Brain Damage In Ischemic Stroke Research Articles

Soluble Triggering Receptors Expressed on Myeloid Cells (sTREM) in Acute Ischemic Stroke: A Potential Pathway of sTREM-1 and sTREM-2 Associated with Disease Severity.

In 2022, stroke emerged as the most significant cerebrovascular disorder globally, causing 6.55 million deaths. Microglia, crucial for CNS preservation, can exacerbate brain damage in ischemic stroke by triggering neuroinflammation. This process is mediated by receptors on microglia, triggering receptors expressed on myeloid cells (TREM-1 and TREM-2), which have contrasting roles in neuroinflammation. In this study, we recruited 38 patients within 4.5 h from the onset of ischemic stroke. The degree of severity was evaluated by means of the National Institutes of Health Stroke Scale (NIHSS) at admission (T0) and after one week of ischemic events (TW) and the Modified Rankin Scale (mRS) at three months. The plasma concentration of TREMs (sTREM) was analyzed by next-generation ELISA at T0 and TW. The sTREM-1 concentrations at T0 were associated with mRS, while the sTREM-2 concentrations at T0 were associated with both the NIHSS at T0 and the mRS. A strong correlation between sTREM-1 and sTREM-2 was observed, suggesting a dependent modulation of the levels. This study provides insights into the potential pathway of TREM-1 and TREM-2 as a future biomarker for stratifying high-risk patients with ischemic stroke.

Abstract 105: Neutrophil Cracr2a Is Crucial For Calcium Signaling And Enhances Neutrophil Adhesive Function Under Thromboinflammatory Conditions

Ca 2+ release-activated channel regulator 2A (CRACR2A) is a Ca 2+ -binding protein that facilitates store-operated Ca 2+ entry during T cell activation. However, it is unknown whether neutrophil CRACR2A regulates neutrophil functions in inflammation. Our confocal intravital microscopy (IVM) revealed that compared to wild-type (WT) mice, myeloid-specific cracr2a conditional knockout (CKO, cracr2a fl/fl;Lyz-cre ) mice exhibited a significant increase in neutrophil rolling and a decrease in neutrophil adhesion, crawling, and transmigration on ear and cremaster venules in TNF-α-induced inflammation. Deletion of neutrophil cracr2a impaired β2 integrin function without affecting the expression of L-selectin, PECAM-1, and JAM-A. Neutrophil cracr2a increased Ca 2+ mobilization after stimulation with fMLP, A23187, and thapsigargin by its rapid interaction with STIM1. Due to the lack of CRACR2A inhibitors, we generated CRACR2A-deleted HL60 cells using CRISPR/Cas9. CRACR2A deletion in differentiated HL60 cells recapitulated the defects seen in cracr2a-null neutrophils, including reduced β2 integrin function and Ca 2+ mobilization after agonist stimulation. Since excessively recruited neutrophils cause brain damage in ischemic stroke, we examined whether neutrophil cracr2a contributes to stroke pathology. Using a transient middle cerebral artery occlusion (tMCAO)-induced ischemic stroke model, we found that compared to WT mice, cracr2a CKO mice exhibited improved neurological deficits and reduced infarct volume. Longitudinal 4D IVM of mouse brain revealed that neutrophil cracr2a is crucial for neutrophil adhesion and transmigration on cerebral vessels 16-24 hours after tMCAO and upregulated αMβ2 integrin on adherent neutrophils. Further, single-cell behavior analysis of 4D IVM showed that a proinflammatory subset of neutrophils arise in WT mice, but not cracr2a CKO mice, after tMCAO. We observed that the number of circulating pro-inflammatory neutrophils was markedly decreased 24 hours after tMCAO in cracr2a CKO mice compared with WT mice. Our findings indicate that neutrophil CRACR2A is a key regulator for Ca 2+ signaling and enhances neutrophil pro-inflammatory and adhesive activities in vascular inflammation and ischemic stroke.

Pathogenetic scenarios of the development of brain ischemia and the main elements of the ischemic cascade

Ischemic stroke is one of the leading causes of disability and mortality. The key approaches to the treatment of this pathology are thrombolytic therapy and thromboaspiration. Despite their proven efficacy, the application of these methods is limited by a relatively narrow therapeutic window. There remains a great relevance in the further study of the molecular mechanisms of ischemic- reperfusion brain damage within the framework of the ischemic cascade concept. The impact on the mechanisms of induction and implementation of various types of programmed cell death with the help of pharmacological agents is a promising approach to reduce brain damage in ischemic stroke. This review considers the key processes leading to irreversible damage to neurons and their death. The mechanisms of formation and the role of excitotoxicity, calcium overload, oxidative and nitrosyl stress, mitochondrial dysfunction, as well as the triggering of signaling pathways of apoptosis and aseptic inflammation are considered in detail. The issue of various variants of cell death against the background of these pathological processes is discussed. Along with the consideration of signaling pathways that contribute to the progression of brain damage, special attention is paid to the activation of protective signaling mechanisms during ischemia, which provide an increase in cell resistance to death.

Blocking cerebral lymphatic system reduces central and peripheral inflammatory response in ischemic stroke

Reduced blood supply to the brain activates the intracranial inflammatory response, a key contributor to secondary brain damage in ischemic stroke. Post-stroke, activation of peripheral immune cells leads to systemic inflammatory responses. Usingin vivo approaches, we investigated meningeal lymphatics’ role in central immune cell infiltration and peripheral immune cell activation. The bilateral deep cervical lymph nodes (dCLNs) were removed 7 days before right middle cerebral artery occlusion in Sprague Dawley (SD) rats. At 3, 24, and 72 h post-intervention, brain immune cell infiltration and microglial and astrocyte activation were measured, while immune cells were classified in the spleen and blood. Inflammatory factor levels in peripheral blood were analyzed. Simultaneously, reverse verification was conducted by injecting AAV-vascular endothelial growth factor C (AAV-VEGFC) adenovirus into the lateral ventricle 14 days before middle cerebral artery occlusion (MCAO) induction to enhance meningeal lymph function. Blocking meningeal LVs in MCAO rats significantly reduced infarct area and infiltration, and inhibited microglia and pro-inflammatory astrocytes activation. After removing dCLNs, CD4+ T lymphocytes, CD8+ T lymphocytes, B lymphocytes, macrophages, and neutrophils in the spleen and blood of MCAO rats decreased significantly at different time points. The levels of inflammatory factors IL-6, IL-10, IL-1β, and TNF-α in plasma decreased significantly. Tests confirmed the results, and AAV-VEGFC-induced MCAO rats provided reverse validation.

Identification of Withanolide G as a Potential Inhibitor of Rho-associated Kinase-2 Catalytic Domain to Confer Neuroprotection in Ischemic Stroke

Background: Cerebral stroke is one of the leading causes of death and disability in a large number of patients globally. Brain damage in ischemic stroke is led by a complex cascade of events. The Rho-associated kinase-2 (ROCK2) has a significant role in cerebral vasospasm, vascular remodeling, and inflammation. It is activated in cerebral ischemia and its inhibition leads to a neuroprotective effect. Objective: The present study is designed to identify potential inhibitors of ROCK2 using a molecular docking approach. Method: We docked phytochemicals of Withania somnifera (WS) into the catalytic site of ROCK2 and compared results with inhibitor Y-27632. ADME and drug-likeness properties of WS phytochemicals were also analyzed. Results: Results suggest that 11 phytochemicals exhibited higher binding affinity toward the ROCK2 catalytic domain compared to the Y-27632 inhibitor. Among these phytochemicals, Withanolide G formed H-bonding and established hydrophobic contacts with key catalytic domain residues of ROCK2. Conclusion: Our findings suggest that Withanolide G has the potential to inhibit the action of ROCK2 and can be developed as a neurotherapeutic agent to combat cerebral ischemic insult.

Pre-ischemic exercise prevents inflammation and apoptosis by inhibiting MAPK pathway in ischemic stroke.

Mitogen-activated protein kinase (MAPK) pathway is a major mechanism of acute brain damage in ischemic stroke. Pre-ischemic exercise is an effective method to reduce ischemic injury. However, the regulation by pre-ischemic exercise of MAPK pathway and associated mechanisms in animal models remains unclear. In this study, Male SD rats were randomly divided into sham group, middle cerebral artery occlusion (MCAO) group, and exercise plus MCAO (EX + MCAO) group for 21 days, and then was established by MCAO. Longa score was used to measure neurological deficits at 0, 1, 2, and 3 days after MCAO. Hematoxylin and eosin staining was used to observe the brain injury. The expression of MAPK pathway was quantified by western blot. The M1 microglia protein was quantified by western blot and immunofluorescence, and the level of inflammatory factor was measured by enzyme-linked immunosorbent assay. TUNEL staining and western blot were used to measure apoptosis. In the current study, we observed that pre-ischemic exercise effectively decreased infarct volume, neurological deficit score and brain injury in MCAO rats through suppressing the activation of p-JNK and p-ERK1/2. Further investigation revealed that pre-ischemic exercise decreased M1 microglia activation and the serum level of TNF-α and IL-1β. In addition, the increased number of TUNEL-positive cells and Bax/Bcl-2 ratio also were reversed by pre-ischemic exercise. Pre-ischemic exercise can alleviate inflammatory response and apoptosis by inhibiting the MAPK pathway in MCAO rats.

Activation of Swell1 in microglia suppresses neuroinflammation and reduces brain damage in ischemic stroke

Cl− movement and Cl−-sensitive signal pathways contributes to the survival and switch of inflammatory phenotype of microglia and are believed to play a key role in the inflammatory brain injury after ischemic stroke. Here, we demonstrated an important role of Cl− transmembrane transporter Swell1, in the survival and M2-like polarization of microglia in ischemic stroke. Knockdown or overexpression of Swell1 in cultured microglia inhibited or increased hypotonic-activated Cl− currents, respectively, and these changes were completely blocked by the volume-regulated anion channels (VRACs) inhibitor DCPIB. Swell1 conditional knock-in mice promoted microglia survival in ischemic brain region and resulted in significant reductions in neural cell death, infarction volume and neurological deficits following transient middle cerebral artery occlusion (tMCAO). Using gene manipulating technique and pharmacological inhibitors, we further revealed that Swell1 opening led to SGK1 (a Cl−-sensitive kinase)-mediated activation of FOXO3a/CREB as well as WNK1 (another Cl−-sensitive kinase)-mediated SPAK/OSR1-CCCs activation, which promoted microglia survival and M2-like polarization, thereby attenuating neuroinflammation and ischemic brain injury. Taken together, our results demonstrated that Swell1 is an essential component of microglia VRACs and its activation protects against ischemic brain injury through promoting microglia survival and M2-like polarization.

Effects of Mdivi-1 on Neural Mitochondrial Dysfunction and Mitochondria-Mediated Apoptosis in Ischemia-Reperfusion Injury After Stroke: A Systematic Review of Preclinical Studies.

This systematic review sought to determine the effects of Mitochondrial division inhibitor-1 (Mdivi-1) on neural mitochondrial dysfunction and neural mitochondria-mediated apoptosis in ischemia/reperfusion (I/R) injury after ischemic stroke. Pubmed, Web of Science, and EMBASE databases were searched through July 2021. The studies published in English language that mentioned the effects of Mdivi-1 on neural mitochondrial dysfunction and neural mitochondria-mediated apoptosis in I/R-induced brain injury were included. The CAMARADES checklist (for in vivo studies) and the TOXRTOOL checklist (for in vitro studies) were used for study quality evaluation. Twelve studies were included (median CAMARADES score = 6; TOXRTOOL scores ranging from 16 to 18). All studies investigated neural mitochondrial functions, providing that Mdivi-1 attenuated the mitochondrial membrane potential dissipation, ATP depletion, and complexes I-V abnormalities; enhanced mitochondrial biogenesis, as well as inactivated mitochondrial fission and mitophagy in I/R-induced brain injury. Ten studies analyzed neural mitochondria-mediated apoptosis, showing that Mdivi-1 decreased the levels of mitochondria-mediated proapoptotic factors (AIF, Bax, cytochrome c, caspase-9, and caspase-3) and enhanced the level of antiapoptotic factor (Bcl-2) against I/R-induced brain injury. The findings suggest that Mdivi-1 can protect neural mitochondrial functions, thereby attenuating neural mitochondria-mediated apoptosis in I/R-induced brain injury. Our review supports Mdivi-1 as a potential therapeutic compound to reduce brain damage in ischemic stroke (PROSPERO protocol registration ID: CRD42020205808).Systematic Review Registration: [https://www.crd.york.ac.uk/prospero/], identifier [CRD42020205808].

Inhibition of microRNA-9-5p and microRNA-128-3p can inhibit ischemic stroke-related cell death in vitro and in vivo.

Ischemic stroke is the major form of stroke and is accentuated by multiple comorbidities. It has been previously shown that different microRNAs (miRNAs) regulate separate aspects of ischemic stroke. Differential miRNA expression analysis in cerebrospinal fluid of stroke patients had revealed upregulation of miR-124-3p, miR-9-3p, miR-9-5p, and miR-128-3p. However, whether the overexpression is correlative or causative was not known. Here, using an in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) neuronal cell model, we saw OGD/R-induced injury was associated with significant upregulation of the aforementioned four miRNAs. Target gene prediction using in situ algorithms and gene set enrichment analysis revealed significant enrichment of FOXO and Relaxin signaling pathways and regulatory processes associated with endothelial cell migration, which are all known to associate with apoptotic pathways. In situ protein-protein interaction network analysis confirmed the findings of gene set enrichment analysis. TUNEL analysis showed that OGD/R-induced injury resulted in significant apoptosis, which was significantly inhibited in neuronal cells pretransfected with inhibitors of either miR-9-5p or miR-128-3p. Further testing in an in vivo middle cerebral artery occlusion (MCAO) mouse model of ischemic stroke showed that inhibiting miR-9-5p or miR-128-3p significantly decreases MCAO-induced infraction volume and inhibited apoptotic response as revealed by decreased cleaved Caspase-3 protein expression in immunohistochemical analysis. Combined inhibition of miR-9-5p and miR-128-3p resulted in a synergistic decrease in cell death and infraction volume in vitro and in vivo, respectively. Cumulatively, our results provide critical knowledge about the mechanism by which elevated miR-9-5p and miR-128-3p causes brain damage in ischemic stroke and provides evidence of them being attractive therapeutic targets.

Abstract TP433: Effect of Vitamin D and Marine Omega-3 Fatty Acid Supplements on Stroke Outcomes

Introduction: Low serum 25-hydroxyvitamin D (25(OH)D) and low marine omega-3 fatty acid (n-3) intake are associated with increased stroke risk in observational studies. Among stroke patients, low serum 25(OH)D at admission predicts poor outcomes and animal studies suggest that higher n-3 intake may diminish brain damage in ischemic stroke. However, few studies have examined effects of vitamin D or n-3 supplements on stroke outcomes. Hypothesis: We hypothesized that vitamin D (cholecalciferol, 2,000 IU/day) or n-3 (840 mg/day of EPA/DHA [ratio of 1.3:1]) supplementation initiated prior to stroke would reduce risk of functional limitations or physical disability after stroke compared to placebo. Methods: We used data from the completed VITamin D and OmegA-3 TriaL (VITAL) which randomized 25,871 men aged ≥50 years and women aged ≥55 years without cardiovascular disease or cancer at baseline and followed them for incident events over 5.3 years. Individuals experiencing a stroke were mailed a questionnaire to assess functional limitations (with the physical performance scale adapted from Nagi) and physical disability (with the modified Katz Activities of Daily Living and Rosow-Breslau Functional Health scales). We used logistic regression to analyze associations between randomized treatment assignments and limitations on each scale. Results: 290 individuals experienced their first stroke during the trial (including 42 fatal strokes). 197 stroke survivors completed the stroke outcomes questionnaire a mean of 1.6 years after diagnosis. We observed no associations between randomized treatment to vitamin D and functional limitations (odds ratio (OR)=1.01, 95% confidence interval (CI): 0.52, 1.97) or physical disability on the Rosow-Breslau (OR=1.03, 95% CI: 0.31, 3.42) or Katz (OR=0.92, 95% CI: 0.50, 1.67) scales. Although not statistically significant, those randomized to n-3 had lower risk of functional limitations (OR=0.55, 95% CI: 0.28, 1.09) and physical disability on the Katz scale (OR=0.32, 95% CI: 0.50, 1.67) but not on the Rosow-Breslau scale (OR=1.03, 95% CI: 0.31, 3.42). Conclusion: Supplementation with vitamin D or omega-3 fatty acids prior to stroke did not result in significantly improved outcomes after stroke among older individuals.

Computational Insights on Small Molecule Binding to the Hv1 Proton Channel

Hv1 is a voltage-gated proton channel whose main function is to extrude excess protons from the cell. It appears to be involved in a variety of maladies (e.g., breast cancer metastasis, brain damage in ischemic stroke). Despite extensive recent research, details of the gating mechanism, proton permeation pathway, and exquisite selectivity of Hv1 remain elusive. We have previously developed an atomistic model of human Hv1 in its open state using molecular dynamics (MD) simulations on the microsecond timescale. This model has been validated by comparison with electrophysiology data. Further investigation of the model with respect to small molecule inhibitor binding may further support the structure and lead to the design and development of effective channel blockers. The objective of this work is to understand and improve the binding of small molecules that have been experimentally shown to block Hv1. Our approach utilizes the docking of these inhibitors to our open state model, atomistic molecular dynamics simulations, and alchemical free energy calculations. Here, we present results on the relative binding free energies of one prototypical Hv1 inhibitor with comparison to experimental mutagenesis and electrophysiology data.

Neuroprotective activity of orexin system in ischemic stroke

The protective potential of orexin system is a field of interest in the search of the new methods to diminish brain damage in ischemic stroke. The cytoprotective potential of orexins in hypoxic damage is associated with their antioxidant, anti-inflammatory and anti-apoptotic properties and with their ability to activate proliferation and normalize metabolism. Even though today little is known about the role of orexins in memory and pain in ischemic stroke, the common features of the pathogenesis of these disruptions and the mechanisms of orexin-associated protection could suggest the opportunity to use of orexins for correction of these complications following ischemic stroke. Further studies of the orexin-associated neuroprotection could become the further step on the way to the new therapeutic approaches in ischemic stroke.

Pathogenetic aspects of the development of acute focal cerebral ischemia

Current concepts on the main mechanisms of brain damage in ischemic stroke are considered. Chemical regulation of physiological and pathological processes of maintaining cellular pool is supported by a multistep system that included compounds of different structure and complexity. A complex assessment and comparison of the processes taking place during the development of acute local cerebral ischemia (necrosis, apoptosis, autoimmune inflammatory reaction, neuroplasticity) can help in the objectification and prognosis of individual characteristics of the course and outcome of ischemic stroke. Understanding of the cascade of events that occur during the acute ischemic damage is critical for determining current and future diagnostic and therapeutic approaches.

Prognostic significance of Ischemia Modified Albumin and role of high sensitive Creactive protein levels in acute ischemic stroke patients

Introduction: Acute ischemic stroke (AIS) is one of the leading cause of death worldwide and the main causes of long-term disability. Brain CT shows no abnormality in the early event of AIS and the infarct may not be seen reliably for 24-48 hours. Oxidative stress is proposed as a fundamental mechanism of brain damage in ischemic stroke. Earlier it was reported that free radicals alters the N-terminus of the albumin which decreases its binding capacity for metals and is termed as Ischemia Modified Albumin (IMA). Recently it was found that IMA is increased in cerebrovascular diseases and may serve as biomarker for early identification of acute stroke. There is little information regarding the use of IMA for the prognosis of AIS. There is growing evidence of the importance of high sensitive C-reactive protein (hs-CRP) in Acute Ischemic Stroke. Therefore, the current study aims to determine the prognostic evaluation of time course of serum level of IMA and hs-CRP in Acute ischemic Stroke patients. Material and Method: 56 AIS patients diagnosed (clinically/ radiologically) of either sex and >18 years of age were taken as cases. 56 age and sex matched healthy subjects were taken as controls. Serum IMA levels were estimated by spectrophotometric method and hs-CRP levels were estimated by Auto-analyzer. Results: There was a significant increase in IMA levels in AIS patients at the time of admission (488.82±24.84) and on 3rd day (397.17±32.44) when compared to control (324.27±36.45) group. It was found to be statistically significant (P < 0.001). There was a significant increase in Hs-CRP levels in AIS patients at the time of admission (9.55±1.63) than in control (0.95±0.40) group. It was found to be statistically significant (P < 0.001). Conclusion: Estimation of serum IMA in AIS patients has both diagnostic and prognostic application. Routine hs-CRP measurement will be a useful tool for identifying AIS patients, in order to plan aggre

The Voltage-Gated Proton Channel: A Riddle, Wrapped in a Mystery, inside an Enigma.

The main properties of the voltage-gated proton channel (HV1) are described in this review, along with what is known about how the channel protein structure accomplishes its functions. Just as protons are unique among ions, proton channels are unique among ion channels. Their four transmembrane helices sense voltage and the pH gradient and conduct protons exclusively. Selectivity is achieved by the unique ability of H3O(+) to protonate an Asp-Arg salt bridge. Pathognomonic sensitivity of gating to the pH gradient ensures HV1 channel opening only when acid extrusion will result, which is crucial to most of its biological functions. An exception occurs in dinoflagellates in which influx of H(+) through HV1 triggers the bioluminescent flash. Pharmacological interventions that promise to ameliorate cancer, asthma, brain damage in ischemic stroke, Alzheimer's disease, autoimmune diseases, and numerous other conditions await future progress.

A Truncated Fragment of Src Protein Kinase Generated by Calpain‐mediated Cleavage is a Mediator of Neuronal Death in Excitotoxicity.

Excitotoxicity resulting from overstimulation of glutamate receptors is a major cause of neuronal death in cerebral ischemic stroke. The overstimulated ionotropic glutamate receptors exert their neurotoxic effects in part by overactivation of calpains, which induce neuronal death by catalyzing limited proteolysis of specific cellular proteins. Here, we report that in cultured cortical neurons and in vivo in a rat model of focal ischemic stroke, the tyrosine kinase Src is cleaved by calpains at a site in the N‐terminal unique domain. This generates a truncated Src fragment of ~52 kDa, which we localized predominantly to the cytosol. A cell membrane‐permeable fusion peptide derived from the unique domain of Src prevents calpain from cleaving Src in neurons and protects against excitotoxic neuronal death. To explore the role of the truncated Src fragment in neuronal death, we expressed a recombinant truncated Src fragment in cultured neurons and examined how it affects neuronal survival. Expression of this fragment, which lacks the myristoylation motif and unique domain, was sufficient to induce neuronal death. Furthermore, inactivation of the prosurvival kinase Akt is a key step in its neurotoxic signaling pathway. Because Src maintains neuronal survival, our results implicate calpain cleavage as a molecular switch converting Src from a promoter of cell survival to a mediator of neuronal death in excitotoxicity. Besides unveiling a new pathological action of Src, our discovery of the neurotoxic action of the truncated Src fragment suggests new therapeutic strategies with the potential to minimize brain damage in ischemic stroke

Nanomedical Approach to Monitor the Central Role of NO/ONOOImbalance in Ischemic Stroke Brain Damage – The Effects of Statins and Heme Oxygenase-1

Background: There are conflicting reports concerning the role of nitric oxide (NO) and oxidative stress in brain ischemic damage. In the present study, a nanomedical approach was utilized to elucidate the role of NO and peroxynitrite (ONOO - ) imbalance in ischemic stroke. Methods: Nanosensors (diameter ~ 200 nm, detection limit of 10-9 molL-1, response time ~10 μs) were used to monitor in situ the concentration of NO and ONOO - . Adult male Sprague Dawley rats were given permanent middle cerebral artery occlusion (pMCAO) for 3 h, 12 h or 24 h. [NO]/[ONOO - ] was measured in striatum, along with, constitutive nitric oxide synthase (cNOS) enzymes and heme oxygenase-1 (HO-1) expression and infarct volume. The [NO]/[ONOO - ] was also monitored in pre-treated animals with simvastatin and atorvastatin in the presence of the cNOS inhibitor L-NAME. The effect of modulators of cNOS or NADPH oxidase (sepiapterin, PEG-SOD, VAS2870 and IN-7) on the [NO]/[ONOO - ] was elucidated. Results: After 3 h of ischemia, NO decreased from 400 ± 20 nmolL-1 to 217 ± 11 nmolL-1 and ONOO - increased from 150 ± 9 nmolL-1 to 244 ± 9 nmolL-1.The [NO]/[ONOO - ] balance shifted from 2.67 ± 0.06 to 0.89 ± 0.07 after 3 h of ischemia, indicating severe uncoupling of cNOS. The [NO]/[ONOO - ] imbalance shifted with time of ischemia and correlated directly with the increase in infarct volume and expression of cNOS and HO-1. Treatment with simvastatin or atorvastatin partially, but significantly, restored [NO]/[ONOO - ] balance and decreased infarct size in ischemic brain. Also, modulators of cNOS an NADPH restored [NO]/[ONOO - ]. Conclusions: The imbalance between cytoprotective NO and cytotoxic ONOO - directly correlates with brain damage in ischemic stroke. The [NO]/[ONOO - ] imbalance reflects on the level of uncoupled cNOS and the nitroxidative stress. [NO]/[ONOO - ] imbalance increases cNOS and HO-1, which contributes to or prevents further brain damage, respectively. Balancing [NO]/[ONOO - ] is the determinant in preventing or mollifying brain damage. Simvastatin or atorvastatin shifts favorably [NO]/[ONOO - ], and may provide prophylactic treatment strategy for ischemic stroke.

Gua Lou Gui Zhi decoction suppresses LPS-induced activation of the TLR4/NF-κB pathway in BV-2 murine microglial cells

Toll-like receptor4 (TLR4)/nuclear factor-κB (NF-κB) signaling-mediated neuroinflammation contributes to secondary brain damage in ischemic stroke; therefore, anti-inflammatory therapy via suppression of the TLR4/NF-κB pathway could be a promising strategy for the treatment of stroke and post-stroke disabilities. Gua Lou Gui Zhi decoction (GLGZD) has long been used in China to clinically treat dysfunction after stroke such as muscular spasticity, but the precise mechanisms are largely unknown. In the present study, we evaluated the anti-inflammatory effect of GLGZD and investigated the underlying molecular mechanisms using lipopolysaccharide (LPS)-stimulated BV-2 microglial cells as an invitro inflammatory model of neural cells. We found that GLGZD inhibited the inflammatory response in microglial cells as it significantly reduced LPS-induced expression of pro-inflammatory nitric oxide, tumour necrosis factor-α, interleukin (IL)-6 and IL-1β in BV-2 cells, in a dose-dependent manner. In addition, GLGZD treatment significantly decreased the protein expression of TLR4 and myeloid differentiation factor88, inhibited the phosphorylation of IκB and blocked the nuclear translocation of NF-κB in BV-2 cells, demonstrating its inhibitory effect on the activation of TLR4/NF-κB signaling. Collectively, our findings suggest that inhibition of the inflammatory response via suppression of the TLR4/NF-κB pathway may be one of the mechanisms through which GLGZD ameliorates the damage in ischemic cerebral tissues.

A Truncated Fragment of Src Protein Kinase Generated by Calpain-mediated Cleavage Is a Mediator of Neuronal Death in Excitotoxicity

Excitotoxicity resulting from overstimulation of glutamate receptors is a major cause of neuronal death in cerebral ischemic stroke. The overstimulated ionotropic glutamate receptors exert their neurotoxic effects in part by overactivation of calpains, which induce neuronal death by catalyzing limited proteolysis of specific cellular proteins. Here, we report that in cultured cortical neurons and in vivo in a rat model of focal ischemic stroke, the tyrosine kinase Src is cleaved by calpains at a site in the N-terminal unique domain. This generates a truncated Src fragment of ~52 kDa, which we localized predominantly to the cytosol. A cell membrane-permeable fusion peptide derived from the unique domain of Src prevents calpain from cleaving Src in neurons and protects against excitotoxic neuronal death. To explore the role of the truncated Src fragment in neuronal death, we expressed a recombinant truncated Src fragment in cultured neurons and examined how it affects neuronal survival. Expression of this fragment, which lacks the myristoylation motif and unique domain, was sufficient to induce neuronal death. Furthermore, inactivation of the prosurvival kinase Akt is a key step in its neurotoxic signaling pathway. Because Src maintains neuronal survival, our results implicate calpain cleavage as a molecular switch converting Src from a promoter of cell survival to a mediator of neuronal death in excitotoxicity. Besides unveiling a new pathological action of Src, our discovery of the neurotoxic action of the truncated Src fragment suggests new therapeutic strategies with the potential to minimize brain damage in ischemic stroke.

Blockade of CXCR1/2 chemokine receptors protects against brain damage in ischemic stroke in mice

OBJECTIVE:Ischemic stroke may result from transient or permanent reductions of regional cerebral blood flow. Polymorphonuclear neutrophils have been described as the earliest inflammatory cells to arrive in ischemic tissue. CXCR1/2 receptors are involved in the recruitment of these cells. However, the contribution of these chemokine receptors during transient brain ischemia in mice remains poorly understood. In this work, we investigated the effects of reparixin, an allosteric antagonist of CXCR1/2 receptors, in a model of middle cerebral artery occlusion and reperfusion in mice.METHODS:C57BL/6J male mice treated with reparixin or vehicle were subjected to a middle cerebral artery occlusion procedure 1 h after the treatment. Ninety minutes after ischemia induction, the monofilament that prevented blood flow was removed. Twenty-four hours after the reperfusion procedure, behavioral changes, including motor signs, were analyzed with the SmithKline/Harwell/Imperial College/Royal Hospital/Phenotype Assessment (SHIRPA) battery. The animals were sacrificed, and brain tissue was removed for histological and biochemical analyses. Histological sections were stained with hematoxylin and eosin, neutrophil infiltration was estimated by myeloperoxidase activity and the inflammatory cytokine IL-1β was measured by ELISA.RESULTS:Pre-treatment with reparixin reduced the motor deficits observed in this model of ischemia and reperfusion. Myeloperoxidase activity and IL-1β were reduced in the reparixin-treated group. Histological analysis revealed that ischemic injury was also attenuated by reparixin pre-treatment.CONCLUSIONS:Our results suggest that the blockade of the CXCR1/2 receptors by reparixin promotes neuroprotective effects by reducing the levels of polymorphonuclear infiltration in the brain and the tissue damage associated with middle cerebral artery occlusion and reperfusion.