Cell Death In Cerebral Ischemia Research Articles

Protection of blood-brain barrier by endothelial DAPK1 deletion after stroke

Death-associated protein kinase (DAPK) 1 is a critical mediator for neuronal cell death in cerebral ischemia, but its role in blood-brain barrier (BBB) disruption is incompletely understood. Here, we found that endothelial-specific deletion of Dapk1 using Tie2 Cre protected the brain of Dapk1fl/fl mice against middle cerebral artery occlusion (MCAO), characterized by mitigated Evans blue dye (EBD) extravasation, reduced infarct size and improved behavior. In vitro experiments also indicated that DAPK1 deletion inhibited oxygen-glucose deprivation (OGD)-induced tight junction alteration between cerebral endothelial cells (CECs). Mechanistically, we revealed that DAPK1-DAPK3 interaction activated cytosolic phospholipase A2 (cPLA2) in OGD-stimulated CECs. Our results thus suggest that inhibition of endothelial DAPK1 specifically prevents BBB damage after stroke.

How cytosolic compartments play safeguard functions against neuroinflammation and cell death in cerebral ischemia.

Ischemic stroke is the second leading cause of mortality and disability globally. Neuronal damage following ischemic stroke is rapid and irreversible, and eventually results in neuronal death. In addition to activation of cell death signaling, neuroinflammation is also considered as another pathogenesis that can occur within hours after cerebral ischemia. Under physiological conditions, subcellular organelles play a substantial role in neuronal functionality and viability. However, their functions can be remarkably perturbed under neurological disorders, particularly cerebral ischemia. Therefore, their biochemical and structural response has a determining role in the sequel of neuronal cells and the progression of disease. However, their effects on cell death and neuroinflammation, as major underlying mechanisms of ischemic stroke, are still not understood. This review aims to provide a comprehensive overview of the contribution of each organelle on these pathological processes after ischemic stroke.

Administration of metformin in clinical medicine; an updated mini-review on current findings

Metformin is in the biguanide class that has been considered as the treatment for insulin resistance diabetes and polycystic ovarian disease. Many mechanisms have been suggested for it such as inhibition of the mitochondrial respiratory chain and mitochondrial glycerophosphate dehydrogenase, activation of adenosine monophosphate-activated protein kinase, inhibition of glucagon-induced elevation of cyclic adenosine monophosphate with reduced activation of protein kinase A, an effect on gut microbiota and activation of adenosine monophosphateactivated protein kinase. Metformin is a suppressor for transforming growth factor-β1 via directly binding and interact with transforming growth factor-β1 receptor. Lactic acidosis is one of the adverse and noxious effects of metformin. Nowadays, metformin has an important role in inflammation pathways and antioxidant pathways that can prevent or decrease kidney fibrosis, cardiac remodeling in hypertensive heart disease, and cell death in cerebral ischemia, kidney crystal formation, immunological diseases and cancer. Although there has been strong evidence for the potential harm caused by metformin, several studies have shown beneficial effects for it. Hence, it is necessary to revision and modification in contraindications for prescription of this drug

Pre-treatment with metformin activates Nrf2 antioxidant pathways and inhibits inflammatory responses through induction of AMPK after transient global cerebral ischemia.

Global cerebral ischemia arises in patients who have a variety of clinical conditions including cardiac arrest, shock and asphyxia. In spite of advances in understanding of the brain ischemia and stroke etiology, therapeutic approaches to improve ischemic injury still remain limited. It has been established that metformin can attenuate cell death in cerebral ischemia. One of the main functions of metformin is proposed to be conducted via AMP-activated protein kinase (AMPK)-dependent pathway in the experimental cerebral ischemia model. It is also established that metformin can suppress inflammation and activate Nuclear factor erythroid 2-related factor (Nrf2) pathways in neurons. In the current study, the role of metformin in regulating inflammatory and antioxidant pathways in the global cerebral ischemia was investigated. Our results indicated that pretreatment of rats by metformin attenuated cellular levels of nuclear factor-κB, Tumor Necrosis Factor alpha and Cyclooxygenase-2 which are considered as three important proteins involved in the inflammation pathway. Pretreatment by metformin increased the level of Nrf2 and heme oxygenase-1 in the hippocampus of ischemic rats compared with untreated ischemic group. Moreover, pretreatment by metformin enhanced the level of glutathione and catalase activities compared with them in ischemic group. Such protective changes detected by metformin pretreatment were reversed by injecting compound c, an AMPK inhibitor. These findings suggested that metformin might protect cells through modulating inflammatory and antioxidant pathways via induction of AMPK. However, more experimental and clinical trial studies regarding neuroprotective potential of metformin and the involved mechanisms, especially in the context of cerebral ischemic injuries, are necessary.

NAD+ treatment prevents rotenone-induced apoptosis and necrosis of differentiated PC12 cells

Nicotinamide adenine dinucleotide (NAD+) plays critical roles in not only energy metabolism and mitochondrial functions, but also calcium homeostasis and immunological functions. It has been reported that NAD+ administration can reduce ischemic brain damage. However, the mechanisms underlying the protective effects remain unclear. Because mitochondrial impairments play a key role in the cell death in cerebral ischemia, in this study we tested our hypothesis that NAD+ can decrease mitochondrial damage-induced cell death using differentiated PC12 cells as a cellular model. We found that NAD+ can decrease both early-stage and late-stage apoptosis, as well as necrosis of rotenone-treated PC12 cells, as assessed by FACS-based Annexin V/AAD assay. We also found that NAD+ treatment can restore the intracellular NAD+ levels of the rotenone-treated cells. Moreover, NAD+ treatment can prevent rotenone-induced mitochondria depolarization. In summary, our study has provided first direct evidence that NAD+ treatment can prevent rotenone-induced apoptosis and necrosis. Our study has also indicated that NAD+ treatment can prevent mitochondrial damage-induced cell death, which may at least partially result from its protective effects on rotenone-induced mitochondrial depolarization. Because both mitochondrial damage and apoptosis play key roles in multiple neurological disorders, our study has highlighted the therapeutic potential of NAD+ for brain ischemia and other neurological diseases.

TRPM7 in cerebral ischemia and potential target for drug development in stroke

Searching for effective pharmacological agents for stroke treatment has largely been unsuccessful. Despite initial excitement, antagonists for glutamate receptors, the most studied receptor channels in ischemic stroke, have shown insufficient neuroprotective effects in clinical trials. Outside the traditional glutamate-mediated excitotoxicity, recent evidence suggests few non-glutamate mechanisms, which may also cause ionic imbalance and cell death in cerebral ischemia. Transient receptor potential melastatin 7 (TRPM7) is a Ca(2+) permeable, non-selective cation channel that has recently gained attention as a potential cation influx pathway involved in ischemic events. Compelling new evidence from an in vivo study demonstrated that suppression of TRPM7 channels in adult rat brain in vivo using virally mediated gene silencing approach reduced delayed neuronal cell death and preserved neuronal functions in global cerebral ischemia. In this review, we will discuss the current understanding of the role of TRPM7 channels in physiology and pathophysiology as well as its therapeutic potential in stroke.

Ca 2+ and acidosis synergistically lead to the dysfunction of cortical GABAergic neurons during ischemia

Cell death in cerebral ischemia is presumably initiated by neural excitotoxicity resulted from the dysfunction of inhibitory neurons in early stage. Molecular processes underlying the ischemic injury of inhibitory neurons remain to be elusive, which we investigated by biochemical manipulations with cellular imaging and patch clamp at GFP-labeled GABAergic cells in cortical slices. Ischemia induces Ca 2+ elevation, acidosis and dysfunction in GABAergic cells. An elevation of cytoplasmic Ca 2+ or H + impairs the encoding of action potentials in these neurons. The effects of Ca 2+ and H + are additive in nature and occlude ischemic outcomes. Ischemia impairs spike production through prolonging spike refractory periods and raising threshold potentials. Therefore, calcium toxicity and acidosis during ischemia synergistically impair the dynamics of sodium channels and function of cortical GABAergic neurons, which lead to neural excitotoxicity. Our results also suggest that the cocktail therapeutics is needed to prevent neuronal death from ischemia.

Commentary [Research Highlights

Drug delivery to the central nervous system remains a vexing problem because of the presence of the blood-brain barrier (BBB), whose endothelial cell tight junctions limit the paracellular flux of hydrophilic molecules. One solution may be vector-mediated delivery to the brain. This employs chimeric peptide technology, wherein the drug is conjugated to a molecular carrier of protein nature (e.g. cationized albumin or a transferrin receptor antibody). Transferrin receptor antibodies selectively target BBB endothelium due to the high levels of receptor expressed by these cells, thereby triggering receptor-mediated transport across the BBB via transcytosis. Chitosan, a naturally occurring, abundant, and biocompatible polysaccharide has found widespread pharmaceutical application. In particular, the cationic nature of chitosan facilitates its interaction with negative charges on the brain endothelium, Building on their previous work on brain-deliverable chitosan-polyethylene glycol nanoparticles functionalized with monoclonal anti-transferrin antibody, Karatas and colleagues have now designed caspase-3 inhibitor-loaded chitosan nanospheres conjugated with an anti-mouse transferrin receptor monoclonal antibody that selectively recognizes the transferrin receptor type 1 on the cerebral vasculature. Caspase-3 has been implicated in neuronal cell death in cerebral ischemia, as well as in the pathophysiology of other neurological disorders. When given intravenously, the caspase-3-loaded nanospheres were rapidly transported across the BBB and dose-dependently decreased infarct volume, neurological deficit, and ischemiainduced caspase-3 activity in mice subjected to focal cerebral ischemia/reperfusion. Because significant opening of the BBB starts only 6 h after ischemia/reperfusion, the rapid transfer of nanospheres to the brain was not likely caused by an increased permeability of the BBB. Similarly, nanospheres inhibited physiological caspase-3 activity during development in the neonatal mouse cerebellum on postnatal day 17, after closure of the BBB. These observations convincingly demonstrate that transferrin monoclonal antibody-conjugated chitosan nanospheres are an efficient delivery system for bringing neuroprotective, small peptide drugs into the brain. This opens intriguing new avenues for the treatment of central nervous system disorders in which the administration of therapeutic agents is handicapped by insufficient diffusion across the BBB. Further studies are eagerly awaited to see if this technology will be applicable to polypeptide factors, for example, brain-derived neurotrophic factor and ciliary neurotrophic factor.

Genomics of Ischemia

The quest to understand the neurobiology of ischemic brain injury has relied on the investigational techniques available over the years. Historically, morbid anatomy was the approach used through the midportion of the last century.1 The establishment of the Journal of Neurochemisty in 1956 marks the assent of tools to understanding brain injury beyond the whole organ level. Cellular and molecular approaches supervened especially after the discovery of apoptosis or programmed cell death by Kerr and colleagues in 1972,2 which lead to a focus on cellular and molecular cell death mechanisms. We now know that ischemic cell death is accompanied by transcriptional and translational events that may modulate ischemic injury. The induction of death-inducing gene products, such as the caspases, and the regulation of prosurvival gene products, such as antiapoptotic bcl-2 family genes, are now well known.3 More broad-based approaches to gene modulation in disease, such as …

Involvement of oxidative stress in the rapid Akt1 regulating a JNK scaffold during ischemia in rat hippocampus

It has been well documented that the activation of Akt1 and JNK pathways are involved in the neuronal cell death in cerebral ischemia. In this study, we describe a novel interaction between Akt1 and JNK interacting protein 1 (JIP-1). We first detected the interaction of Akt1 and JIP-1 in hippocampus at various time points of ischemia. In the basal state, JIP-1 bind to Akt1, MLK3 at maximum while JIP-1 binds to JNK3 at minimum. Ischemia stimulus decreased the Akt1–JIP-1 interaction and concomitantly increased association between JIP-1 and JNK3. While MLK3 binding to JIP-1 decreased, similar to Akt1–JIP-1 interaction during ischemia. These results indicated that Akt1 interaction with JIP-1 inhibited JIP-1-mediated potentiation of JNK activity by decreasing JIP-1 binding to specific JNK pathway kinases. Akt1 binding to JIP-1 acts as a regulatory gate preventing JNK activation, which is opened under conditions ischemia injury. Administration of antioxidant N-acetylcysteine (NAC) can obviously affected the level of MLK3, JNK3 and Akt1 binding to JIP-1 and JNK3 activation in the hippocampus at 15 min ischemia. The findings suggest that Akt1 regulating JNK scaffold and then regulating JNK activation were closely associated with reactive oxygen species (ROS) during cerebral ischemia.

Exciting, Radical, Suicidal

It is now widely accepted that not all brain cells die immediately after stroke. Surrounding a core of severe and rapid tissue injury, brain cell death evolves more slowly in a heterogeneous area that has been called the penumbra.1,2 In 1977, Astrup et al provided one of the first experimental demonstrations of the penumbra in a baboon model of cerebral artery occlusion.3 This region of brain in acute ischemic stroke was found to be electrically silent but sufficiently active metabolically to sustain membrane potentials. Neurons within the penumbra are functionally impaired but not yet dead. Without reperfusion, the penumbra collapses, brain cells die, and the lesion expands. Although the precise timing and cellular pathways involved are not fully understood, it is believed that mechanisms actively promoting cell death are triggered after stroke. Remarkable progress has been made in dissecting these mechanisms over the past 3 decades. Three major pathways have emerged: excitotoxicity, oxidative stress, and apoptosis, and they are inextricably linked. Here, we explore the notion that stroke is most fruitfully investigated by an integrative “systems biology” approach that encompasses cell death and survival signaling within all components of the neurovascular unit. How do brain cells die after stroke? A large body of data suggest that it may be exciting (glutamate and excitotoxicity), radical (oxidative stress and free radicals), and suicidal (apoptotic-like pathways).4–6 Simply put, when brain fails to generate sufficient ATP, such as after oxygen and glucose deprivation, energy failure occurs and ionic gradients are lost. Glutamate is released, reuptake processes are impaired, and this excitatory amino acid binds to its postsynaptic receptors and promotes excessive calcium entry and calcium release. Calcium-dependent synthases and proteases contribute to cell and tissue demise by degrading key cytoskeletal and enzymatic proteins, and generating nitric oxide and peroxynitrite. Mitochondrial functions …

The neuroprotective effects of K252a through inhibiting MLK3/MKK7/JNK3 signaling pathway on ischemic brain injury in rat hippocampal CA1 region

It has been well documented that the activation of c-Jun N-terminal protein kinase (JNK) pathway and caspase-3 signal are involved in the delayed neuronal cell death in cerebral ischemia. In this study, we first detected the activation pattern of JNK signaling including mixed lineage kinase (MLK)3, mitogen-activated protein kinase kinase (MKK)7 and JNK3 in hippocampal CA1 and CA3/DG regions at various time points after 15 min of ischemia. These results indicated that cerebral ischemia induced the continuous activation of MLK3/MKK7/JNK3 cascade, which all had two active waves only in the CA1 region. We also detected the phosphorylation of JNK substrates c-Jun and Bcl-2, and the activation of a key protease of caspase-3 in CA1 region, which only had one active peak, respectively. Because K252a has recently been shown to be a potent inhibitor of MLK3 activity both in vivo and in vitro, we further examined the possible effects and mechanism of this interesting drug in cerebral ischemia. In our present paper, we found that administration of K252a 20 min prior to ischemia inhibited MLK3/MKK7/JNK3 signaling, Bcl-2 phosphorylation, the activation of c-Jun and caspase-3, but had no significant effects on these protein expressions. Additionally, pretreatment of K252a significantly increased the number of the surviving CA1 pyramidal cells at 5 days of reperfusion. Our results suggest that K252a play a neuroprotective role in ischemic injury via inhibition of the JNK pathway, involving the death effector of caspase-3. Thus, JNK signaling may eventually emerge as a prime target for novel therapeutic approaches to treatment of ischemic stroke, and K252a may serve as a potential and important neuroprotectant in therapeutic aspect in ischemic stroke.

Oxyresveratrol ( trans-2,3′,4,5′-tetrahydroxystilbene) is neuroprotective and inhibits the apoptotic cell death in transient cerebral ischemia

Oxidative stress is one of the major pathological factors in the cascade that leads to cell death in cerebral ischemia. Here, we investigated the neuroprotective effect of a naturally occurring antioxidant, oxyresveratrol, to reduce brain injury after cerebral stroke. We used the transient rat middle cerebral artery occlusion (MCAO) model of brain ischemia to induce a defined brain infarction. Oxyresveratrol was given twice intraperitoneally: immediately after occlusion and at the time of reperfusion. Oxyresveratrol (10 or 20 mg/kg) significantly reduced the brain infarct volume by approximately 54% and 63%, respectively, when compared to vehicle-treated MCAO rats. Also, the neurological deficits as assessed by different scoring methods improved in oxyresveratrol-treated MCAO rats. Histological analysis of apoptotic markers in the ischemic brain area revealed that oxyresveratrol treatment diminished cytochrome c release and decreased caspase-3 activation in MCAO rats. Also, staining for apoptotic DNA showed that the number of apoptotic nuclei in ischemic brain was reduced after oxyresveratrol treatment as compared to the vehicle-treated MCAO rats. This dose-dependent neuroprotective effect of oxyresveratrol in an in vivo stroke model demonstrates that this drug may prove to be beneficial for a therapeutic strategy to limit brain injury in acute brain ischemia.

Prospects for antiapoptotic drug therapy of neurodegenerative diseases

The evidence for a role of apoptosis in the neurodegenerative diseases, Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS), and in the more acute conditions of cerebral ischemia, traumatic brain injury (TBI), and spinal cord injury (SCI) is reviewed with regard to potential intervention by means of small antiapoptotic molecules. In addition, the available animal models for these diseases are discussed with respect to their relevance for testing small antiapoptotic molecules in the context of what is known about the apoptotic pathways involved in the diseases and the models. The principal issues related to pharmacotherapy by apoptosis inhibition, i.e., functionality of rescued neurons and potential interference with physiologically occurring apoptosis, are pointed out. Finally, the properties of a number of small antiapoptotic molecules currently under clinical investigation are summarized. It is concluded that the evidence for a role of apoptosis at present is more convincing for PD and ALS than for AD. In PD, damage to dopaminergic neurons may occur through oxidative stress and/or mitochondrial impairment and culminate in activation of an apoptotic, presumably p53-dependent cascade; some neurons experiencing energy failure may not be able to complete apoptosis, end up in necrosis and give rise to inflammatory processes. These events are reasonably well reflected in some of the PD animal models, notably those involving 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and rotenone. In sporadic ALS, an involvement of pathways involving p53 and Bcl-2 family members appears possible if not likely, but is not established. The issue is important for the development of antiapoptotic compounds for the treatment of this disease because of differential involvement of p53 in different mutant superoxide dismutase (SOD) mice. Most debated is the role of apoptosis in AD; this implies that little is known about potentially involved pathways. Moreover, there is a lack of suitable animal models for compound evaluation. Apoptosis or related phenomena are likely involved in secondary cell death in cerebral ischemia, TBI, and SCI. Most of the pertinent information comes from animal experiments, which have provided some evidence for prevention of cell death by antiapoptotic treatments, but little for functional benefit. Much remains to be done in this area to explore the potential of antiapoptotic drugs. There is a small number of antiapoptotic compounds in clinical development. With some of them, evidence for maintenance of functionality of the rescued neurons has been obtained in some animal models, and the fact that they made it to phase II studies in patients suggests that interference with physiological apoptosis is not an obligatory problem. The prospect that small antiapoptotic molecules will have an impact on the therapy of neurodegenerative diseases, and perhaps also of ischemia and trauma, is therefore judged cautiously positively.

Hypoxia-inducible factor-1alpha accumulation in the brain after experimental intracerebral hemorrhage.

Hypoxia-inducible factor-1 (HIF-1), a transcription factor composed of HIF-1alpha and HIF-1beta protein subunits, has been implicated in cellular protection and cell death in cerebral ischemia. The extent to which HIF-1 plays a role in brain pathology during intracerebral hemorrhage (ICH) is unknown. This study determined whether HIF-1alpha is upregulated at different time points in a rat model of ICH and the role of thrombin and red blood cell lysis in upregulation. Recently, thrombin has been implicated as a nonhypoxic regulator of HIF-1alpha in cultured smooth-muscle cells. Male Sprague-Dawley rats received intracerebral infusions of saline, autologous whole blood, blood plus hirudin, thrombin, thrombin plus hirudin, or lysed erythrocytes. Rats were killed at different time points for Western blot analysis, immunohistochemistry, immunofluorescent double staining, and reverse transcription polymerase chain reaction measurements of HIF-1alpha. HIF-1alpha protein levels increased without changing HIF-1alpha messenger RNA levels after intracerebral infusions of blood, thrombin, and lysed erythrocytes. HIF-1alpha positive cells, which proved to be neurons, were found in the brain after ICH. Hirudin, a specific thrombin inhibitor, reduced HIF-1alpha upregulation in response to both thrombin and blood. This study demonstrates that perihematomal HIF-1alpha protein is upregulated after ICH. This phenomenon is an early response of brain parenchyma to the clot. Thrombin and erythrocyte lysate are involved in HIF-1alpha upregulation through reducing HIF-1alpha degradation.

Bcl-2 family gene products in cerebral ischemia and traumatic brain injury.

The proto-oncogene bcl-2 plays a key role in regulating programmed cell death in neurons. The present review discusses the mechanisms by which bcl-2 family genes regulate programmed cell death, and their role in controlling cell death in cerebral ischemia and traumatic brain. Expression of several bcl-2 family members is altered in brain tissues after ischemia and trauma, suggesting that bcl-2 family genes could play a role in determining the fate of injured neurons. Furthermore, alteration of expression of bcl-2 family genes using transgenic approaches, viral vectors, or anti-sense oligonucleotides modifies neuronal cell death and neurological outcome after injury. These data suggest that the activity of bcl-2 family gene products participates in determining cellular and neurologic outcomes in ischemia and trauma. Strategies that either mimic the death-suppressor effects or inhibit the death-promoter effects of bcl-2 family gene products may improve outcome after ischemia and trauma.

Apoptosis in a neonatal rat model of cerebral hypoxia-ischemia.

The mechanisms of excitotoxic cell death in cerebral ischemia are poorly understood. In addition to necrosis, apoptotic cell death may occur. The purpose of this study was to determine whether an established model of cerebral hypoxia-ischemia in the neonatal rat demonstrates any features of apoptosis. Seven-day-old neonatal rats underwent bilateral, permanent carotid ligation followed by 1 hour of hypoxia, and their brains were examined 1, 3, and 4 days after hypoxia-ischemia. The severity of ischemic damage was assessed in the dentate gyrus and frontotemporal cortex by light microscopy. Immunocytochemistry was performed to detect the cleavage of actin by caspases, a family of enzymes activated in apoptosis. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) reactivity was examined in the cortical infarction bed and dentate gyrus. Neonatal rat brain DNA was run on agarose gel electrophoresis to detect DNA fragmentation. Ethidium bromide-staining and electron microscopy were used to determine whether apoptotic bodies, 1 of the hallmarks of apoptosis, were present. The frontotemporal cortex displayed evidence of infarction, and in most rats the dentate gyrus showed selective, delayed neuronal death. Immunocytochemistry demonstrated caspase-related cleavage of actin. TUNEL and DNA electrophoresis provided evidence of DNA fragmentation. Ethidium bromide-staining and electron microscopy confirmed the presence of chromatin condensation and apoptotic bodies. Features of apoptosis are present in the described model of cerebral hypoxia-ischemia. Apoptosis may represent a mode of ischemic cell death that could be the target of novel treatments that could potentially expand the therapeutic window for stroke.

Apoptosis and ischemic infarct

Apoptosis and programmed cell death have attracted major scientific interest during the last few years. Apoptosis can be differentiated from necrosis by means of morphological, biochemical, molecular and pharmacological parameters. Several molecular pathways leading to apoptosis have been elucidated over the last few years. Apoptosis is not only important for physiological cell turnover, but also plays a role in many pathological processes. Recently, a number of laboratories have found evidence of apoptotic cell death in cerebral ischemia in animal models. Quantitatively, apoptosis seems to be important in the border zone of the infarction, the so-called penumbra. Since parts of the apoptotic cascade can be inhibited, pharmacological treatment seems feasible. The so-called caspase inhibitors are promising therapeutic agents for the treatment of acute stroke and may be administered with other neuroprotective medications in humans in the near future.