animals, retinas were subjected to propodium iodide, TUNEL staining or Western Blotting and probed with anti‐LC‐3 antibody. Results: Retinal ischemia resulted in selective neuronal degeneration of the inner layers. Pretreatment with Nec‐1 led to significant preservation in thickness and histoarchitecture of the inner retina and functional improvement compared with vehicle‐treated controls. Pretreatment with Nec‐1i did not provide histological or functional protection. Post‐treatment with Nec‐1 also significantly attenuated the ERG b‐wave reduction compared with ischemic vehicle controls. Nec‐1 had no effect on the number of caspase or TUNEL‐labelled cells in the ischemic retina but did inhibit the induction of LC‐3 II and reduced the number of PI‐labelled cells after ischemia. Conclusion: Necroptosis is an important mode of neuronal cell death and involves autophagy in a model of retinal ischemia. © 2009 Wiley‐Liss, Inc.     

毛柳苷在脑缺血再灌注后抑制神经元自噬及神经保护作用研究

目的 通过动物模型和体外细胞培养探索毛柳苷（salidroside，SAL）对脑缺血再灌注损伤的神经保护作用及其可能机制。方法 36只雄性C57BL/6J小鼠随机分为假手术组（12只）、溶剂组（12只）和SAL组（12只）。线栓法制作小鼠右侧大脑中动脉闭塞模型，缺血1 h后再灌注，而后即刻尾静脉注射150 μL SAL（剂量10 mg/kg，每只实际用量0.22 mg），溶剂组注射同等体积磷酸盐缓冲液，假手术组不给药。缺血再灌注后24 h进行小鼠神经功能缺损评分，神经元特异核蛋白（neuronal nuclei，NeuN）染色统计脑梗死率。取溶剂组小鼠全脑冰冻切片，自噬标记物微管相关蛋白1轻链3B（microtubule associated protein 1 light chain 3 beta，LC3B）分别与NeuN、胶质纤维酸性蛋白（glial fibrillary acidic protein，GFAP）免疫荧光共染，观察LC3B与梗死周边区神经元、星形胶质细胞共定位情况，以明确缺血再灌注后24 h产生自噬活动的主要部位。自噬标记物苄氯素1（Beclin-1）、自噬相关蛋白3（autophagy related protein 3，Atg3）分别与NeuN免疫荧光共染，进一步观察梗死周边区神经元自噬水平变化情况，并统计Beclin-1+/NeuN+、Atg3+/NeuN+细胞在单位面积（1 mm2）内的个数。原代大鼠神经元培养10 d后进行氧糖剥夺（oxygen glucose deprivation，OGD），将细胞分为以下6组：对照（control，CON）组、OGD 1 h后复氧1 h（OGD 1 h）组、OGD 1 h后复氧4 h（OGD 4 h）组、SAL组、OGD 1 h后复氧1 h/全程SAL治疗（OGD+SAL 1 h）组、OGD 1 h后复氧4 h/全程SAL治疗（OGD+SAL 4 h）组，CON组和SAL组不进行OGD，SAL剂量50 μmol/L。采用免疫印迹法检测各组细胞中自噬标记物LC3B、Beclin-1、Atg3、Atg5和Atg7的相对表达量；CON组、OGD 4 h组细胞进行LC3B、NeuN免疫荧光共染，观察OGD后神经元内LC3B表达变化情况，并统计LC3B+/NeuN+细胞在单位面积（1 mm2）内的个数。结果 缺血再灌注后24 h，SAL组小鼠神经功能缺损评分（5.8±1.4分 vs. 7.1±1.4分，P=0.0332）和脑梗死率（28.7%±9.7% vs. 39.9%±9.4%，P=0.0038）均低于溶剂组。溶剂组LC3B与梗死周边区神经元有共定位，与星形胶质细胞无共定位。SAL组Beclin-1+/NeuN+（312.4±45.6个/平方毫米 vs. 471.2±50.3个/平方毫米，P=0.0121）、Atg3+/NeuN+（322.5±26.5个/平方毫米 vs. 491.3±42.1个/平方毫米，P=0.0013）细胞数量少于溶剂组。大鼠原代神经元免疫印迹结果显示，OGD 1 h组（1.096±0.004 vs. 1.000±0.000，P=0.0174）、OGD 4 h组（1.213±0.019 vs. 1.000±0.000，P<0.0001）LC3B相对表达量高于CON组；OGD+SAL 4 h组LC3B、Beclin-1、Atg3相对表达量低于OGD 4 h组（0.833±0.029 vs. 1.213±0.019，P<0.0001，0.579±0.081 vs. 1.152±0.144，P<0.0001，0.726±0.182 vs. 1.091±0.177，P=0.0211）；OGD+SAL 4 h组LC3B、Beclin-1相对表达量低于OGD+SAL 1 h组（0.833±0.029 vs. 1.046±0.063，P<0.0001；0.579±0.081 vs. 0.921±0.09，P=0.0030）。CON组LC3B+/NeuN+细胞数少于OGD 4 h组（51.9±18.7个/平方毫米 vs. 584.2±34.5个/平方毫米，P=0.0002）。结论 SAL可能通过抑制神经元自噬在急性脑缺血再灌注损伤中发挥神经保护作用。     

Role of salt-induced kinase 1 in androgen neuroprotection against cerebral ischemia

Androgens within physiological ranges protect castrated male mice from cerebral ischemic injury. Yet, underlying mechanisms are unclear. Here, we report that, after middle cerebral artery occlusion (MCAO), salt-induced kinase 1 (SIK1) was induced by a potent androgen—dihydrotestosterone (DHT) at protective doses. To investigate whether SIK1 contributes to DHT neuroprotection after cerebral ischemia, we constructed lentivirus-expressing small interference RNA (siRNA) against SIK1. The SIK1 knockdown by siRNA exacerbated oxygen–glucose deprivation (OGD)-induced cell death in primary cortical neurons, suggesting that SIK1 is an endogenous neuroprotective gene against cerebral ischemia. Furthermore, lentivirus-mediated SIK1 knockdown increased both cortical and striatal infarct sizes in castrated mice treated with a protective dose of DHT. Earlier studies show that SIK1 inhibits histone deacetylase (HDAC) activities by acting as a class IIa HDAC kinase. We observed that SIK1 knockdown decreased histone H3 acetylation in primary neurons. The SIK1 siRNA also exacerbated OGD-induced neuronal death in the presence of trichostatin A (TSA), an HDAC inhibitor, and decreased histone H3 acetylation at 4 hours reoxygenation in TSA-treated neurons. Finally, we showed that DHT at protective doses prevented ischemia-induced histone deacetylation after MCAO. Our finding suggests that SIK1 contributes to neuroprotection by androgens within physiological ranges by inhibiting histone deacetylation.     

Micro‐RNA‐30a regulates ischemia‐induced cell death by targeting heat shock protein HSPA5 in primary cultured cortical neurons and mouse brain after stroke

Micro‐RNAs (miRs) have emerged as key gene regulators in many diseases, including stroke. We recently reported that miR‐30a protects N2A cells against ischemic injury, in part through enhancing beclin 1‐mediated autophagy. The present study explores further the involvement of miR‐30a in ischemia‐induced apoptosis and its possible mechanisms in primary cortical neurons and stroked mouse brain. We demonstrate that miR‐30a level is significantly decreased in cortical neurons after 1‐hr oxygen–glucose deprivation (OGD)/24‐hr reoxygenation. Overexpression of miR‐30a aggravated the OGD‐induced neuronal cell death, whereas inhibition of miR‐30a attenuated necrosis and apoptosis as determined by 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐di‐phenyl‐2H‐tetrazolium bromide, lactate dehydrogenase, TUNEL, and cleaved caspase‐3. The amount of HSPA5 protein, which is predicted to be a putative target of miR‐30a by TargetScan, could be reduced by pre‐miR‐30a, whereas it was increased by anti‐miR‐30a. Furthermore, the luciferase reporter assay confirmed that miR‐30a directly binds to the predicted 3′‐UTR target sites of the hspa5 gene. The cell injury regulated by miR‐30a in OGD‐treated cells could be aggravated by HSPA5 siRNA. We also observed an interaction of HSPA5 and caspase‐12 by coimmunoprecipitation and speculate that HSPA5 might be involved in endoplasmic reticulum stress‐induced apoptosis. In vivo, reduced miR‐30a increased the HSPA5 level and attenuated ischemic brain infarction in focal ischemia‐stroked mice. Downregulation of miR‐30a could prevent neural ischemic injury through upregulating HSPA5 protein expression, and decreased ER stress‐induced apoptosis might be one of the mechanisms underlying HSPA5‐mediated neuroprotection. © 2015 Wiley Periodicals, Inc.     

Increased p38 mitogen-activated protein kinase signaling is involved in the oxidative stress associated with oxygen and glucose deprivation in neonatal hippocampal slice cultures

The pathological basis of neonatal hypoxia–ischemia (HI) brain damage is characterized by neuronal cell loss. Oxidative stress is thought to be one of the main causes of HI‐induced neuronal cell death. The p38 mitogen‐activated protein kinase (MAPK) is activated under conditions of cell stress. However, its pathogenic role in regulating the oxidative stress associated with HI injury in the brain is not well understood. Thus, this study was conducted to examine the role of p38 MAPK signaling in neonatal HI brain injury using neonatal rat hippocampal slice cultures exposed to oxygen/glucose deprivation (OGD). Our results indicate that OGD led to a transient increase in p38 MAPK activation that preceded increases in superoxide generation and neuronal death. This increase in neuronal cell death correlated with an increase in the activation of caspase‐3 and the appearance of apoptotic neuronal cells. Pre‐treatment of slice cultures with the p38 MAPK inhibitor, SB203580, or the expression of an antisense p38 MAPK construct only in neuronal cells, through a Synapsin I‐1‐driven adeno‐associated virus vector, inhibited p38 MAPK activity and exerted a neuroprotective effect as demonstrated by decreases in OGD‐mediated oxidative stress, caspase activation and neuronal cell death. Thus, we conclude that the activation of p38 MAPK in neuronal cells plays a key role in the oxidative stress and neuronal cell death associated with OGD.     

p53 induction contributes to excitotoxic neuronal death in rat striatum through apoptotic and autophagic mechanisms

The present study sought to investigate mechanisms by which p53 induction contributes to excitotoxic neuronal injury. Rats were intrastriatally administered the N‐methyl‐d ‐aspartate (NMDA) receptor agonist quinolinic acid (QA), the changes in the expression of p53 and its target genes involved in apoptosis and autophagy, including p53‐upregulated modulator of apoptosis (PUMA), Bax, Bcl‐2, damage‐regulated autophagy modulator (DRAM) and other autophagic proteins including microtubule‐associated protein 1 light chain 3 (LC3) and beclin 1 were assessed. The contribution of p53‐mediated autophagy activation to apoptotic death of striatal neurons was assessed with co‐administration of the nuclear factor‐kappaB (NF‐κB) inhibitor SN50, the p53 inhibitor Pifithrin‐alpha (PFT‐α) or the autophagy inhibitor 3‐methyladenine (3‐MA). The increased formation of autophagosomes and secondary lysosomes were observed with transmission electron microscope after excitotoxin exposure. QA induced increases in the expression of p53, PUMA, Bax and a decrease in Bcl‐2. These changes were significantly attenuated by pre‐treatment with SN50, PFT‐α or 3‐MA. SN50, PFT‐α or 3‐MA also reversed QA‐induced upregulation of DRAM, the ratio of LC3‐II/LC3‐I and beclin 1 protein levels in the striatum. QA‐induced internucleosomal DNA fragmentation and loss of striatal neurons were robustly inhibited by SN50, PFT‐α or 3‐MA. These results suggest that overstimulation of NMDA receptors can induce NF‐κB‐dependent expression of p53. p53 participates in excitotoxic neuronal death probably through both apoptotic and autophagic mechanisms.     

Thrombin in ischemic neuronal death

Thrombin plays a role in cerebral ischemia as rats subjected to focal cerebral ischemia were protected by the intracerebral injection of hirudin, a selective thrombin inhibitor. To separate the roles of thrombin in cell death and in coagulation, we have used an in vitro approach to test the effect of hirudin and of protease nexin-1 (PN-1), a cerebral thrombin inhibitor, on neuronal ischemia. Rat organotypic hippocampal slice cultures were subjected to oxygen (5%) and glucose (1 mmol/L) deprivation (OGD) during 30 min. Hirudin or PN-1 administered after OGD significantly prevented neuronal death in the CA1 region. After 24 h, there was a marked increase in thrombin immunoreactivity on Western blots. Thrombin therefore contributes to ischemic damage in neural tissue in vitro.     

The involvement of cellular prion protein in the autophagy pathway in neuronal cells

Apoptosis and autophagy are main mechanisms of neuronal death involved in prion diseases. Serum deprivation can induce both pathways to cell death in various types of cells. To investigate whether PrP(C) is involved in autophagy pathway, we analyzed the level of microtubule-associated protein 1 light chain 3 (LC3), an autophagy marker, by monitoring the conversion from LC3-I into LC3-II in Zürich I Prnp(-/-) hippocampal neuronal cells. We found that the expression level of LC3-II was increased in Prnp(-/-) compared to wild-type cells under serum deprivation. In electron microscopy, increased accumulation of autophagosomes in Prnp(-/-) cells was correlated with the increase in levels of LC3-II. Interestingly, this up-regulated autophagic activity was retarded by the introduction of PrP(C) into Prnp(-/-) cells but not by the introduction of PrP(C) lacking octapeptide repeat region. Thus, the octapeptide repeat region of PrP(C) may play a pivotal role in the control of autophagy exhibited by PrP(C) in neuronal cells.     

去铁敏预处理通过增加缺氧诱导因子1α和促红细胞生成素的表达减轻大鼠缺血性脑损伤

目的观察去铁敏（Desferoxamine,DFO）预处理后大鼠脑组织和体外培养神经元中缺氧诱导因子1α（hypoxia inducible factor1α,HIF-1α）和促红细胞生成素（erythropoietin,EPO）表达的变化,探讨预处理是否对体内及体外的脑缺血损伤的具有保护效应。方法去铁敏预处理大鼠后不同时间点制作大脑中动脉阻塞（middle cerebral artery occlusion,MCAO）模型,术后24h后处死动物。采用神经功能评分（neurological severity scores,NSS）和计算梗死体积（TTC染色）评价DFO的脑保护效应,细胞活力测定评价DFO对缺氧缺糖条件下（oxygen-glucosede privation,OGD）皮层神经元的保护效应。免疫荧光染色检测HIF-1α和EPO蛋白表达情况。结果与生理盐水对照组比较,去铁敏预处理后2d,MCAO大鼠出现梗塞面积缩小,神经功能损伤减轻,在预处理后3d达到高峰,7d仍然有效,14d去铁敏预处理的保护效应消失。去铁敏对OGD神经元同样具有神经保护作用：与未进行预处理的神经元细胞相比,预处理后8h的细胞活力增加23％,12h增加34％,24h增加40％,36h增加48％,48h增加56％（P〈0．05）。免疫荧光染色发现,大鼠脑组织的HIF-1α和EPO在去铁敏预处理后3d及7d表达上调;皮层神经元细胞的HIF-1α和EPO在去铁敏预处理后36h及48h表达上调。结论去铁敏预处理有确切有效的脑保护效应,不仅可以预防脑缺血损伤,对体外培养的OGD皮层神经元细胞损伤也具有保护作用,其机制可能与脑神经细胞的HIF-1α和EPO蛋白表达增加有关。     

Cell death in primary cultures of mouse neurons and astrocytes during exposure to and ‘recovery’ from hypoxia, substrate deprivation and simulated ischemia

Effects of hypoxia, substrate deprivationand simulated ischemia (combined hypoxia and substrate deprivation) on cell survival during the insult itself and during a 24 h ‘recovery’ period were studied in primary cultures of mouse astrocytes and in cerebral cortical neuronal-astrocytic co-cultures. Cell death was determined by release of the cytosolic high molecular enzyme lactate dehydrogenase (LDH) as well as morphologically (retention of staining with rhodamine 123 and lack of staining with propidium iodide as an indicator of live cells). Glutamate concentrations were measured in the incubation media at the end of the metabolic insults. Astrocytes were very resistant to hypoxia, but less so to simulated ischemia; under both conditions the glutamate concentrations in the media remained low. Cerebral cortical neurons were almost equally susceptible to damage by hypoxia and by stimulated ischemia, although hypoxia had a faster deleterious effects on some of the neurons and simulated ischemia during a long-term insult (9 h) killed all neurons, whereas a non-negligible neuronal subpopulation survived 9 h of hypoxia. Neuronal cell death after long-term hypoxia (but not after simulated ischemia) was correlated with high concentrations of glutamate in the incubation media. After certain insults, most notably relatively short lasting simulated ischemia (3 h) in neurons (which caused no increased cell death during the insult), there was a large release of LDH during the ‘recovery’ period.     

Regional and time‐dependent neuroprotective effect of hypothermia following oxygen‐glucose deprivation

The neuroprotective effect of hypothermia has been demonstrated in in vivo and in vitro models of cerebral ischemia. In regard to the hippocampus, previous studies have mainly focused on CA1 pyramidal neurons, which are very vulnerable to ischemia. But the dentate gyrus (DG), in which neuronal proliferation occurs, can also be damaged by ischemia. In this study, we explored the neuroprotective effect of postischemic hypothermia in different areas of the hippocampus after mild or severe ischemia. Organotypic hippocampal slice cultures were prepared from 6‐ to 8‐day‐old rats and maintained for 12 days. Cultures were exposed to 25 or 35 min of oxygen and glucose deprivation (OGD). Neuronal damage was quantified after 6, 24, 48, and 72 h by propidium iodide fluorescence. Mild hypothermia (33°C) was induced 1 h after the end of OGD and was maintained for a period of 24 h. Short OGD produced delayed neuronal damage in the CA1 area and in the DG and to a lesser extend in the CA3 area. Damage in CA1 pyramidal cells was totally prevented by hypothermia whereas neuroprotection was limited in the DG. Thirty‐five‐minute OGD induced more rapid and more severe cell death in the three regions. In this case, hypothermia induced 1 h after OGD was unable to protect CA1 pyramidal cells whereas hypothermia induced during OGD was able to prevent cell loss. This study provides evidence that neuroprotection by hypothermia is limited to specific areas and depends on the severity of the ischemia. © 2014 Wiley Periodicals, Inc.     

Postconditioning mitigates cell death following oxygen and glucose deprivation in PC12 cells and forebrain reperfusion injury in rats

Postconditioning mitigates ischemia‐induced cellular damage via a modified reperfusion procedure. Mitochondrial permeability transition (MPT) is an important pathophysiological change in reperfusion injury. This study explores the role of MPT modulation underlying hypoxic postconditioning (HPoC) in PC12 cells and studies the neuroprotective effects of ischemic postconditioning (IPoC) on rats. Oxygen‐glucose deprivation (OGD) was performed for 10 hr on PC12 cells. HPoC was induced by three cycles of 10‐min reoxygenation/10‐min rehypoxia after OGD. The MPT inhibitor N‐methyl‐4‐isoleucine cyclosporine (NIM811) and the MPT inducer carboxyatractyloside (CATR) were administered to selective groups before OGD. Cellular death was evaluated by flow cytometry and Western blot analysis. JC‐1 fluorescence signal was used to estimate the mitochondrial membrane potential (△Ψ_m). Transient global cerebral ischemia (tGCI) was induced via the two‐vessel occlusion and hypotension method in male Sprague Dawley rats. IPoC was induced by three cycles of 10‐sec reperfusion/10‐sec reocclusion after index ischemia. HPoC and NIM811 administration attenuated cell death, cytochrome c release, and caspase‐3 activity and maintained △Ψ_m of PC12 cells after OGD. The addition of CATR negated the protection conferred by HPoC. IPoC reduced neuronal degeneration and cytochrome c release and cleaved caspase‐9 expression of hippocampal CA1 neurons in rats after tGCI. HPoC protected PC12 cells against OGD by modulating the MPT. IPoC attenuated degeneration of hippocampal neurons after cerebral ischemia. © 2014 Wiley Periodicals, Inc.     

Expression of the RNA-binding protein TIAR is increased in neurons after ischemic cerebral injury

T-cell restricted intracellular antigen-related protein (TIAR) is an RNA recognition motif-type RNA-binding protein that has been implicated in the apoptotic death of T-lymphocytes and retinal pigment epithelial cells. Western blots prepared with a monoclonal antibody against TIAR showed expression in normal rat hippocampus, and induction by 15 min of global cerebral ischemia. This increased expression was evident at 8 hr after ischemia and maximal at 24 hr, whereas expression at 72 hr was reduced below basal levels. Expression of TIAR protein was also increased in parietal cortex 6 and 24 hr after 90 min of focal cerebral ischemia induced by middle cerebral artery (MCA) occlusion, as well as in cultured cortical neurons and astroglia after exposure to hypoxia in vitro. Immunocytochemistry showed that increased expression of TIAR occurred mainly in the CA1 sector of hippocampus 24 hr after global ischemia, and in cortical and striatal neurons 24 hr after 20 or 90 min of focal ischemia. Double-labeling studies showed that TIAR protein expression was co-localized with DNA damage in neuronal cells. The findings suggest that TIAR may be involved in neuronal cell death after cerebral ischemic injury.     

A dietary polyphenol resveratrol acts to provide neuroprotection in recurrent stroke models by regulating AMPK and SIRT1 signaling,thereby reducing energy requirements during ischemia

Polyphenol resveratrol (RSV) has been associated with Silent Information Regulator T1 (SIRT1) and AMP‐activated protein kinase (AMPK) metabolic stress sensors and probably responds to the intracellular energy status. Our aim here was to investigate the neuroprotective effects of RSV and its association with SIRT1 and AMPK signaling in recurrent ischemia models. In this study, elderly male Wistar rats received a combination of two mild transient middle cerebral artery occlusions (tMCAOs) as an in vivo recurrent ischemic model. Primary cultured cortical neuronal cells subjected to combined oxygen–glucose deprivation (OGD) were used as an in vitro recurrent ischemic model. RSV administration significantly reduced infarct volumes, improved behavioral deficits and protected neuronal cells from cell death in recurrent ischemic stroke models in vivo and in vitro. RSV treatments significantly increased the intracellular NAD⁺/NADH ratio, AMPK and SIRT1 activities, decreased energy assumption and restored cell energy ATP level. SIRT1 and AMPK inhibitors and specific small interfering RNA (siRNA) for SIRT1 and AMPK significantly abrogated the neuroprotection induced by RSV. AMPK‐siRNA and inhibitor decreased SIRT1 activities; however, SIRT1‐siRNA and inhibitor had no impact on phospho‐AMPK (p‐AMPK) levels. These results indicated that the neuroprotective effects of RSV increased the intracellular NAD⁺/NADH ratio as well as AMPK and SIRT1 activities, thereby reducing energy ATP requirements during ischemia. SIRT1 is a downstream target of p‐AMPK signaling induced by RSV in the recurrent ischemic stroke model.