Suppression of REDD1 attenuates oxygen glucose deprivation/reoxygenation-evoked ischemic injury in neuron by suppressing mTOR-mediated excessive autophagy

Cerebral ischemia/reperfusion (I/R) typically occurs after mechanical thrombectomy to treat ischemic stroke, generation of reactive oxygen species (ROS) after reperfusion may result in neuronal insult, ultimately leading to disability and death. Regulated in development and DNA damage responses 1 (REDD1) is a conserved stress response protein under various pathogenic conditions. Recent research confirms the controversial role of REDD1 in injury processes. Nevertheless, the role of REDD1 in cerebral I/R remains poorly defined. In the current study, increased expression of REDD1 was observed in neurons exposed to simulated I/R via oxygen glucose deprivation/reoxygenation (OGD/R) treatment. Knockdown of REDD1 enhanced OGD/R-inhibited cell viability, but suppressed lactate dehydrogenase (LDH) release in neurons upon OGD/R. Simultaneously, suppression of REDD1 also antagonized OGD/R-evoked cell apoptosis, Bax expression, and caspase-3 activity. Intriguingly, REDD1 depression abrogated neuronal oxidative stress under OGD/R condition by suppressing ROS, MDA generation, and increasing antioxidant SOD levels. Further mechanism analysis corroborated the excessive activation of autophagy in neurons upon OGD/R with increased expression of autophagy-related LC3 and Beclin-1, but decreased autophagy substrate p62 expression. Notably, REDD1 inhibition reversed OGD/R-triggered excessive neuronal autophagy. More importantly, depression of REDD1 also elevated the expression of p-mTOR. Preconditioning with mTOR inhibitor rapamycin engendered not only a reduction in mTOR activation, but also a reactivation of autophagy in REDD1 knockdown-neurons upon OGD/R. In addition, blocking the mTOR pathway muted the protective roles of REDD1 inhibition against OGD/R-induced neuron injury and oxidative stress. Together these data suggested that REDD1 may regulate I/R-induced oxidative stress injury in neurons by mediating mTOR-autophagy signaling, supporting a promising therapeutic strategy against brain ischemic diseases.     

Down-Regulation of miRNA-30a Alleviates Cerebral Ischemic Injury Through Enhancing Beclin 1-Mediated Autophagy

The understanding of molecular mechanism underlying ischemia/reperfusion-induced neuronal death and neurological dysfunction may provide therapeutic targets for ischemic stroke. The up-regulated miRNA-30a among our previous identified 19 MicroRNAs (miRNAs) in mouse brain after 6 h middle cerebral artery occlusion (MCAO) could negatively regulate Beclin 1 messenger RNA (mRNA) resulting in decreased autophagic activity in tumor cells and cardiomyocytes, but its role in ischemic stroke is unclear. In this study, the effects of miRNA-30a on ischemic injury in N2A cells and cultured cortical neurons after oxygen glucose deprivation (OGD), and mouse brain with MCAO-induced ischemic stroke were evaluated. The results showed that miRNA-30a expression levels were up regulated in the brain of mice after 6 h MCAO without reperfusion, but significantly down regulated in the peri-infarct region of mice with 1 h MCAO/24 h reperfusion and in N2A cells after 1 h OGD/6–48 h reoxygenation. Both the conversion ratio of microtubule-associated protein 1 light chain 3 (LC3)-II/LC3-I and Beclin 1 protein level increased in N2A cells and cultured cortical neurons following 1 h OGD/24 h reoxygenation. The down-regulated miRNA-30a could attenuate 1 h OGD/24 h reoxygenation-induced ischemic injury in N2A cells and cultured cortical neurons through enhancing Beclin 1-mediated autophagy, as miRNA-30a recognized the 3′-untranslated region of beclin 1 mRNA to negatively regulate Beclin 1-protein level via promoting beclin 1 messenger RNA (mRNA) degradation, and Beclin 1 siRNA abolished anti-miR-30a-induced neuroprotection in 1 h OGD/24 h reoxygenation treated N2A cells. In addition, anti-miR-30a attenuated the neural cell loss and improved behavioral outcome of mice with ischemic stroke. These results suggested that down-regulation of miRNA-30a alleviates ischemic injury through enhancing beclin 1-mediated autophagy, providing a potential therapeutic target for ischemic stroke.     

Silibinin activates AMP-activated protein kinase to protect neuronal cells from oxygen and glucose deprivation-re-oxygenation

In this study, we explored the cytoprotective potential of silibinin against oxygen–glucose deprivation (OGD)-induced neuronal cell damages, and studied underling mechanisms. In vitro model of ischemic stroke was created by keeping neuronal cells (SH-SY5Y cells and primary mouse cortical neurons) in an OGD condition followed by re-oxygenation. Pre-treatment of silibinin significantly inhibited OGD/re-oxygenation-induced necrosis and apoptosis of neuronal cells. OGD/re-oxygenation-induced reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) reduction were also inhibited by silibinin. At the molecular level, silibinin treatment in SH-SY5Y cells and primary cortical neurons led to significant AMP-activated protein kinase (AMPK) signaling activation, detected by phosphorylations of AMPKα1, its upstream kinase liver kinase B1 (LKB1) and the downstream target acetyl-CoA Carboxylase (ACC). Pharmacological inhibition or genetic depletion of AMPK alleviated the neuroprotective ability of silibinin against OGD/re-oxygenation. Further, ROS scavenging ability by silibinin was abolished with AMPK inhibition or silencing. While A-769662, the AMPK activator, mimicked silibinin actions and suppressed ROS production and neuronal cell death following OGD/re-oxygenation. Together, these results show that silibinin-mediated neuroprotection requires activation of AMPK signaling.     

NOX2 deficiency ameliorates cerebral injury through reduction of complexin II-mediated glutamate excitotoxicity in experimental stroke

Although NADPH oxidase (NOX)-mediated oxidative stress is considered one of the major mechanisms triggering the pathogenic actions of ischemic stroke and very recent studies have indicated that NADPH oxidase is a major source of reactive oxygen species (ROS) production controlling glutamate release, how neuronal NADPH oxidase activation is coupled to glutamate release is not well understood. Therefore, in this study, we used an in vivo transient middle cerebral artery occlusion model and in vitro primary cell cultures to test whether complexins, the regulators of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes necessary for vesicle fusion, are associated with NOX2-derived ROS and contribute to glutamate-mediated excitotoxicity in ischemic stroke. In this study, we first identified the upregulation of complexin II in the ischemic brain and evaluated its potential role in ischemic stroke showing that gene silencing of complexin II ameliorated cerebral injury as evidenced by reduced infarction volume, neurological deficit, and neuron necrosis accompanied by decreased glutamate levels, consistent with the results from NOX2^−/− mice with ischemic stroke. We further demonstrated that complexin II expression was mediated by NOX2 in primary cultured neurons subjected to oxygen–glucose deprivation (OGD) and contributed to OGD-induced glutamate release and neuron necrosis via SNARE signaling. Taken together, these findings for the first time provide evidence that complexin II is a central target molecule that links NADPH oxidase-derived ROS to glutamate-mediated neuronal excitotoxicity in ischemic stroke.     

Ischemic Preconditioning Mediates Neuroprotection against Ischemia in Mouse Hippocampal CA1 Neurons by Inducing Autophagy

The hippocampal CA1 region is sensitive to hypoxic and ischemic injury but can be protected by ischemic preconditioning (IPC). However, the mechanism through which IPC protects hippocampal CA1 neurons is still under investigation. Additionally, the role of autophagy in determining the fate of hippocampal neurons is unclear. Here, we examined whether IPC induced autophagy to alleviate hippocampal CA1 neuronal death in vitro and in vivo with oxygen glucose deprivation (OGD) and bilateral carotid artery occlusion (BCCAO) models. Survival of hippocampal neurons increased from 51.5% ± 6.3% in the non-IPC group (55 min of OGD) to 77.3% ± 7.9% in the IPC group (15 min of OGD, followed by 55 min of OGD 24 h later). The number of hippocampal CA1 layer neurons increased from 182 ± 26 cells/mm² in the non-IPC group (20 min of BCCAO) to 278 ± 55 cells/mm² in the IPC group (1 min × 3 BCCAO, followed by 20 min of BCCAO 24 h later). Akt phosphorylation and microtubule-associated protein light chain 3 (LC3)-II/LC3-I expression were increased in the preconditioning group. Moreover, the protective effects of IPC were abolished only by inhibiting the activity of autophagy, but not by blocking the activation of Akt in vitro. Using in vivo experiments, we found that LC3 expression was upregulated, accompanied by an increase in neuronal survival in hippocampal CA1 neurons in the preconditioning group. The neuroprotective effects of IPC on hippocampal CA1 neurons were completely inhibited by treatment with 3-MA. In contrast, hippocampal CA3 neurons did not show changes in autophagic activity or beneficial effects of IPC. These data suggested that IPC may attenuate ischemic injury in hippocampal CA1 neurons through induction of Akt-independent autophagy.     

N-Myc Downstream-Regulated Gene 2 (Ndrg2) Is Involved in Ischemia–Hypoxia-Induced Astrocyte Apoptosis: a Novel Target for Stroke Therapy

Nearly all clinical trials that have attempted to develop effective strategies against ischemic stroke have failed, excluding those for thrombolysis, and most of these trials focused only on preventing neuronal loss. However, astrocytes have gradually become a target for neuroprotection in stroke. In previous studies, we showed that the newly identified molecular N-myc downstream-regulated gene 2 (Ndrg2) is specifically expressed in astrocytes in the brain and involved in some neurodegenerative diseases. However, the role of NDRG2 in ischemic stroke remained unclear. In this study, we investigated the role of NDRG2 in middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia and in oxygen–glucose deprivation (OGD)-induced cellular apoptosis in the M1800 astrocyte cell line. NDRG2 mRNA and protein expression began to increase at 6 and 2 h after reperfusion and peaked at 24 h in the ischemic penumbra and in M1800 cells, as detected by RT-PCR and Western blotting. Double immunofluorescence staining showed that the number of apoptotic cells increased as the NDRG2-positive signal increased and that the NDRG2 signal was sometimes co-localized with TUNEL-positive cells and translocated from the cytoplasm to the nucleus in both the ischemic penumbra and the M1800 cells. Using a lentivirus, we successfully constructed two stable astrocytic cell lines in which NDRG2 expression was significantly up- or down-regulated. NDRG2 silencing had a proliferative effect and reduced the percentage of apoptotic cells, reactive oxygen species (ROS) production, and cleaved Caspase-3 protein expression following OGD, whereas NDRG2 over-expression had the opposite effects. In conclusion, NDRG2 is involved in astrocyte apoptosis following ischemic–hypoxic injury, and inhibiting NDRG2 expression significantly reduces ROS production and astrocyte apoptosis. These findings provide insight into the role of NDRG2 in ischemic–hypoxic injury and provide potential targets for future clinical therapies for stroke.     

白藜芦醇对氧糖剥夺／再灌注损伤的PCI2细胞的保护作用及其作用机制

目的：探讨白藜芦醇对氧糖剥夺／再灌注（OGD／R）损伤的PCI2细胞的保护作用及其机制。方法：体外培养PCI2细胞,分为对照组,白藜芦醇组,OGD／R组及OGD／R＋白藜芦醇组。以改良的噻唑蓝法测定细胞活性,采用AnnexinV—FITC／PI双染法检测细胞的凋亡率,用双氯罗丹明（DHR）检测细胞内活性氧簇（Ros）的水平,采用蛋白印迹法（westemblot）分析SIRTl的蛋白表达情况。结果：与对照组相比,经过OGD／R损伤后,细胞活力显著降低。而在OGD／R的同时给予10μmol／L的白藜芦醇处理。可以明显提高细胞活力。流式细胞仪检测发现,10μmol／L的白藜芦醇可以显著地减少OGD／R引起的细胞凋亡,抑制细胞内的ROS产生。westemblot的结果提示,与对照组比较,白藜芦醇可提高SIRTl的蛋白表达水平。结论：白藜芦醇可以通过抑制ROS的产生和上调SIRTl的表达等机制而发挥其对抗氧糖剥夺／再灌注损伤的神经保护性作用。     

Brain edema induced by in vitro ischemia: causal factors and neuroprotection

Decreased cerebral blood flow, hence decreased oxygen and glucose, leads to ischemic brain injury via complex pathophysiological events, including excitotoxicity, mitochondrial dysfunction, increased intracellular Ca2+, and reactive oxygen species (ROS) generation. Each of these could also contribute to cerebral edema, which is the primary cause of patient mortality after stroke. In vitro brain slices are widely used to study ischemia. Here we introduce a slice model to investigate ischemia-induced edema. Significant water gain was induced in coronal slices of rat brain by 5 min of oxygen and glucose deprivation (OGD) at 35 degrees C, with progressive edema formation after return to normoxic, normoglycemic medium. Edema increased with increasing injury severity, determined by OGD duration (5-30 min). Underlying factors were assessed using glutamate-receptor antagonists (AP5/CNQX), blockade of mitochondrial permeability transition [cyclosporin A (CsA) versus FK506], inhibition of Na+/Ca2+ exchange (KB-R7943), and ROS scavengers (ascorbate, Trolox, dimethylthiourea, Tempol). All agents except KB-R7943 and FK506 significantly attenuated edema when applied after OGD; KB-R7943 was effective when applied before OGD. Significantly, complete prevention of ischemia-induced edema was achieved with a cocktail of AP5/CNQX, CsA and Tempo applied after OGD, which demonstrates the involvement of multiple, additive mechanisms. The efficacy of this cocktail further shows the potential value of combination therapies for the treatment of cerebral ischemia.     

Intrinsic Effects of Gold Nanoparticles on Oxygen–Glucose Deprivation/Reperfusion Injury in Rat Cortical Neurons

This study aimed to investigate the potential effects of gold nanoparticles (Au-NPs) on rat cortical neurons exposed to oxygen–glucose deprivation/reperfusion (OGD/R) and to elucidate the corresponding mechanisms. Primary rat cortical neurons were exposed to OGD/R, which is commonly used in vitro to mimic ischemic injury, and then treated with 5- or 20-nm Au-NPs. We then evaluated cell viability, apoptosis, oxidative stress, and mitochondrial respiration in these neurons. We found that 20-nm Au-NPs increased cell viability, alleviated neuronal apoptosis and oxidative stress, and improved mitochondrial respiration after OGD/R injury, while opposite effects were observed for 5-nm Au-NPs. In terms of the underlying mechanisms, we found that Au-NPs could regulate Akt signaling. Taken together, these results show that 20-nm Au-NPs can protect primary cortical neurons against OGD/R injury, possibly by decreasing apoptosis and oxidative stress, while activating Akt signaling and mitochondrial pathways. Our results suggest that Au-NPs may be potential therapeutic agents for ischemic stroke.

     

(S)-ZJM-289, a nitric oxide-releasing derivative of 3-n-butylphthalide, protects against ischemic neuronal injury by attenuating mitochondrial dysfunction and associated cell death

Pharmacological compounds that release nitric oxide (NO) have been recognized as the potential therapeutic agents for acute stroke. (S)-ZJM-289 is a novel NO-releasing derivative of 3-n-butylphthalide (NBP) with enhanced anti-platelet and anti-thrombotic actions. The present study was performed to investigate the neuroprotective effects and related mechanisms of (S)-ZJM-289 on ischemic neuronal injury in vitro and in vivo. Primary cortical neuronal cultures were exposured to oxygen-glucose deprivation followed by recovery (OGD/R), a model of ischemia-like injury, and treated with (S)-ZJM-289 before OGD. In vitro results showed that (S)-ZJM-289 attenuated OGD/R-induced neuronal injury, which was associated with the maintenance of mitochondrial integrity and function by alleviating intracellular calcium overload and reactive oxygen species (ROS) accumulation, preventing mitochondrial membrane depolarization and preserving respiratory chain complexes activities. Moreover, (S)-ZJM-289 treatment suppressed mitochondrial release of cytochrome c (cyt c) and nuclear translocation of apoptosis-inducing factor (AIF), thereby blocking mitochondria-mediated cell death, which may be partially mediated by up-regulation of Hsp70. The neuroprotection by (S)-ZJM-289 was also studied using a model of middle cerebral artery occlusion (MCAO). Oral administration of (S)-ZJM-289 at the onset of reperfusion for 3d significantly reduced the brain infarct size, improved neurological deficit and prevented neuronal loss and apoptosis. In current study, (S)-ZJM-289 appears to be more potent in ischemic neuroprotection than NBP, in particular at the lower doses, which may be due to the synergistic action of NBP and NO. These findings point to that (S)-ZJM-289 could be an attractive alternative to NBP in preventing the process of ischemia/reperfusion (I/R) injury.     

自噬在高压氧预处理预防脊髓缺血再灌注损伤中 的作用机制研究*

目的:研究自噬在高压氧预处理预防脊髓缺血再灌注损伤中的机制。方法:新生大鼠脊髓神经元原代培养,分为对照组(氧糖剥夺)和高压氧(HBO)预处理组。通过应用免疫组织化学、Western blot分析两组LC3-Ⅱ与凋亡相关分子Beclin-1,Bcl-2,Casp-ase-3的表达变化。结果:发现重复高压氧预处理对氧糖剥夺诱导原代培养的脊髓神经元损伤具有明显的保护作用。免疫组化和Western blot显示与对照组相比高压氧预处理显著增加脊髓神经元细胞Bcl-2的表达,降低Beclin-1,Caspase-3以及自噬的特异性标记蛋白LC3-Ⅱ的表达。氧糖剥夺后对照组与高压氧组相比,LDH释放量明显增多(P<0.05)。结论:HBO预处理通过调节自噬减轻缺血再灌注损伤,为HBO预处理神经保护提供一条新的作用机制。     

Che-1通过抑制自噬减轻氧糖剥夺所致神经元损伤的研究

目的:研究Che-1蛋白对氧糖剥夺(Oxygen glucose deprivation, OGD)所致神经元损伤的保护作用及机制。方法:OGD处理神经元后,采用免疫荧光染色和免疫印迹法检测Che-1蛋白的表达;慢病毒转染神经元实现Che-1过表达,检测乳酸脱氢酶(Lactate dehydrogenase, LDH)释放量和流式细胞术检测神经元凋亡反映OGD所致神经元损伤程度,采用免疫荧光染色和免疫印迹法检测神经元自噬;使用自噬激动剂雷帕霉素(Rapamycin)处理神经元,并通过检测LDH释放量和流式细胞术研究自噬在Che-1保护作用中的作用。结果:免疫荧光结果显示,OGD后神经元Che-1蛋白表达明显增高;免疫印迹结果显示,OGD后6至48 h神经元Che-1蛋白表达明显增高;慢病毒转染过表达Che-1蛋白后,OGD所致神经元LDH释放量明显减低,且OGD所致神经元凋亡明显减少;过表达Che-1蛋白可显著减少OGD所致神经元Beclin1和LC3II的表达;自噬激动剂Rapamycin可逆转Che-1对OGD所致神经元损伤的保护作用。结论:过表达Che-1蛋白可通过抑制神经元自噬对OGD所致神经元损伤发挥保护作用。     

Cover Image,Volume 234, Number 8, August 2019

KCNQ/M potassium channels play a vital role in neuronal excitability; however, it is required to explore their pharmacological modulation on N-Methyl- d -aspartic acid receptors (NMDARs)-mediated glutamatergic transmission of neurons upon ischemic insults. In the current study, both presynaptic glutamatergic release and activities of NMDARs were measured by NMDAR-induced miniature excitatory postsynaptic currents (mEPSCs) in cultured cortical neurons of C57 mice undergoing oxygen and glucose deprivation (OGD) or OGD/reperfusion (OGD/R). The KCNQ/M-channel opener, retigabine (RTG), suppressed the overactivation of postsynaptic NMDARs induced by OGD and then NO transient; RTG also decreased OGD-induced neuronal death measured with MTT assay, suggesting the beneficial role of KCNQ/M-channels for the neurons exposed to ischemic insults. However, when the neurons exposed to the subsequent reperfusion, KCNQ/M-channels played a differential role from its protective effect. OGD/R increased presynaptic glutamatergic release, which was further augmented by RTG or decreased by KCNQ/M-channel blocker, XE991. Reactive oxygen species (ROS) were produced partly in a NO-dependent manner. In addition, XE991 decreased neuronal injuries upon reperfusion measured with DCF and PI staining. Meanwhile, the addition of RTG upon OGD or XE991 upon reperfusion can reverse OGD or OGD/R-reduced mitochondrial membrane potential. Our present study indicates the dual role of KCNQ/M-channels in OGD and OGD/R, which will decide the fate of neurons. Provided that activation of KCNQ/M-channels has differential effects on neuronal injuries during OGD or OGD/R, we propose that therapy targeting KCNQ/M-channels may be effective for ischemic injuries but the proper timing is so crucial for the corresponding treatment.     

Tetramethylpyrazine Protects Against Oxygen-Glucose Deprivation-Induced Brain Microvascular Endothelial Cells Injury via Rho/Rho-kinase Signaling Pathway

Tetramethylpyrazine (TMP, also known as Ligustrazine), which is isolated from Chinese Herb Medicine Ligustium wollichii Franchat (Chuan Xiong), has been widely used in China for the treatment of ischemic stroke by Chinese herbalists. Brain microvascular endothelial cells (BMECs) are the integral parts of the blood–brain barrier (BBB), protecting BMECs against oxygen-glucose deprivation (OGD) which is important for the treatment of ischemic stroke. Here, we investigated the protective mechanisms of TMP, focusing on OGD-injured BMECs and the Rho/Rho-kinase (Rho-associated kinases, ROCK) signaling pathway. The model of OGD-injured BMECs was established in this study. BMECs were identified by von Willebrand factor III staining and exposed to fasudil, or TMP at different concentrations (14.3, 28.6, 57.3 µM) for 2 h before 24 h of OGD injury. The effect of each treatment was examined by cell viability assays, measurement of intracellular reactive oxygen species (ROS), and transendothelial electric resistance and western blot analysis (caspase-3, endothelial nitric oxide synthase (eNOS), RhoA, Rac1). Our results show that TMP significantly attenuated apoptosis and the permeability of BMECs induced by OGD. In addition, TMP could notably down-regulate the characteristic proteins in Rho/ROCK signaling pathway such as RhoA and Rac1, which triggered abnormal changes of eNOS and ROS, respectively. Altogether, our results show that TMP has a strong protective effect against OGD-induced BMECs injury and suggest that the mechanism might be related to the inhibition of the Rho/ROCK signaling pathway.     

Hydroxysafflor Yellow A Protects PC12 Cells Against the Apoptosis Induced by Oxygen and Glucose Deprivation

Hydroxysafflor yellow A (HSYA) was reported neuroprotective under several ischemic models in vivo. In this study, the direct effect of HSYA against oxygen–glucose deprivation (OGD) inducing acute neuronal injury and the underling mechanisms in vitro were investigated. Four-hour oxygen and glucose deprivation (OGD) followed by 20 h reperfusion (adding back oxygen and glucose, OGD-R) was used to induce in vitro ischemia reperfusion injury in differentiated rat pheochromocytoma PC12 cells. HSYA (1, 10, and 100 μmol/l) was added to the cultures 30 min prior to the ischemic insult and was present during OGD and reoxygenation phases. The survival rate of PC12 cells was detected by MTT assay. The contents of malondialdehyde (MDA), superoxide dismutase (SOD) activity were elevated by biochemical method. Hoechst 33258 staining and flow cytometric analysis were used to detect apoptosis; western blotting was used to detect the expression of Bcl-2, Bax, and Cytochrome C protein. The activity of caspase-3 was assessed by colorimetry. HSYA concentration-dependently attenuated neuronal damage with characteristics of increasing injured neuronal absorbance of MTT, decreasing cell apoptosis, and antagonizing decreases in SOD activity and increase in MDA level induced by OGD-R. Moreover, the down-regulation of Bcl-2, up-regulation of Bax and the release of mitochondrial cytochrome c to cytosol and the consequent activation of caspase-3 were reversed by HSYA in a dose-dependent manner. These results suggest that apoptosis is an important characteristic of OGD-R-induced PC 12 death and that treatment of PC12 cells with HSYA can block OGD-R-induced apoptosis through suppression of intracellular oxidative stress and mitochondria dependent caspase cascade.     

Beneficial Effect of Astragalosides on Stroke Condition Using PC12 Cells under Oxygen Glucose Deprivation and Reperfusion

Astragalosides (AST) are reported to be neuroprotective in focal cerebral ischemic models in vivo. In this study, the direct effect of AST against oxygen and glucose deprivation (OGD) including neuronal injury and the underlying mechanisms in vitro were investigated. 5 h OGD followed by 24 h of reperfusion [adding back oxygen and glucose (OGD-R)] was used to induce in vitro ischemia reperfusion injury in differentiated rat pheochromocytoma PC12 cells. AST (1, 100, and 200 µg/mL) were added to the culture after 5 h of the OGD ischemic insult and was present during the reoxygenation phases. A key finding was that OGD-R decreased cell viability, increased lactate dehydrogenase, increased reactive oxygen species, apoptosis, autophagy, functional impairment of mitochondria, and endoplasmic reticulum stress in PC12 cells, all of which AST treatment significantly reduced. In addition, AST attenuated OGD-R-induced cell loss through P38 MAPK activation a neuroprotective effect blunted by SB203580, a specific inhibitor of P38 MAPK. Our data suggest that both apoptosis and autophagy are important characteristics of OGD-R-induced PC12 death and that treating PC12 cells with AST blocked OGD-R-induced apoptosis and autophagy by suppressing intracellular oxidative stress, functional impairment of mitochondria, and endoplasmic reticulum stress. Our data provide identification of AST that can concomitantly inhibit multiple cells death pathways following OGD injuries in neural cells.     

Orexin-A protects against cerebral ischemia-reperfusion injury by inhibiting excessive autophagy through OX1R-mediated MAPK/ERK/mTOR pathway

Orexin A (OXA) is a neuroprotective peptide that exerts protective effects on multiple physiological and pathological processes. Activation of autophagy is linked to the occurrence of cerebral ischemia–reperfusion injury (CIRI); however, its function remains incompletely understood. In this study, OXA was sought to exert its neuroprotective role by regulating autophagy in oxygen and glucose deprivation and reoxygenation (OGD/R) model and middle cerebral artery occlusion (MCAO) model of rats, and to elucidate the underlying molecular mechanisms. Acridine orange (AO) staining was used to evaluate autophagic vacuoles. Cell viability was measured by CCK8. The levels of p-ERK_1/2, t-ERK_1/2, p-mTOR, LC3B, Beclin 1, and p62 were evaluated by western blotting. Apoptosis rate was detected by Hoechst 33342 staining and Terminal deoxynucleotidyltransferase–mediated dUTP nick-end labeling (TUNEL). OXA treatment alleviated neuronal apoptosis and significantly inhibited autophagy activity. Mechanistically, OXA exerted its neuroprotective effects in vivo and in vitro by suppressing over-activated autophagy by modulating OX1R-mediated MAPK/ERK/mTOR pathway. The results of this study elucidate the roles of autophagy in CIRI and the mechanisms underlying the neuroprotective action of OXA. Our findings could facilitate the development of novel therapeutics for ischemic stroke.     

Quantitative imaging of mitochondrial and cytosolic free zinc levels in an in vitro model of ischemia/reperfusion

The role of zinc ion in cytotoxicity following ischemic stroke, prolonged status epilepticus, and traumatic brain injury remains controversial, but likely is the result of mitochondrial dysfunction. We describe an excitation ratiometric fluorescence biosensor based on human carbonic anhydrase II variants expressed in the mitochondrial matrix, permitting free zinc levels to be quantitatively imaged therein. We observed an average mitochondrial matrix free zinc concentration of 0.2 pM in the PC12 rat pheochromacytoma cell culture line. Cytoplasmic and mitochondrial free zinc levels were imaged in a cellular oxygen glucose deprivation (OGD) model of ischemia/reperfusion. We observed a significant increase in mitochondrial zinc 1 h following 3 h OGD, at a time point when cytosolic zinc levels were depressed. Following the increase, mitochondrial zinc levels returned to physiological levels, while cytosolic zinc increased gradually over a 24 h time period in viable cells. The increase in intramitochondrial zinc observed during reoxygenation after OGD may contribute to bioenergetic dysfunction and cell death that occurs with both in vitro and in vivo models of reperfusion.     

Pharmacological induction of ischemic tolerance in hippocampal slices by sarcosine preconditioning

Brain ischemic tolerance is a protective mechanism induced by a preconditioning stimulus, which prepare the tissue against harmful insults. Preconditioning with N-methyl-d-aspartate (NMDA) agonists induces brain tolerance and protects it against glutamate excitotoxicity. Recently, the glycine transporters type 1 (GlyT-1) have been shown to potentiate glutamate neurotransmission through NMDA receptors suggesting an alternative strategy to protect against glutamate excitotoxicity. Here, we evaluated the preconditioning effect of sarcosine pre-treatment, a GlyT-1 inhibitor, in rat hippocampal slices exposed to ischemic insult. Sarcosine (300mg/kg per day, i.p.) was administered during seven consecutive days before induction of ischemia in hippocampus by oxygen/glucose deprivation (OGD). To access the damage caused by an ischemic insult, we evaluated cells viability, glutamate release, nitric oxide (NO) production, lactate dehydrogenase (LDH) levels, production of reactive oxygen species (ROS), and antioxidant enzymes as well as the impact of oxidative stress in the tissue. We observed that sarcosine reduced cell death in hippocampus submitted to OGD, which was confirmed by reduction on LDH levels in the supernatant. Cell death, glutamate release, LDH levels and NO production were reduced in sarcosine hippocampal slices submitted to OGD when compared to OGD controls (without sarcosine). ROS production was reduced in sarcosine hippocampal slices exposed to OGD, although no changes were found in antioxidant enzymes activities. This study demonstrates that preconditioning with sarcosine induces ischemic tolerance in rat hippocampal slices submitted to OGD.