Xiaokun Geng

Neuroprotective Effect of Acute Ethanol Administration in a Rat With Transient Cerebral Ischemia

Background and Purpose— Ethanol consumption is inversely associated with the risk of ischemic stroke, suggesting a neuroprotective effect. In a rat model of transient cerebral ischemia, we identified ethanol as a possible treatment for acute ischemic stroke. Methods— Sprague-Dawley rats were subjected to middle cerebral artery occlusion for 2 hours. Five sets of experiments were conducted: to determine the dose–response effect of ethanol on brain infarction and functional outcome; to determine whether combining ethanol and hypothermia produces synergistic neuroprotection; to determine the therapeutic windows of opportunity for ethanol in stroke; to test whether ethanol promotes intracerebral hemorrhage in a hemorrhagic or ischemic stroke or after administration of thrombolytics; and to test the affect of ethanol on hypoxia-inducible factor-1&agr; protein expression. Results— Ethanol at 1.5 g/kg reduced infarct volume and behavioral dysfunction when administered at 2, 3, or 4 hours after middle cerebral artery occlusion. The protective effect of ethanol was not improved when paired with hypothermia. Ethanol did not promote cerebral hemorrhage in hemorrhagic or ischemic stroke in combination with recombinant tissue-type plasminogen activator or urokinase. Ethanol treatment (1.5 g/kg) increased protein levels of hypoxia-inducible factor-1&agr; at 3 hours postreperfusion. Conclusions— Ethanol exerts a strong neuroprotective effect when administered up to 4 hours after ischemia, increases expression of hypoxia-inducible factor-1&agr;, and does not promote intracerebral hemorrhage when used with thrombolytics. Ethanol is a potential neuroprotectant for acute ischemic stroke.

Remote Ischemic Preconditioning-Mediated Neuroprotection against Stroke is Associated with Significant Alterations in Peripheral Immune Responses.

Remote ischemic preconditioning (RIPC) of a limb is a clinically feasible strategy to protect against ischemia–reperfusion injury after stroke. However, the mechanism underlying RIPC remains elusive.

Effect of remote ischemic postconditioning on an intracerebral hemorrhage stroke model in rats

Abstract Background and purpose: While recent studies suggest that remote ischemic postconditioning (RIP) therapy may be of benefit to patients with acute ischemic stroke, RIP’s effects on intracerebral hemorrhage (ICH) still remains unclear. In the present study, the use of RIP in a rat model ICH was investigated to elucidate any potential beneficial or detrimental effects as determined by motor testing, blood brain barrier integrity, and brain water content, as well as aquaporin-4 (AQP-4) and matrix metalloproteinase-9 (MMP-9) expression. Methods: ICH was induced in Sprague–Dawley rats and they were randomized into either a control (n = 24) or RIP treatment (n = 24) group. RIP was performed by repetitive, brief occlusion and release of the bilateral femoral arteries. Functional outcome in each group was assessed by neurologic deficits on vibrissae-elicited forelimb placing test and a 12-point outcome scale. At 72 hours, brain blood volume, water content, blood–brain barrier (BBB) permeability, and protein expression of AQP-4 and MMP-9 were determined. Results: This collagenase model yielded well-defined striatal hematomas. Vibrissae-elicited forelimb placement was significantly (P<0·01) affected by ICH. However, there was no significant difference between the RIP and control groups at either 24 or 72 hours. A 12-point neurological deficit score also failed to differentiate between the RIP and control. There were no significant differences between the two groups in cerebral blood volumes, brain water content, Evans blue extravasations, and expressions of AQP-4 and MMP-9. Conclusions: Although RIP did not show a beneficial effect in our ICH model, treatment with RIP did not exacerbate ICH.

Neuroprotection conferred by post-ischemia ethanol therapy in experimental stroke: an inhibitory effect on hyperglycolysis and NADPH oxidase activation

Ethanol provides neuroprotection following ischemia/reperfusion. This study assessed ethanols effect on hyperglycolysis and NADPH oxidase (NOX) activation. Adult, male Sprague–Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 2 h. Three sets of experiments were conducted to determine ethanols effect on (i) conferring neuroprotection by measuring infarct volume and neurological deficits 24 h post reperfusion; (ii) cerebral glucose metabolism and lactic acidosis by measuring brain and blood glucose concentrations and protein expression of glucose transporter 1 and 3 (GLUT1, GLUT3), phosphofructokinase (PFK), as well as lactic acidosis by measuring lactate dehydrogenase (LDH), and lactate; and (iii) nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activation by detecting enzymatic activity and subunit expression at 3 h after reperfusion. When administered upon reperfusion, ethanol (1.5 g/kg) reduced infarct volume by 40% (p < 0.01) and neurological deficits by 48% at 24 h post reperfusion while reducing (p < 0.01) elevations in glycolytic protein expression and lactate levels during early reperfusion (3 h). Ethanol increased the reductions in cerebral glucose concentration at 3 h post reperfusion by 64% (p < 0.01) while enhancing (p < 0.01) post stroke blood glucose concentration, suggesting a reduced cellular glucose uptake and utilization. Ethanol decreased (p < 0.01) stroke‐induced NOX activation by reducing enzymatic activity and gp91phox expression by 45% and 38%, respectively. Post‐ischemia ethanol treatment exerts neuroprotection through attenuation of hyperglycolysis and associated NOX activation. Because of the lack of associated hypoglycemia and selectivity toward decreasing cerebral metabolism, further investigation of ethanols use as a post‐stroke therapy, especially in the context of hyperglycemia, seems warranted.

Reduced apoptosis by combining normobaric oxygenation with ethanol in transient ischemic stroke.

BACKGROUND AND PURPOSE The effect of normobaric oxygen (NBO) on apoptosis remains controversial. The present study evaluated the effect of NBO on ischemia-induced apoptosis and assessed the potential for improved outcomes by combining NBO administration with another neuroprotective agent, ethanol, in a rat stroke model. METHODS Rats were subjected to right middle cerebral artery occlusion (MCAO) for 2h. At the onset of reperfusion, ischemic animals received either NBO (2h duration), an intraperitoneal injection of ethanol (1.0g/kg), or both NBO and ethanol. Extent of brain injury was determined by infarct volume, neurological deficit, and apoptotic cell death. Expression of pro- and anti-apoptotic proteins was evaluated through Western immunoblotting. RESULTS Given alone, NBO and ethanol each slightly (p<0.05) reduced infarct volume to 38% and 37%, respectively, as compared to the impressive reduction of 51% (p<0.01) seen with combined NBO-ethanol administration. Neurologic deficits were also significantly reduced by 48% with combined NBO-ethanol therapy, as compared to lesser reductions of 24% and 23% with NBO or ethanol, respectively. Combined NBO-ethanol therapy decreased apoptotic cell death by 49%, as compared to 31% with NBO and 30% with ethanol. Similarly, combination therapy significantly increased expression of anti-apoptotic factors (Bcl-2 and Bcl-xL) and significantly reduced expression of pro-apoptotic proteins (BAX, Caspase-3, and AIF), as compared to the minimal or nil protein expression changes elicited by NBO or ethanol alone. CONCLUSIONS In rats subjected to ischemic stroke, NBO administration salvages ischemic brain tissue through evidenced decrease in apoptotic cell death. Combined NBO therapy with ethanol administration greatly improves both degree of neuroprotection and associated apoptosis.

Synergetic Neuroprotection of Normobaric Oxygenation and Ethanol in Ischemic Stroke Through Improved Oxidative Mechanism

Background and Purpose— Normobaric oxygenation (NBO) and ethanol both provide neuroprotection in stroke. We evaluated the enhanced neuroprotective effect of combining these 2 treatments in a rat stroke model. Methods— Sprague-Dawley rats were subjected to middle cerebral artery occlusion for 2 hours. Reperfusion was then established and followed by treatment with either (1) an intraperitoneal injection of ethanol (1.0 g/kg), (2) NBO treatment (2-hour duration), or (3) NBO plus ethanol. The extent of brain injury was determined by infarct volume and motor performance. Oxidative metabolism was determined by ADP/ATP ratios, reactive oxygen species levels, nicotinamide adenine dinucleotide phosphate oxidase activity, and pyruvate dehydrogenase activity. Protein expression of major nicotinamide adenine dinucleotide phosphate oxidase subunits (p47phox, gp91phox, and p67phox) and the enzyme pyruvate dehydrogenase was evaluated through Western immunoblotting. Results— NBO and ethanol monotherapies each demonstrated reductions as compared to stroke without treatment in infarct volume (36.7% and 37.9% vs 48.4%) and neurological deficits (score of 6.4 and 6.5 vs 8.4); however, the greatest neuroprotection (18.8% of infarct volume and 4.4 neurological deficit) was found in animals treated with combination therapy. This neuroprotection was associated with the largest reductions in ADP/ATP ratios, reactive oxygen species levels, and nicotinamide adenine dinucleotide phosphate oxidase activity, and the largest increase in pyruvate dehydrogenase activity. Conclusions— Combination therapy with NBO and ethanol enhances the neuroprotective effect produced by each therapy alone. The mechanism behind this synergistic action is related to changes in cellular metabolism after ischemia reperfusion. NBO plus ethanol is attractive for clinical study because of its ease of use, tolerability, and tremendous neuroprotective potential in stroke.

NOX Activation by Subunit Interaction and Underlying Mechanisms in Disease

Nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase (NOX) is an enzyme complex with the sole function of producing superoxide anion and reactive oxygen species (ROS) at the expense of NADPH. Vital to the immune system as well as cellular signaling, NOX is also involved in the pathologies of a wide variety of disease states. Particularly, it is an integral player in many neurological diseases, including stroke, TBI, and neurodegenerative diseases. Pathologically, NOX produces an excessive amount of ROS that exceed the body’s antioxidant ability to neutralize them, leading to oxidative stress and aberrant signaling. This prevalence makes it an attractive therapeutic target and as such, NOX inhibitors have been studied and developed to counter NOX’s deleterious effects. However, recent studies of NOX have created a better understanding of the NOX complex. Comprised of independent cytosolic subunits, p47-phox, p67-phox, p40-phox and Rac, and membrane subunits, gp91-phox and p22-phox, the NOX complex requires a unique activation process through subunit interaction. Of these subunits, p47-phox plays the most important role in activation, binding and translocating the cytosolic subunits to the membrane and anchoring to p22-phox to organize the complex for NOX activation and function. Moreover, these interactions, particularly that between p47-phox and p22-phox, are dependent on phosphorylation initiated by upstream processes involving protein kinase C (PKC). This review will look at these interactions between subunits and with PKC. It will focus on the interaction involving p47-phox with p22-phox, key in bringing the cytosolic subunits to the membrane. Furthermore, the implication of these interactions as a target for NOX inhibitors such as apocynin will be discussed as a potential avenue for further investigation, in order to develop more specific NOX inhibitors based on the inhibition of NOX assembly and activation.

Effect of Normobaric Oxygen Therapy in a Rat Model of Intracerebral Hemorrhage

Background and Purpose— Normobaric oxygen (NBO) therapy may be neuroprotective in acute ischemic stroke. However, how NBO may affect intracerebral hemorrhage is unclear. We tested NBO in a rat model of striatal intracerebral hemorrhage. Methods— Intracerebral hemorrhage was induced by stereotactic injection of collagenase Type VII (0.5 U) into the right striatum of male Sprague-Dawley rats. One hour later, rats were randomized into controls (n=13) versus NBO treatment (n=13). NBO was applied for 2 hours. Hemorrhagic blood volume, brain water content, and neurological outcomes (forelimb placement test, forelimb asymmetry, neuroscore) were quantified at 72 hours. Experiments were repeated in a second independent laboratory to assess reproducibility in neurological outcomes (n=10 per group). Results— NBO did not worsen hemorrhage severity or brain edema. There were no significant differences in hemorrhagic blood volumes (control, 6.4±0.9 &mgr;L versus NBO, 7.0±2.1 &mgr;L; P=0.18) or brain water content (control, 81.9%±1.1% versus NBO, 81.6%±0.5%; P=0.58). NBO did not affect any of the neurological outcome tests in the primary or secondary studies. Conclusions— NBO therapy may not worsen outcomes in intracerebral hemorrhage.

Preconditioning in neuroprotection: From hypoxia to ischemia

Sublethal hypoxic or ischemic events can improve the tolerance of tissues, organs, and even organisms from subsequent lethal injury caused by hypoxia or ischemia. This phenomenon has been termed hypoxic or ischemic preconditioning (HPC or IPC) and is well established in the heart and the brain. This review aims to discuss HPC and IPC with respect to their historical development and advancements in our understanding of the neurochemical basis for their neuroprotective role. Through decades of collaborative research and studies of HPC and IPC in other organ systems, our understanding of HPC and IPC-induced neuroprotection has expanded to include: early- (phosphorylation targets, transporter regulation, interfering RNA) and late- (regulation of genes like EPO, VEGF, and iNOS) phase changes, regulators of programmed cell death, members of metabolic pathways, receptor modulators, and many other novel targets. The rapid acceleration in our understanding of HPC and IPC will help facilitate transition into the clinical setting.

Endovascular Hypothermia in Acute Ischemic Stroke Pilot Study of Selective Intra-Arterial Cold Saline Infusion

Background and Purpose— We conducted a pilot feasibility and safety study of selective brain cooling with intra-arterial infusion of cold saline combined with endovascular reperfusion for acute ischemic stroke. Methods— Patients with large-vessel occlusion within 8 hours after symptom onset were enrolled. All patients received intra-arterial recanalization combined with infusion of cold isotonic saline (4°C) in the ischemic territory through the angiographic catheter. Results— Twenty-six patients underwent the procedure, which was technically successful in all. The temperature of ischemic cerebral tissue was decreased by at least 2°C during infusion of the cold solution, and systemic temperature was mildly reduced (maximum 0.3°C). No obvious complications related to intra-arterial hypothermia were observed. Conclusions— Selective brain cooling by intra-arterial infusion of cold saline combined with endovascular recanalization therapy in acute ischemic stroke seems feasible and safe.