Exacerbation Of Ischemic Injury Research Articles

Abstract WP322: GluN2A-NMDAR Mediated Increased Neuronal Prostaglandin E2 Release and Microglial Activation Exacerbates Ischemic Brain Injury Under Hyperhomocysteinemic Condition

Hyperhomocysteinemia (Hhcy), a metabolic disorder is recognized as a risk factor for ischemic stroke. However, the impact of Hhcy on stroke outcome is not known. Using a rodent model of experimental stroke, we recently showed that predisposition to Hhcy exacerbates ischemic brain injury. The findings also showed that Hhcy triggers GluN2A subunit containing NMDA receptor (GluN2A-NMDAR) stimulation that act in conjunction with ischemia-induced pathways involving GluN2B subunit containing NMDAR (GluN2B-NMDAR) stimulation to enhance stroke pathology. The current study evaluated whether GluN2A-NMDAR-mediated increased expression of cyclooxygenase-2 (COX-2) and excessive release of the pro-inflammatory mediator prostaglandin E2 (PGE2) from neurons results in increased microglial activation and exacerbated ischemic brain injury. HHcy was induced by implantation of Alzet pumps with L-homocysteine in adult male Wistar rats. Cerebral ischemia was induced by middle cerebral artery occlusion (60 min) in control and Hhcy rats. In a subset of Hhcy rats, NVP-AAM077, a pharmacological inhibitor of GluN2A-NMDAR was injected intravenously at the onset of stroke. At specified times following reperfusion, brain tissues were processed for FluroJade staining, immunoblotting with COX2 antibody, immunohistochemistry with COX2/NeuN and Iba1 antibodies, prostaglandin E2 (PGE2) assay and RT-PCR. FluroJade staining shows an early onset and exacerbation of ischemic injury in Hhcy rats. This involves increased expression of COX-2 in neurons, release of PGE2, and a shift in microglial morphology from ramified state to active amoeboid state, 6h after reperfusion. Administration of NVP-AAM077 attenuates the increase in COX2 expression and PGE2 levels as well as microglial activation. The findings demonstrate that an early onset of neuroinflammatory response mediated through GluN2A-NMDAR stimulation could contribute to the exacerbation of ischemic brain injury under Hhcy conditions. As GluN2A-NMDAR stimulation is primarily involved in promoting synaptic plasticity, neuronal growth, and survival, establishing their role in promoting neuroinflammatory response could lead to a paradigm shift in our understanding of the consequences of NMDAR signaling.

Abstract WP217: Key Role Of Translocator Protein (TSPO) In Stress-aggravated Ischemic Injury After Stroke

Introduction: Ischemic stroke is the leading cause of death and disability worldwide. It was recently reported that mental stress could aggravate stroke damage. However, the causal relationship between mental stress and stroke outcomes remained unclear. In current study, we determined whether mitochondria dysfunction was induced by mental stress and thus enhanced brain damage after ischemic stroke. The translocator protein (TSPO) is a transmembrane protein located on the outer mitochondria membrane (OMM) and induced mitochondrial dysfunction. This study focused on the key role of TSPO in stress aggravated ischemia/reperfusion injury. Methods: Six-week-old male C57BL/6J mice were divided into four groups: control, chronic restraint stress (CRS), TSPO antagonist (PK11195, 3mg/kg, i.p.) and TSPO antagonist+CRS. Mice in CRS were placed in ventilated 50mL tubes 6 hours/day for 28 days. On the day 29 th , behavioral tests were conducted to measure the depressive-like levels. Mice then were subjected to middle cerebral artery occlusion (MCAO). At 6/24 hours after MCAO, TTC staining and modified scoring systems were applied to analyze infarct volume and neurological deficits, and the expression of TSPO was detected using immunofluorescence. Apoptotic cell death and apoptotic proteins (Bcl-2, Bax and caspase-3/9) were measured by TUNEL and Western blot. Effects of TSPO on mitochondrial membrane potential (MMP) and mito-ROS were measured by JC-1 staining and live cell imaging dyes (mitoSOX and DCFDA) in vitro . Results: As depressive model, CRS aggravated infarct volume and neurological deficits after MCAO, with increased levels of apoptosis (cell death and protein expressions), in association with increased TSPO levels. TSPO antagonist could decrease stress-aggravated stroke injury and apoptotic cell death, indicating TSPO was involved in stress related stroke outcomes. In vitro , MMP was increased and mito-ROS was decreased after TSPO siRNA transfection, suggesting TSPO may induce mitochondria deficits through producing more mito-ROS. Conclusions: Mental stress by CRS exacerbated ischemic injury after stroke. Importantly, the results showed that TSPO may play a key role in inducing mitochondrial dysfunction in stress-aggravated stroke damage.

Possible Engagement of Nicotinic Acetylcholine Receptors in Pathophysiology of Brain Ischemia-Induced Cognitive Impairment

Post-stroke disabilities like cognitive impairment impose are complex conditions with great economic burdens on health care systems. For these comorbidities, no effective therapies have been identified yet. Nicotinic acetylcholine receptors (nAChRs) are multifunctional receptors participating in various behavioral and neurobiological functions. During brain ischemia, the increased glutamate accumulation leads to neuronal excitotoxicity as well as mitochondrial dysfunction. These abnormalities then cause the increased levels of oxidants, which play key roles in neuronal death and apoptosis in the infarct zone. Additionally, recall of cytokines and inflammatory factors play a prominent role in the exacerbation of ischemic injury. As well, neurotrophic factors' insufficiency results in synaptic dysfunction and cognitive impairments in ischemic brain. Of note, nAChRs through various signaling pathways can participate in therapeutic approaches such as cholinergic system's stimulation, and reduction of excitotoxicity, inflammation, apoptosis, oxidative stress, mitochondrial dysfunction, and autophagy. Moreover, the possible roles of nAChRs in neurogenesis, synaptogenesis, and stimulation of neurotrophic factors expression have been reported previously. On the other hand, the majority of the above-mentioned mechanisms were found to be common in both brain ischemia pathogenesis and cognitive function tuning. Therefore, it seems that nAChRs might be known as key regulators in the control of ischemia pathology, and their modulation could be considered as a new avenue in the multi-target treatment of post-stroke cognitive impairment.

The Acid Sphingomyelinase/ Ceramide System as Target for Ischemic Stroke Therapies.

In this review, we summarize implications of the acid sphingomyelinase/ ceramide system in ischemic stroke. Acid sphingomyelinase catalyzes the formation of the bioactive sphingolipid ceramide which coalesces into membrane platforms and has a pivotal role in inflammation, cell signaling and death. Cerebral ischemia increases acid sphingomyelinase activity and elevates brain ceramide levels, which has been associated with the exacerbation of ischemic injury and deterioration of stroke outcome. In view of the fact that lowering acid sphingomyelinase activity and ceramide level was shown to protect against ischemic injury and ameliorate neurological deficits, the acid sphingomyelinase/ ceramide system might represent a promising target for stroke therapies.

VEGF Stimulates the ERK 1/2 Signaling Pathway and Apoptosis in Cerebral Endothelial Cells After Ischemic Conditions

Cerebral endothelial cells that line microvessels play an important role in maintaining blood flow homeostasis within the brain-forming part of the blood-brain barrier. These cells are injured by hypoxia-induced reperfusion, leading to blood-brain barrier breakdown and exacerbation of ischemic injury. We investigated the roles of vascular endothelial growth factor (VEGF) and the downstream extracellular signal-regulated kinase (ERK) protein after oxygen-glucose deprivation (OGD) in primary endothelial cells. Primary mouse endothelial cells were isolated and subjected to OGD. Western analysis of VEGF and ERK 1/2 protein levels was performed. Cells were transfected with VEGF small interference RNA. A terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL) assay and DNA fragmentation assay were used on mouse endothelial cells that overexpress copper/zinc-superoxide dismutase (SOD1). VEGF protein expression was induced and its receptor, Flk-1, was stimulated by OGD. Phosphorylation of ERK 1/2 protein levels was upregulated. Inhibition of phosphorylated ERK (pERK) expression by U0126 reduced endothelial cell death by OGD. Transfection of small interfering RNA for VEGF also inhibited an increase in pERK, suggesting that VEGF acts via ERK. The TUNEL and DNA fragmentation assays showed a significant decrease in TUNEL-positivity in the SOD1-overexpressing endothelial cells compared with wild-type cells after OGD. Our data suggest that OGD induces VEGF signaling via its receptor, Flk-1, and activates ERK via oxidative-stress-dependent mechanisms. Our study shows that in cerebral endothelial cells the ERK 1/2 signaling pathway plays a significant role in cell injury after OGD.

Abstract 1618: Role of Nitric Oxide in Regulation of Myocardial Glucose Metabolism; A Potential Mechanism of Cardioprotection

Ischemia induces a switch in myocardial substrate utilization from fatty acids to glucose and stimulates nitric oxide (NO) production. Although increased NO synthesis can ameliorate ischemic injury, mechanism by which NO confers cardioprotection remains unclear. Based on our recent observation that increased NO production contributes to ischemic activation of aldose reductase (AR), we tested the hypothesis that NO prevents ischemic injury by increasing reduction of glucose to sorbitol via polyol pathway over its oxidation to lactate in glycolysis, that could prevent acidosis. Our data show that in isolated mouse hearts subjected to 30 min of ischemia, sorbitol levels increased from 43.2 ± 2.4 to 68.7 ± 4.6 nmol/mg (n=6; P < 0.05) and returned to pre-ischemic levels after 30 min of reperfusion. Treatment with eNOS agonist, bradykinin (10 μM), prior to ischemia, increased AR activity from 27.5 ± 0.7 to 35.9 ± 1.4 mU/mg (n=6; P < 0.01) and decreased lactate levels from 3.7 ± 0.6 to 1.5 ± 0.4 mM (n=6; P < 0.05) upon reperfusion. Bradykinin also decreased creatine kinase (CK) and lactate dehydrogenase (LDH) release by 65 ± 7% and 85 ± 9%, respectively. In contrast, NOS inhibitor, L-NAME (0.1 mM) decreased AR activity (22.4 ± 2.0 mU/mg), but increased the activity of glyceraldehyde-3-phosphate dehydrogenase (from 2.4 ± 0.1 to 3.5 ± 0.2 U/mg) and lactate production (5.8 ± 0.9 mM). AR inhibitor, sorbinil (0.1 mM) increased lactate production (5.9 ± 0.1 mM) and exacerbated ischemic injury. Pretreatment with bradykinin normalized lactate levels and reduced injury caused by sorbinil. To examine whether upregulation of AR is sufficient to confer cardioprotection, cardio-specific AR transgenic mice were generated. Echocardiography confirmed, that these mice had normal myocardial function and when subjected to 30 min of coronary occlusion followed by 24 h of reperfusion displayed reduced myocardial infarct size (49.6 ± 4.1 vs 64.6 ± 3.5 % of the area at risk; transgenic vs wild type; n=7; P < 0.01). Collectively, these results show that NO mediates cardioprotection by increasing glucose flux via polyol pathway, which may represent an adaptive mechanism protecting the heart from ischemic injury. This research has received full or partial funding support from the American Heart Association, AHA Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).

Blood Genomics in Human Stroke

Advances in microarray technology and the sequencing of the human genome are opening up new possibilities for applying genomic information in clinical medicine, using information about structural polymorphisms in DNA and changes in gene expression as measured by mRNA. Gene expression profiling studies in cancer samples have led to the identification of clinical signatures that are already being applied in some centers. In stroke, it may be possible to use peripheral blood as a source of mRNA to study gene expression. After stroke, there is a selective recruitment and migration of white blood cells to the ischemic focus in the brain. This appears to involve all white cell types and is believed to impact significantly on tissue and clinical outcome through the exacerbation of ischemic injury, particularly after reperfusion, on the one hand, and conversely contributing to tissue remodeling and repair days to weeks after stroke. The first results from clinical studies in ischemic stroke suggest that a gene expression signature can be demonstrated from peripheral white blood cells and that this represents at least a partial adaptation to the altered cerebral microenvironment. Further studies are indicated to see whether these methods may lead to new management approaches for stroke.

Reductions in mRNA of the neuroprotective agent, neuroserpin, after cerebral ischemia/reperfusion in diabetic rats

The aim of this study is to investigate disturbances in fibrinolytic components in diabetic rats with middle cerebral artery occlusion (MCAO). Comparison of cerebral injury at 23 h after reperfusion indicated that infarction volumes were increased in diabetic rats as compared with normal rats. Cerebral ischemia/reperfusion in normal and diabetic rats was accompanied by increased expression of tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), plasminogen activator inhibitor 1 (PAI-1) and neuroserpin (NSP) mRNA after reperfusion. Most importantly, the expression of NSP mRNA, but not t-PA, u-PA and PAI-1 mRNA, was reduced to undetectable levels at 11 and 23 h after reperfusion in diabetic rats as compared with normal rats. Although activity of PA (t-PA and u-PA) and the ratio of PA/PAI were increased at 5 h after reperfusion in both ischemic brains of diabetic and normal rats, the levels in diabetic rats were lower than that in normal rats. We speculate that the exacerbation of ischemic injury in diabetic rats may be related to the reduction of fibrinolytic component and the neuroprotective role of NSP. Further study of the efficacy of NSP in the pathogenesis and treatment of cerebral ischemia may provide novel insights.

Reduction of neuroprotective role of neuroserpin after cerebral ischemia/reperfusion in diabetic rats

Background: To investigate disturbances in fibrinolytic components in diabetic rats with middle cerebral artery occlusion. Methods: Middle cerebral artery occlusion in diabetic (induced by streptozotocin ) and control rats was performed using intraluminal procedure. Cerebral infarction volume was detected with TTC staining. The mRNA of tissue plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), plasminogen activator-1 (PAI-1), neuroserpin (NSP) was detected with RT-PCR. PA and PAI activity was determined with spectrophotometric methods. Results: Comparison of cerebral injury at 23 hours after reperfusion indicated that infarction volumes were increased in diabetic rats as compared to normal rats. Cerebral ischemia/reperfusion in normal and diabetic rats was accompanied by increased expression of t-PA, u-PA, PAI-1 and NSP mRNA at 1, 2, 5, 11, and 23 hours after reperfusion. Expression of NSP mRNA, but not t-PA, u-PA and PAI-1 mRNA, reduced to undetectable levels at 11 and 23 hours after reperfusion in diabetic rats, compared to normal rats. In ischemic brains of diabetic rats and normal rats, activity of PA and ratio of PA/PAI was significantly reduced at 5 hours after reperfusion, and that of diabetic rats reduced more. Conclusion: The exacerbation of ischemic injury in diabetic rats may related to reduction of fibrinolytic components and neuroprotective role of NSP.

Potassium channel activators cromakalim and celikalim (WAY-120,491) fail to decrease myocardial infarct size in the anesthetized canine.

The cardioprotective effects of the K channel activator drugs celikalim (WAY-120,491) and cromakalim were studied in a canine model of myocardial infarction consisting of 90 min of ischemia and 5 h of reperfusion. Intracoronary infusion of cromakalim and celikalim at 0.2 microgram/kg/min beginning 10 min before occlusion of the left circumflex coronary artery and continuing throughout the duration of the reperfusion period appeared to exacerbate ischemic injury. Infarct size (percent of risk area) was 27.7 +/- 5.6% in vehicle control animals (n = 5), 40.3 +/- 6.2% for cromakalim (n = 5) and 55.7 +/- 6.4% (p less than 0.05 vs. vehicle) for celikalim-treated animals (n = 5). When these compounds were administered intravenously, using doses shown to increase total coronary flow in nonoccluded control animals, no exacerbation of ischemic injury was observed. Anatomic infarct size was 32.8 +/- 7.1% for vehicle animals (n = 5) and 32.6 +/- 13.3 and 30.9 +/- 9.8% for cromakalim- (n = 6) and celikalim-treated (n = 5) animals, respectively. Intravenous diltiazem decreased myocardial infarct size to 16.3 +/- 7.3% (n = 5) of area at risk (p = NS vs. vehicle). The anatomic area at risk was similar in all three treatment groups, and no significant differences in rate-pressure product were observed. Results of this study suggest that K-channel-activating drugs such as cromakalim and celikalim may not be effective agents in the acute therapeutic management of myocardial ischemic injury.