Huishan Du

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.

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.

Ethanol and Normobaric Oxygen: Novel Approach in Modulating Pyruvate Dehydrogenase Complex After Severe Transient and Permanent Ischemic Stroke

Background and Purpose— Ischemic stroke induces metabolic disarray. A central regulatory site, pyruvate dehydrogeanse complex (PDHC) sits at the cross-roads of 2 fundamental metabolic pathways: aerobic and anaerobic. In this study, we combined ethanol (EtOH) and normobaric oxygen (NBO) to develop a novel treatment to modulate PDHC and its regulatory proteins, namely pyruvate dehydrogenase phosphatase and pyruvate dehydrogenase kinase, leading to improved metabolism and reduced oxidative damage. Methods— Sprague–Dawley rats were subjected to transient (2, 3, or 4 hours) middle cerebral artery occlusion followed by 3- or 24-hour reperfusion, or permanent (28 hours) middle cerebral artery occlusion without reperfusion. At 2 hours after the onset of ischemia, rats received either an intraperitoneal injection of saline, 1 dose of EtOH (1.5 g/kg) for 2- and 3-hour middle cerebral artery occlusion, 2 doses of EtOH (1.5 g/kg followed by 1.0 g/kg in 2 hours) in 4 hours or permanent middle cerebral artery occlusion, and EtOH+95% NBO (at 2 hours after the onset of ischemia for 6 hours) in permanent stroke. Infarct volumes and neurological deficits were examined. Oxidative metabolism and stress were determined by measuring ADP/ATP ratio and reactive oxygen species levels. Protein levels of PDHC, pyruvate dehydrogenase kinase, and pyruvate dehydrogenase phosphatase were assessed. Results— EtOH induced dose-dependent neuroprotection in transient ischemia. Compared to EtOH or NBO alone, NBO+EtOH produced the best outcomes in permanent ischemia. These therapies improved brain oxidative metabolism by decreasing ADP/ATP ratios and reactive oxygen species levels, in association with significantly raised levels of PDHC and pyruvate dehydrogenase phosphatase, as well as decreased pyruvate dehydrogenase kinase. Conclusions— Both EtOH and EtOH+NBO treatments conferred neuroprotection in severe stroke by affecting brain metabolism. The treatment may modulate the damaging cascade of metabolic events by bringing the PDHC activity back to normal metabolic levels.

Splenic responses in ischemic stroke: new insights into stroke pathology.

In the past decade, the significant contribution of the spleen to ischemic brain damage has gained considerable attention in stroke research. As the largest natural reservoir of immune cells, the spleen establishes critical connections with the ischemic brain during the progression of stroke and mobilizes its cells to the site of injury. Multiple “alarm” signals released from the injured brain are essential for the initiation of brain–spleen communication. Spleen‐derived cells, including neutrophils, lymphocytes, and monocytes/macrophages, are known to contribute significantly to ischemic brain damage. Understanding the dynamic splenic responses to stroke will not only provide insights into the evolvement of ischemic brain injury but will also identify potential targets for stroke treatment. Here, we review recent studies on the functions of the spleen in ischemic stroke. We have included a discussion of several therapeutic strategies that target splenic responses and reduce acute ischemic brain damage in preclinical studies. Future investigations on the effects of the spleen on long‐term stroke recovery are highly warranted.

Curcumin Protects against Ischemic Stroke by Titrating Microglia/Macrophage Polarization

Stroke is the most common type of cerebrovascular disease and is a leading cause of disability and death. Ischemic stroke accounts for approximately 80% of all strokes. The remaining 20% of strokes are hemorrhagic in nature. To date, therapeutic options for acute ischemic stroke are very limited. Recent research suggests that shifting microglial phenotype from the pro-inflammatory M1 state toward the anti-inflammatory and tissue-reparative M2 phenotype may be an effective therapeutic strategy for ischemic stroke. The dietary phytochemical curcumin has shown promise in experimental stroke models, but its effects on microglial polarization and long-term recovery after stroke are unknown. Here we address these gaps by subjecting mice to distal middle cerebral artery occlusion (dMCAO) and administering curcumin intraperitoneally (150 mg/kg) immediately after ischemia and 24 h later. Histological studies revealed that curcumin post-treatment significantly reduced cerebral ischemic damage 3 days after dMCAO. Sensorimotor functions—as measured by the adhesive removal test and modified Garcia scores—were superior in curcumin-treated mice at 3, 5, 7 and 10 days after stroke. RT-PCR measurements revealed an elevation of M2 microglia/macrophage phenotypic markers and a reduction in M1 markers in curcumin-treated brains 3 days after dMCAO. Immunofluorescent staining further showed that curcumin treatment significantly increased the number of CD206+Iba1+ M2 microglia/macrophages and reduced the number of CD16+Iba1+ M1 cells 10 days after stroke. In vitro studies using the BV2 microglial cell line confirmed that curcumin inhibited lipopolysaccharide (LPS) and interferon-γ (IFN-γ)-induced M1 polarization. Curcumin treatment concentration-dependently reduced the expression of pro-inflammatory cytokines, including TNF-α, IL-6 and IL-12p70, in the absence of any toxic effect on microglial cell survival. In conclusion, we demonstrate that curcumin has a profound regulatory effect on microglial responses, promoting M2 microglial polarization and inhibiting microglia-mediated pro-inflammatory responses. Curcumin post-treatment reduces ischemic stroke-induced brain damage and improves functional outcomes, providing new evidence that curcumin might be a promising therapeutic strategy for stroke.

Screening circular RNA expression patterns following focal cerebral ischemia in mice

Circular RNAs (circRNAs) have been demonstrated to act as microRNA (miRNA) sponges and they play important roles in regulating gene expression through a circRNA-miRNA-gene pathway. The specific roles of circRNAs in the pathogenesis of cerebral ischemia, however, are still unclear. Thus, the aim of this study is to determine circRNA expression profiles in the ischemic brain after stroke, which was induced by 45 min of transient middle cerebral artery occlusion (MCAO). The results from the circRNA microarrays revealed that 1027 circRNAs were significantly altered 48 hours after reperfusion in the ischemic brain compared with the sham group. Among them, 914 circRNAs were significantly upregulated, and the remaining 113 were significantly downregulated. In addition, the expressions of the three selected circRNAs, mmu_circRNA_40001, mmu_circRNA_013120, and mmu_circRNA_40806, were verified using quantitative real-time polymerase chain reaction (qRT-PCR). After predicting their target genes, the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses were further used to predict the associated significant cell signaling pathways and functions. The results show that the most enriched pathways are associated with the Rap1 signaling pathway and the Hippo signaling pathway, which regulate cell survival and death. Finally, we constructed an interaction network of circRNA-miRNA-target genes, including 13 miRNAs and their corresponding genes, indicating that changes in circRNA are associated with genes related with brain injury and recovery. In conclusion, circRNAs are complicated in the pathological development of brain injury after stroke, suggesting novel diagnostic and therapeutic targets for stroke therapy.

Omega-3 fatty acid supplement prevents development of intracranial atherosclerosis.

OBJECTIVES Intracranial atherosclerotic stenosis (ICAS) is one of the most common causes of stroke worldwide and, in particular, has been implicated as a leading cause of recurrent ischemic stroke. We adapted a rat model of atherosclerosis to study brain intracranial atherosclerosis, and further investigated the effect of omega-3 fatty acids (O3FA) in attenuating development of ICAS. MATERIALS AND METHODS Adult male Sprague-Dawley rats were divided into control normal-cholesterol or high-cholesterol diet groups with or without O3FA for up to 6weeks. During the first 2weeks, NG-nitro-l-arginine methyl ester (l-NAME, 3mg/mL) was added to the drinking water of the high-cholesterol groups. The rats received supplementation with O3FA (5mg/kg/day) by gavages. Blood lipids including low density lipoprotein (LDL), cholesterol (CHO), triglycerides (TG) and high density lipoprotein (HDL) were measured at 3 and 6weeks. The lumen of middle cerebral artery (MCA) and the thickness of the vessel wall were assessed. Inflammatory molecular markers were assessed by Western blot. RESULTS A high-cholesterol diet exhibited a significant increase in the classic blood markers (LDL, CHO, and TG) for atherosclerosis, as well as a decrease in HDL. These markers were found to be progressively more severe with time. Lumen stenosis and intimal thickening were increased in MCA. O3FA showed attenuation of blood lipids with an absence of morphological changes. O3FA significantly reduced the inflammatory marker CD68 in MCA and prevented monocyte chemotactic protein (MCP-1) and interferon-γ (IFN-γ) expression in the brain. O3FA similarly decreased inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL-6), markers affiliated with monocyte activity in atherosclerosis. Furthermore, O3FA significantly inhibited the expression of vascular cell adhesion molecule-1 (VCAM-1), a marker for endothelial activation. Lastly, O3FA increased ATP-binding cassette transporter A1 (ABCA1) protein expression via silent information regulator 1 (SIRT1) activation, thus increasing cholesterol efflux from macrophages to HDL. CONCLUSIONS Long-term O3FA dietary supplementation prevents the development of intracranial atherosclerosis. This O3FA effect appears to be mediated by its prevention of macrophage infiltration into the vessel wall, therefore reducing inflammation and intimal thickening. While similar effects in humans need to be determined, O3FA dietary supplement shows promising results in the prevention of ICAS.

Motor Imagery-Based Rehabilitation: Potential Neural Correlates and Clinical Application for Functional Recovery of Motor Deficits after Stroke

Motor imagery (MI), defined as the mental implementation of an action in the absence of movement or muscle activation, is a rehabilitation technique that offers a means to replace or restore lost motor function in stroke patients when used in conjunction with conventional physiotherapy procedures. This article briefly reviews the concepts and neural correlates of MI in order to promote improved understanding, as well as to enhance the clinical utility of MI-based rehabilitation regimens. We specifically highlight the role of the cerebellum and basal ganglia, premotor, supplementary motor, and prefrontal areas, primary motor cortex, and parietal cortex. Additionally, we examine the recent literature related to MI and its potential as a therapeutic technique in both upper and lower limb stroke rehabilitation.