Neuroprotection in ischemia: blocking calcium-permeable acid-sensing ion channels

Ca2+ toxicity remains the central focus of ischemic brain injury. The mechanism by which toxic Ca2+ loading of cells occurs in the ischemic brain has become less clear as multiple human trials of glutamate antagonists have failed to show effective neuroprotection in stroke. Acidosis is a common feature of ischemia and is assumed to play a critical role in brain injury; however, the mechanism(s) remain ill defined. Here, we show that acidosis activates Ca2+ -permeable acid-sensing ion channels (ASICs), inducing glutamate receptor-independent, Ca2+ -dependent, neuronal injury inhibited by ASIC blockers. Cells lacking endogenous ASICs are resistant to acid injury, while transfection of Ca2+ -permeable ASIC1a establishes sensitivity. In focal ischemia, intracerebroventricular injection of ASIC1a blockers or knockout of the ASIC1a gene protects the brain from ischemic injury and does so more potently than glutamate antagonism. Thus, acidosis injures the brain via membrane receptor-based mechanisms with resultant toxicity of [Ca2+]i, disclosing new potential therapeutic targets for stroke.     

Knockdown of ASIC2a subunit aggravates injury of rat C6 glioma cells in acidosis

Acid-sensing ion channel 1a (ASIC1a) and 2a (ASIC2a) subunits are widely expressed throughout mammalian central nervous system. Activation of Ca²⁺-permeable ASIC1a homomultimers is largely responsible for acidosis-mediated, glutamate receptor-independent, ischemic neuronal injury. The function of ASIC2a in brain ischemia is less known except that transient global ischemia induces ASIC2a protein expression up-regulation in neurons that survived ischemia. Acidosis is assumed to play a critical role in brain ischemia injury. In the present experiment, rat C6 neuroglioma cells were used to explore the function of ASIC2a. MTT and relative LDH release assay revealed that knockdown of ASIC2a could aggravate the acidosis-induced injury of C6 cells. Through changing extracellular Ca²⁺ concentration and measuring intracellular calcium fluorescence intensity, it was found that aggravated damage was due to toxic Ca²⁺ overload via ASICs mechanisms. The current results indicated that, different from ASIC1a, ASIC2a probably played a protective role against the injury induced by extracellular acidosis in C6 cells.     

Mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2 deficiency protects brain from ischemic injury in mice

Mitogen-activated protein (MAP) kinase-activated protein kinase 2 (MK2) is one of several kinases directly regulated by p38 MAP kinase. A role of p38 MAP kinase in ischemic brain injury has been previously suggested by pharmacological means. In the present study, we provide evidence for a role of MK2 in cerebral ischemic injury using MK2-deficient (MK2(-/-)) mice. MK2(-/-) mice subjected to focal ischemia markedly reduced infarct size by 64 and 76% after transient and permanent ischemia, respectively, compared with wild-type mice. Furthermore, MK2(-/-) mice had significant reduction in neurological deficits. Real-time PCR analysis identified a significantly lower expression in interleukin-1beta mRNA (53% reduction) but not in tumor necrosis factor-alpha mRNA in MK2(-/-) mice over wild-type animals after ischemic injury. The significant reduction in interleukin-1beta was also confirmed in MK2(-/-) mice by enzyme-linked immunosorbent assay. The marked neuroprotection from ischemic brain injury in MK2(-/-) mice was not associated with the alteration of hemodynamic or systemic variables, activation of caspase-3, or apoptosis. Our data provide new evidence for the involvement of MAP kinase pathway in focal ischemic brain injury and suggest that this effect might be associated with the expression of interleukin-1beta in the ischemic brain tissue.     

Biochemical and Molecular Characteristics of the Brain with Developing Cerebral Infarction

1. We review the biochemical and molecular changes in brain with developing cerebral infarction, based on recent findings in experimental focal cerebral ischemia.2. Occlusion of a cerebral artery produces focal ischemia with a gradual decline of blood flow, differentiating a severely ischemic core where infarct develops rapidly and an area peripheral to the core where the blood flow reduction is moderate (called penumbra). Neuronal injury in the penumbra is essentially reversible but only for several hours. The penumbra area tolerates a longer duration of ischemia than the core and may be salvageable by pharmacological agents such as glutamate antagonists or prompt reperfusion.3. Upon reperfusion, brain cells alter their genomic properties so that protein synthesis becomes restricted to a small number of proteins such as stress proteins. Induction of the stress response is considered to be a rescue program to help to mitigate neuronal injury and to endow the cells with resistance to subsequent ischemic stress. The challenge now is to determine how the neuroprotection conferred by prior sublethal ischemia is achieved so that rational strategies can be developed to detect and manipulate gene expression in brain cells vulnerable to ischemia.4. Expansion of infarction may be caused by an apoptotic mechanism. Investigation of apoptosis may also help in designing novel molecular strategies to prevent ischemic cell death.5. Ischemia/reperfusion injury is accompanied by inflammatory reactions induced by neutrophils and monocytes/macrophages infiltrated and accumulated in ischemic areas. When the role of the inflammatory/immune systems in ischemic brain injury is revealed, new therapeutic targets and agents will emerge to complement and synergize with pharmacological intervention directed against glutamate and Ca²⁺ neurotoxicity.     

炎症标记物与缺血性中风预后评价

缺血性中风触发的炎症反应是一个级联放大过程,不仅可直接对缺血脑组织造成继发性损伤,还可通过与其他病理生理通路的相互影响、相互促进,共同对缺血后脑组织造成不可逆损伤。因此,采用炎症标记物对脑缺血损伤及其预后进行评价,具有重要临床意义。临床研究发现,多炎症标记物法用于缺血性中风的诊治和预后评价比单炎症标记物法更全面、更准确,故更具明显优势。综述脑缺血引发的炎症机制、脑缺血所致炎症通路与其他病理生理通路( 如氧化应激、细胞凋亡和兴奋性毒性) 的关联以及炎症标记物在缺血性中风预后评价中的应用。     

Cerebral Ischemia Is Exacerbated by Extracellular Nicotinamide Phosphoribosyltransferase via a Non-Enzymatic Mechanism

Intracellular nicotinamide phosphoribosyltransferase (iNAMPT) in neuron has been known as a protective factor against cerebral ischemia through its enzymatic activity, but the role of central extracellular NAMPT (eNAMPT) is not clear. Here we show that eNAMPT protein level was elevated in the ischemic rat brain after middle-cerebral-artery occlusion (MCAO) and reperfusion, which can be traced to at least in part from blood circulation. Administration of recombinant NAMPT protein exacerbated MCAO-induced neuronal injury in rat brain, while exacerbated oxygen-glucose-deprivation (OGD) induced neuronal injury only in neuron-glial mixed culture, but not in neuron culture. In the mixed culture, NAMPT protein promoted TNF-α release in a time- and concentration-dependent fashion, while TNF-α neutralizing antibody protected OGD-induced, NAMPT-enhanced neuronal injury. Importantly, H247A mutant of NAMPT with essentially no enzymatic activity exerted similar effects on ischemic neuronal injury and TNF-α release as the wild type protein. Thus, eNAMPT is an injurious and inflammatory factor in cerebral ischemia and aggravates ischemic neuronal injury by triggering TNF-α release from glia cells, via a mechanism not related to NAMPT enzymatic activity.     

Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes

We describe a new histochemical approach for visualization of phagocytic clearance in focal brain ischemia. The approach permits the study of elimination of dead cells in stroke by waste-management phagocytes of any cellular lineage. Although numerous cells of different origins that are capable of phagocytosis are present in ischemic brain, only part of them actively engulf and digest cell corpses. The selective visualization, quantification and analysis of such active phagocytic waste-management are helpful in assessing brain response to ischemia. Efficient cell death clearance is important for brain recovery from ischemic injury, as it opens the way for the subsequent regenerative processes. The failure to clean the corpses would result in a toxic reaction caused by non-degraded DNA and proteins. The described procedure uses fluorescent probes selectively ligated by a viral topoisomerase to characteristic DNA breaks produced in all phagocytes during engulfment and digestion of cells irreversibly damaged by ischemia. The method is a new tool for the investigation of brain reaction to ischemic injury.     

Effects of Bradykinin Postconditioning on Endogenous Antioxidant Enzyme Activity After Transient Forebrain Ischemia in Rat

Bradykinin is considered an important mediator of the inflammatory response in both the peripheral and the central nervous system and it has attracted recent interest as a potential mediator of brain injury following stroke. Bradykinin is recognized to play an important role in ischemic brain. We investigated the effect of bradykinin postconditioning on ischemic damage after 8 min of ischemia (four-vessel occlusion) and 3 days of reperfusion. Bradykinin was administered after 2 days of reperfusion at a dose of 150 μg/kg (i.p.). Catalase (CAT) activity was significantly increased in all examined regions (cortex, hippocampus and striatum) 3 days after 8 min of ischemia, but postconditioning decreased this activity below the control values. The total activity of superoxide dismutase (SOD) 3 days after ischemia was at control level with or without postconditioning. However, the analysis of individual SODs separately revealed interesting differences; while the activity of CuZnSOD was significantly decreased 3 days after ischemia, the activity of MnSOD was significantly increased compared to control levels. In both cases, postconditioning returned SOD activity to control levels. These findings are interesting because MnSOD is a mitochondrial enzyme and its activity in the cytosol suggests that a possible mechanism of protection provided by postconditioning could include prevention of release of mitochondrial proteins to the cytoplasm, resulting in protection against the mitochondrial pathway of apoptosis. 8 min of ischemia alone caused the degeneration of 52.37% neurons in the hippocampal CA1 region 3 days later. Bradykinin used as postconditioning 2 days after the same interval of ischemia enabled the survival of more than 97% of CA1 neurons. This study demonstrated that bradykinin postconditioning induces protection against ischemic brain injury and promotes neuronal survival.     

远端缺血后处理对脑缺血保护作用的研究进展

近20多年来人们对脑缺血损伤的保护研究很多,但真正能将脑缺血保护从基础研究应用到临床治疗的措施甚少。多数基础研究表明缺血预处理对大鼠脑缺血具有保护作用,然而由于脑缺血的不可预见性,研究者们将目标转向了缺血后处理。远端缺血后处理是指在非缺血器官交替实施短时间的缺血和再灌注后对缺血器官所产生的作用。由于脑组织本身对缺血的敏感,很难控制缺血后处理的程度,因此远端缺血后处理被应用到脑缺血的保护研究具有很强的临床应用价值,其机制可能与氧自由基、神经传导、蛋白质、内质网应激、Akt信号通路、线粒体途径、mitoKATP和阿片受体有关。本文主要就近几年远程缺血后处理对脑缺血保护的概念、实施方法、保护作用及分子机制做一综述。     

Death-associated protein kinase is activated by dephosphorylation in response to cerebral ischemia

Death-associated protein kinase (DAPK) is a calcium calmodulin-regulated serine/threonine protein kinase involved in ischemic neuronal death. In situ hybridization experiments show that DAPK mRNA expression is up-regulated in brain following a global ischemic insult and down-regulated in ischemic tissues after focal ischemia. DAPK is inactive in normal brain tissues, where it is found in its phosphorylated state and becomes rapidly and persistently dephosphorylated and activated in response to ischemia in vivo. A similar dephosphorylation pattern is detected in primary cortical neurons subjected to oxygen glucose deprivation or N-methyl-D-aspartate (NMDA)-induced toxicity. Both a calcineurin inhibitor, FK506, and a selective NMDA receptor antagonist, MK-801, inhibit the dephosphorylation of DAPK after in vitro ischemia. This indicates that DAPK could be activated by NMDA receptor-mediated calcium flux, activation of calcineurin, and subsequent DAPK dephosphorylation. Moreover, concomitantly to dephosphorylation, DAPK is proteolytically processed by cathepsin after ischemia. Furthermore, a selective DAPK inhibitor is neuroprotective in both in vitro and in vivo ischemic models. These results indicate that DAPK plays a key role in mediating ischemic neuronal injury.     

中药通过调节小胶质细胞激活改善缺血性脑卒中

炎症反应是造成脑卒中继发性脑损伤的关键因素之一。小胶质细胞作为脑内免疫细胞,在脑卒中的炎症反应具有重要作用。传统观念认为小胶质细胞促进炎症反应加重脑损伤。近年来的研究发现激活的小胶质细胞还能产生抗炎作用来加速脑损伤修复。因此,目前的研究将小胶质细胞分为促炎的M1型和抗炎的M2型。结合目前缺血性脑卒中的神经保护剂相对较少,靶向调控小胶质细胞的极化可能成为脑卒中新的治疗策略。研究发现中药能够通过抑制M1型小胶质细胞,并促进M2型的小胶质细胞来改善缺血性脑损伤,从而展现出对缺血性脑卒中的治疗潜力。本文综述了中药通过调节小胶质细胞极化表型来治疗脑卒中的相关研究,以期为缺血性脑卒中药物开发提供新的思路。     

17β-Estradiol prevents cell death and mitochondrial dysfunction by an estrogen receptor-dependent mechanism in astrocytes after oxygen-glucose deprivation/reperfusion

17β-Estradiol (E2) has been shown to protect against ischemic brain injury, yet its targets and the mechanisms are unclear. E2 may exert multiple regulatory actions on astrocytes that may greatly contribute to its ability to protect the brain. Mitochondria are recognized as playing central roles in the development of injury during ischemia. Increasing evidence indicates that mitochondrial mechanisms are critically involved in E2-mediated protection. In this study, the effects of E2 and the role of mitochondria were evaluated in primary cultures of astrocytes subjected to an ischemia-like condition of oxygen-glucose deprivation (OGD)/reperfusion. We showed that E2 treatment significantly protects against OGD/reperfusion-induced cell death as determined by cell viability, apoptosis, and lactate dehydrogenase leakage. The protective effects of E2 on astrocytic survival were blocked by an estrogen receptor (ER) antagonist (ICI-182,780) and were mimicked by an ER agonist selective for ERα (PPT), but not by an ER agonist selective for ERβ (DPN). OGD/reperfusion provoked mitochondrial dysfunction as manifested by an increase in cellular reactive oxygen species production, loss of mitochondrial membrane potential, and depletion of ATP. E2 pretreatment significantly inhibited OGD/reperfusion-induced mitochondrial dysfunction, and this effect was also blocked by ICI-182,780. Therefore, we conclude that E2 provides direct protection to astrocytes from ischemic injury by an ER-dependent mechanism, highlighting an important role for ERα. Estrogen protects against mitochondrial dysfunction at the early phase of ischemic injury. However, overall implications for protection against brain ischemia and its complex sequelae await further exploration.     

Protective and regenerative response endogenously induced in the ischemic brain

Neuronal cells are highly vulnerable to ischemic insult. Because adult neurons are highly differentiated and cannot self-propagate, loss of neurons often results in functional deficits in mammalian brains. However, it has recently been shown that neurons and neuronal circuits exhibit protective and regenerative responses in a rodent model of experimental ischemia. At first, neurons respond by producing several protective proteins such as heat shock proteins (HSPs) after sublethal ischemia and then acquire tolerance against a subsequent ischemic insult (ischemic tolerance). Once neurons suffer irreversible injury, two repair processes, neurogenesis and synaptogenesis, are endogenously induced. Neuronal stem and (or) progenitor cells can proliferate in two brain areas in adult animals: the subventricular zone and the subgranular zone in the dentate gyrus. After ischemic insult, these stem (progenitor) cells proliferate and differentiate into neurons in the dentate gyrus of the hippocampus. Reactive synaptogenesis has been also observed in the injured brain following a period of long-term infarction, but it is unclear if it can compensate for disconnected circuits. Understanding the molecular mechanism underlying these protective and regenerative responses will be important in developing a new strategy for aimed at the augmentation of resistance against ischemic insult and the replacement of injured neurons and neuronal circuits.     

Protective Effect of Shikonin in Experimental Ischemic Stroke: Attenuated TLR4, p-p38MAPK, NF-κB, TNF-α and MMP-9 Expression, Up-Regulated Claudin-5 Expression, Ameliorated BBB Permeability

Inflammatory damage plays an important role in cerebral ischemic pathogenesis and represents a new target for treatment of stroke. Shikonin has gained attention for its prominent anti-inflammatory property, but up to now little is known about shikonin treatment in acute ischemic stroke. The aim of this study was to evaluate the potential neuroprotective role of shikonin in cerebral ischemic injury, and investigate whether shikonin modulated inflammatory responses after stroke. Focal cerebral ischemia in male ICR mice was induced by transient middle cerebral artery occlusion. Shikonin (10 and 25 mg/kg) was administered by gavage once a day for 3 days before surgery and another dosage after operation. Neurological deficit, infarct volume, brain edema, blood–brain barrier (BBB) dysfunction, and inflammatory mediators were evaluated at 24 and 72 h after stroke. Compared with vehicle group, 25 mg/kg shikonin significantly improved neurological deficit, decreased infarct volume and edema both at 24 and 72 h after transient ischemic stroke, our data also showed that shikonin inhibited the pro-inflammatory mediators, including TLR4, TNF-α, NF-κB, and phosphorylation of p38MAPK in ischemic cortex. In addition, shikonin effectively alleviated brain leakage of Evans blue, up-regulated claudin-5 expression, and inhibited the over-expressed MMP-9 in ischemic brain. These results suggested that shikonin effectively protected brain against ischemic damage by regulating inflammatory responses and ameliorating BBB permeability.     

Exploring ischemia-induced vascular lesions and potential pharmacological intervention strategies

Structural changes in vessels under the influence of ischemia play an important role in the pathogenesis of many diseases, most important of which are stroke and myocardial infarction or myocardial insult. Over the years, information has been gathered, which implicate a role for ischemic vascular changes in the pathogenesis of crush-syndrome, atherosclerosis and other vascular diseases. When blood vessels are damaged they become unresponsive to a stimulus, which normally elicits vasodilatation and can lead to intraluminal thrombosis and ischemic events. The aim of this review is to explore the structural changes seen in vessels affected by ischemia reperfusion injury. With ischemia, the development of observable changes to vascular structure is multifactorial. One key factor is reperfusion ischemic injury. Moreover, the duration of the ischemic event is an important factor when determining both the prognosis and the type of morphological change that is observable in affected vessel walls. In this regard, the deleterious progression of blood flow impairment and its severity depends on the specific organ involved and the type of tissue affected. Further, there are regional differences within affected tissues and the degree of microvascular injury is well correlated with differences in the nature and severity of the ischemic event. Any method aimed at preventing and treating ischemic reperfusion injuries in vessels, based on these investigations, should likewise be able to decrease the early signs of brain, cerebrovascular and heart injury and preserve normal cellular architecture.     

酸敏感离子通道与脑梗死

急性脑梗死约占全部脑卒中的70%,病死率和致残率高,且极易复发。但目前针对急性脑梗死在时间窗内溶栓、抗凝等治疗手段不能从根本上切实有效地修复受损脑组织,且伴有出血等风险。寻找脑梗死形成发展的原因并予以治疗迫在眉睫。酸中毒是引起缺血性脑损伤的重要机制。大量实验研究表明,酸中毒能加重神经元的缺血性损伤,且其梗死面积与酸中毒的程度直接相关。但缺血产生的酸中毒如何引起神经元损伤的确切机制尚不明确。最近研究发现酸中毒能激活一种在中枢及周围神经中广泛存在的膜通道,即酸敏感离子通道,它对Ca2+通透,能引起细胞内Ca2+超载,同时能激活胞内酶引起细胞内蛋白质、脂类及核酸的降解,加重缺血后脑损伤。本文就酸敏感离子通道1a与脑梗死做一综述。     

酸敏感离子通道与脑梗死

急性脑梗死约占全部脑卒中的70%,病死率和致残率高,且极易复发。但目前针对急性脑梗死在时间窗内溶栓、抗凝等治疗手段不能从根本上切实有效地修复受损脑组织,且伴有出血等风险。寻找脑梗死形成发展的原因并予以治疗迫在眉睫。酸中毒是引起缺血性脑损伤的重要机制。大量实验研究表明,酸中毒能加重神经元的缺血性损伤,且其梗死面积与酸中毒的程度直接相关。但缺血产生的酸中毒如何引起神经元损伤的确切机制尚不明确。最近研究发现酸中毒能激活一种在中枢及周围神经中广泛存在的膜通道,即酸敏感离子通道,它对Ca^2＋通透,能引起细胞内Ca^2＋超载,同时能激活胞内酶引起细胞内蛋白质、脂类及核酸的降解,加重缺血后脑损伤。本文就酸敏感离子通道1a与脑梗死做一综述。     

Role of Superoxide Dismutase in Ischemic Brain Injury: A Study Using SOD-1 Transgenic Mice

1. Nitric oxide radicals (NO) play an important role in the pathophysiology of focal cerebral ischemia.2. Vascular NO can reduce ischemic brain injury by increasing CBF, whereas neuronal NO may mediate neurotoxicity following brain ischemia, mainly by its reaction with superoxide to generate peroxynitrite.3. These findings could contribute to a strategy for the treatment of cerebral ischemia.     

Arterially perfused neurosphere-derived cells distribute outside the ischemic core in a model of transient focal ischemia and reperfusion in vitro

BACKGROUND: Treatment with neural stem cells represents a potential strategy to improve functional recovery of post-ischemic cerebral injury. The potential benefit of such treatment in acute phases of human ischemic stroke depends on the therapeutic viability of a systemic vascular delivery route. In spite of the large number of reports on the beneficial effects of intracerebral stem cells injection in experimental stroke, very few studies demonstrated the effectiveness of the systemic intravenous delivery approach. METODOLOGY/PRINCIPAL FINDINGS: We utilized a novel in vitro model of transient focal ischemia to analyze the brain distribution of neurosphere-derived cells (NCs) in the early 3 hours that follow transient occlusion of the medial cerebral artery (MCA). NCs obtained from newborn C57/BL6 mice are immature cells with self-renewal properties that could differentiate into neurons, astrocytes and oligodendrocytes. MCA occlusion for 30 minutes in the in vitro isolated guinea pig brain preparation was followed by arterial perfusion with 1x10(6) NCs charged with a green fluorescent dye, either immediately or 60 minutes after reperfusion onset. Changes in extracellular pH and K(+) concentration during and after MCAO were measured through ion-sensitive electrodes. CONCLUSION/SIGNIFICANCE: It is demonstrated that NCs injected through the vascular system do not accumulate in the ischemic core and preferentially distribute in non-ischemic areas, identified by combined electrophysiological and morphological techniques. Direct measurements of extracellular brain ions during and after MCA occlusion suggest that anoxia-induced tissue changes, such as extracellular acidosis, may prevent NCs from entering the ischemic area in our in vitro model of transitory focal ischemia and reperfusion suggesting a role played by the surrounding microenviroment in driving NCs outside the ischemic core. These findings strongly suggest that the potential beneficial effect of NCs in experimental focal brain ischemia is not strictly dependent on their homing into the ischemic region, but rather through a bystander mechanism possibly mediated by the release of neuroprotective factors in the peri-infarct region.     

The role of thrombin and thrombin receptors in ischemic,hemorrhagic and traumatic brain injury: deleterious or protective?

In the last two decades it has become apparent that thrombin has many extravascular effects that are mediated by a family of protease-activated receptors (PARs). PAR-1, -3 and -4 are activated via cleavage by thrombin. The importance of extravascular thrombin in modulating ischemic, hemorrhagic and traumatic injury in brain has recently become clear. Thus, in vitro, thrombin at low concentration protects neurons and astrocytes from cell death caused by a number of different insults. In vivo, pretreating the brain with a low dose of thrombin (thrombin preconditioning), attenuates the brain injury induced by a large dose of thrombin, an intracerebral hemorrhage or by focal cerebral ischemia. Thrombin may also be an important mediator of ischemic preconditioning. In contrast, high doses of thrombin kill neurons and astrocytes in vitro and cause disruption of the blood-brain barrier, brain edema and seizures in vivo. This review examines the role of thrombin in brain injury and the molecular mechanisms and signaling cascades involved.