Adam Hafeez

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.

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.

Different expression of ubiquitin C-terminal hydrolase-L1 and αII-spectrin in ischemic and hemorrhagic stroke: Potential biomarkers in diagnosis.

The two primary categories of stroke, ischemic and hemorrhagic, both have fundamentally different mechanisms and thus different treatment options. These two stroke categories were applied to rat models to identify potential biomarkers that can distinguish between them. Ischemic stroke was induced by middle cerebral artery occlusion (MCAO) without reperfusion while hemorrhagic stroke was induced by injecting collagenase IV into the striatum. Brain hemispheres and biofluids were collected at two time points: 3 and 6h after stroke. Known molecules were tested on the rat samples via quantitative immunoblotting (injured brain, CSF) and Banyans proprietary ELISA assays (CSF, serum). The injured brain quantitative analyses revealed that αII-spectrin breakdown products (SBDP150, SBDP145) were strongly increased after 6h ischemia. In CSF, SBDP145 and ubiquitin C-terminal hydrolase-L1 (UCH-L1) levels were elevated after 6h ischemic stroke detected by Western blot and ELISA. In serum UCH-L1 levels were increased after 3 and 6h of ischemia detected by ELISA. However, levels of those proteins in hemorrhagic stroke remain normal. In summary, in both the brain and the biofluids, SBDPs and UCH-L1 were elevated after ischemic but not hemorrhagic stroke. These molecules behaved differently in the two stroke models and thus may be capable of being differentiated.

A neuroproteomic and systems biology analysis of rat brain post intracerebral hemorrhagic stroke

Intracerebral hemorrhage (ICH) is a devastating form of stroke leading to a high rate of death and disability worldwide. Although it has been hypothesized that much of the IHC insult occurs in the subacute period mediated via a series of complex pathophysiological cascades, the molecular mechanisms involved in ICH have not been systematically characterized. Among the best approaches to understand the underlying mechanisms of injury and recovery, protein dynamics assessment via proteomics/systems biology platforms represent one of the cardinal techniques optimized for mechanisms investigation and biomarker identification. A proteomics approach may provide a biomarker focused framework from which to identify candidate biomarkers of pathophysiological processes involved in brain injury after stroke. In this work, a neuroproteomic approach (LC-MS/MS) was applied to investigate altered expression of proteins that are induced in brain tissue 3 h after injury in a rat model of ICH. Data from sham and focal ischemic models were also obtained and used for comparison. Based on the differentially expressed protein profile, systems biology analysis was conducted to identify associated cellular processes and related interaction maps. After LC-MS/MS analysis of the 3 h brain lysates, 86 proteins were differentially expressed between hemorrhagic and sham tissues. Furthermore, 38 proteins were differentially expressed between ischemic and sham tissues. On the level of global pathway analysis, hemorrhagic stroke proteins were shown to be involved in autophagy, ischemia, necrosis, apoptosis, calpain activation, and cytokine secretion. Moreover, ischemic stroke proteins were related to cell death, ischemia, inflammation, oxidative stress, caspase activation and apoptotic injury. In conclusion, the proteomic responses identified in this study provide key information about target proteins involved in specific pathological pathways.

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.

Reduced Apoptosis by Ethanol and Its Association with PKC-δ and Akt Signaling in Ischemic Stroke

Along with thrombolytic therapy, which has a number of limitations, stroke outcome may be improved with neuroprotective therapies that disrupt ischemic cell death. Recent research has shown a neuroprotective role of ethanol administration during ischemic stroke, such as its ability to reduce infarct volume and neurologic deficit. In order to investigate this further, we assessed the hypothesis that ethanols neuroprotective effect is through reduction of apoptosis and the modulation of the important apoptotic PKC-δ and Akt signaling pathway. Ethanol (1.5 g/kg) was given by intraperitoneal injections to 54 Sprague-Dawley rats after 2 hours of middle cerebral artery (MCA) occlusion, followed by 3 or 24 hours of reperfusion. We measured apoptotic cell death, PKC-δ, and Akt mRNA and protein expressions in each of ischemic groups with or without ethanol treatment using ELISA, real-time PCR and Western blot analysis. Our results showed that cell death was significantly increased in rats following 2 hour MCA occlusion and 24 hour reperfusion. Subsequently, cell death was significantly reduced by an administration of ethanol. We further found that ethanol administration, prior to either 3 or 24 hours of reperfusion, significantly decreased the expression of PKC-δ while simultaneously increasing the expression Akt at both mRNA and protein levels at the two points. In conclusion, our study suggests that ethanol administration following ischemic stroke modulates the gene and protein profile in such a way that it increased expression of anti-apoptotic Akt and decreased the pro-apoptotic PKC-δ. This ultimately results in a decrease in neuronal apoptosis, thus conferring neuroprotection.

Neuroprotection by peripheral nerve electrical stimulation and remote postconditioning against acute experimental ischaemic stroke

Abstract Objective: Local electrical stimulation (ES) was reported to protect the brain during ischaemic injury, while the protective effect of limb remote ischaemic postconditioning (RIPostC) was confirmed. The aim of this study was to explore whether remote peripheral nerve ES exerted neuroprotection and whether this procedure shared the same neuroprotective mechanism underlying RIPostC. Methods: Stroke in Sprague-Dawley rats was induced by distal middle cerebral artery occlusion (dMCAO). Rats were divided into five groups: dMCAO, RIPostC, ES, nerve resection (NR) + ES and RIPostC+ES. Twenty-four hours after reperfusion, rats were examined for neurobehavioural function, including forelimb fault placing test, Ludmila Belayev 12 score test, and infarct volume. The expression of Bcl-2 and cleaved-caspase-3 in ischaemic cortex was assessed by Western blot. Results: In forelimb fault placing test, as compared to the highest score in the stroke-only group, RIPostC, ES and RIPostC+ES groups showed a significantly (P < 0.01) lower score. The results were similar for the Ludmila Belayev 12 score test. The infarct volume of the treatment groups also exhibited significant (P < 0.01) reduction as compared to the stroke-only group. The volume of infarct tissue in the combination of RIPostC+ES was significantly less than RIPostC and ES alone (P < 0.05). Furthermore, NR blocked the ESs protection (P < 0.05) as compared to the ES group by using above-mentioned methods. Bcl-2 was upregulated, while cleaved-caspase-3 was downregulated in the experimental groups as compared to the control group. No difference was found among the experimental groups. Discussion: Peripheral nerve ES appears to have a neuroprotective effect in a rat dMCAO model. This effect may indicate a neural protective mechanism underlying beneficial effect of RIPostC.

The correlation of D-dimer levels with patient outcomes in acute ischemic cerebrovascular disease complicating coronary heart disease

Objective: To investigate the correlation between nerve function defect, their degree of recovery, and D-dimer levels in patients with acute ischemic cerebrovascular disease (AICVD) complicating coronary heart disease (CHD) in winter. Methods: From 1 October, 2014 to 31 December, 2014, we enrolled patients in a consecutive manner with AICVD who were hospitalized in the Department of Neurology, Beijing Luhe hospital, Capital Medical University. The patients were selected that had an occurrence of AICVD within the last 14 days. A total of 151 cases were divided into CHD group (n = 77) or non-CHD group (n = 74) based on a diagnosis of CHD. The risk factors, hematological indices associated with the diseases, and the nerve function defect and recovery degrees were compared between the two groups. Moreover, according to the result of the preliminary analysis of the CHD and non-CHD groups, patients were further divided into two subgroups based on whether their D-dimer levels were higher than 0.5 mg/l or not. Finally, the nerve function defect and recovery degrees in each subgroups were compared in pairs. Results: Among the patients consecutively enrolled, the percentage of the patients with CHD was 50.99% (77/151) and non-CHD patients was 49.01%. On admission, there was no significant difference in NIHSS scores between the CHD and non-CHD groups. However, there was a significant difference between the CHD and non-CHD groups when comparing the NIHSS scores on 14th day and the mRs scores on 90 (±7)th day after the initial onset (p = 0.006, 0.005). The D-dimer levels of AICVD complicating CHD patients were higher than those not complicating CHD patients (p = 0.006). Those AICVD patients that complicating CHD with also elevated D-dimer levels had most severe neurologic function deficits on 14th day and worst neural function recoveries on 90 (±7)th day after onset (p = 0.001, <0.001). Conclusions: AICVD patients complicating CHD is very common in clinical practice. The AICVD patients that complicating CHD showed worse outcomes within 90 days after initial onset of stroke. The D-dimer levels of patients with AICVD complicating CHD were higher. Patients in the CHD group, whose D-dimer levels were higher than the normal standard, had worst outcomes. Paying close attention to the stage of the coronary artery disease and indicators of the coagulation-fibrinolysis is beneficial in the optimization of the clinical treatment for AICVD patients. Maybe the results of this study could provide some reference for specific groups of stroke patients to accept anticoagulant therapy.

Acute recanalization of carotid stenosis is not proper: an experimental ischaemic stroke study

Abstract Objectives: In a rat common carotid artery (CCA) stenosis model, the author determined the function of blood–brain barrier (BBB) at different time points and established an optimal time for CCA recanalization in rats with CCA stenosis combined with cerebral infarction. Methods: Common carotid artery severe stenosis combined with cerebral infarction was divided into two groups: CCA stenosis group (n = 48) and CCA stenosis recanalization group (n = 48). Common carotid artery stenosis recanalization was opened at time points of 1, 2, 3, 5, 7 and 14 days. Twenty-fourhours after recanalization, neurological behaviour, motor function, brain water content and immunohistochemistry of laminin and fibronectin were used to assess brain injury. The peak systolic velocity (PSV) determined by colour Doppler flow imaging (CDFI) was used to assess blood flow of the CCA. Results: In contrast to CCA stenosis without recanalization, in which severe neurological deficits and foot fault were observed at 1, 2 and 3 days, significantly less neurological deficits at 14 days and less foot fault placing at 5, 7 and 14 days were observed after recanalization (P < 0.05). Although the brain water content was enhanced in the recanalization group at the stage of 1–3 days (P < 0.05), a decrease in recanalization group at all time points (1–14 days) was found. Being consistent with reduced brain oedema, the expression of laminin and fibronectin gradually increased in both groups. However, at the early phase of 7–14 days (vs acute phase), the levels of basal laminar proteins were significantly (P < 0.05) enhanced by vascular recanalization in both the ischaemic core and penumbra. Peak systolic velocity of CCA after recanalization reached the control level without stenosis. Conclusions: Our study suggests that the optimal time to open the CCA stenosis complicating cerebral infarction is at or after 7 days of CCA stenosis.