Artery Occlusion Model Of Stroke Research Articles

Transient Middle Cerebral Artery Occlusion Model of Stroke.

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies during the acute phase of ischemic stroke. To gain insights into stroke physiopathology and develop innovative therapeutic approaches, in vivo rodent models of stroke play a fundamental role. The availability of genetically modified animals has particularly propelled the use of mice as experimental stroke models. In stroke patients, occlusion of the middle cerebral artery (MCA) is a common occurrence. Consequently, the most prevalent experimental model involves intraluminal occlusion of the MCA, a minimally invasive technique that doesn't require craniectomy. This procedure involves inserting a monofilament through the external carotid artery (ECA) and advancing it through the internal carotid artery (ICA) until it reaches the branching point of the MCA. After a 45 min arterial occlusion, the monofilament is removed to allow reperfusion. Throughout the process, cerebral blood flow is monitored to confirm the reduction during occlusion and subsequent recovery upon reperfusion. Neurological and tissue outcomes are evaluated using behavioral tests and magnetic resonance imaging (MRI) studies.

Decanoic acid mitigates ischemia reperfusion injury by modulating neuroprotective, inflammatory and oxidative pathways in middle cerebral artery occlusion model of stroke in rats

Objective: Amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) is an ionotropic transmembrane receptor for glutamate. AMPA receptor blockers have been reported to prevent neurological damage and enhance the post stroke recovery in rats. Decanoic acid, a medium-chain fatty acid, has been reported to exhibit non-competitive AMPA receptor antagonism. This study evaluated the effect of decanoic acid administered before and after ischemia reperfusion injury on neurological damage and post stroke recovery in rats. Methods: Middle cerebral artery occlusion (MCAo) was performed by using the intraluminal method to induce focal cerebral ischemia. Decanoic acid (120 mg/kg) was administered orally for 1 day (5-10 min post reperfusion) in one group and for 2 days (24 h pre and 5-10 min post reperfusion) in the other group. Effect on neurological damage and post stroke recovery was assessed by neurobehavioral parameters, MRI and TTC staining along with inflammatory, oxidative, apoptotic, and neuroprotective biomarkers. Results: Decanoic acid significantly reduced the MCAo induced neurological damage and infarct size. Decanoic acid treatment increased the motor coordination and grip strength. Furthermore, levels of inflammatory (TNFα, IL-1β and IL-6), oxidative stress (MDA), apoptotic (TUNEL positive cells) and neurological injury (GFAP) biomarkers were reduced after decanoic acid treatment. Anti-inflammatory cytokine (IL-10) and neuroprotective markers (NT-3, BDNF and TrkB) were found to be significantly increased with decanoic acid treatment. Conclusion: This study showed protective effects of decanoic acid against ischemia reperfusion injury in rats. Anti-inflammatory, antioxidant, neuroprotective, and anti-apoptotic properties may be responsible for the beneficial effects of decanoic acid observed in the study.

Factor VII activating protease (FSAP) inhibits the outcome of ischemic stroke in mouse models.

The outcome of ischemic stroke can be improved by further refinements of thrombolysis and reperfusion strategies. Factor VII activating protease (FSAP) is a circulating serine protease that could be important in this context. Its levels are raised in patients as well as mice after stroke and a single nucleotide polymorphism (SNP) in the coding sequence, which results in an inactive enzyme, is linked to an increased risk of stroke. In vitro, FSAP cleaves fibrinogen to promote fibrinolysis, activates protease-activated receptors, and decreases the cellular cytotoxicity of histones. Based on these facts, we hypothesized that FSAP can be used as a treatment for ischemic stroke. A combination of tissue plasminogen activator (tPA), a thrombolytic drug, and recombinant serine protease domain of FSAP (FSAP-SPD) improved regional cerebral perfusion and neurological outcome and reduced infarct size in a mouse model of thromboembolic stroke. FSAP-SPD also improved stroke outcomes and diminished the negative consequences of co-treatment with tPA in the transient middle cerebral artery occlusion model of stroke without altering cerebral perfusion. The inactive MI-isoform of FSAP had no impact in either model. FSAP enhanced the lysis of blood clots in vitro, but in the tail transection model of hemostasis, FSAP-SPD treatment provoked a faster clotting time indicating that it also has pro-coagulant actions. Thus, apart from enhancing thrombolysis, FSAP has multiple effects on stroke progression and represents a promising novel therapeutic strategy in the treatment of ischemic stroke.

Abstract 15: The Novel Complement C3ar Antagonist Jr14a Is Neuroprotective In Stroke

Background: Stroke is the third leading cause of disability and mortality in the world. The c omplement C3a receptor plays a prominent role in post stroke brain inflammation. We and others have reported that either genetic deficiency of complement C3a receptor (C3aR) or its pharmacological inhibition in rodents protect against cerebral ischemia. The existing C3aR antagonist (SB290157) is limited by its reported agonist effect in different model systems. Hypothesis: The novel highly-selective C3aR antagonist JR14a confers robust neuroprotection in stroke Methods: Mouse primary brain endothelial cells (cell biologics Inc. IL, USA) were seeded at a density of 5000 cells/well in 96-well plates and cultured until 80-90% confluency. Endothelial cytotoxicity was tested using an LDH assay following 24 hours of exposure to JR14a. Oxygen-glucose deprivation (OGD) was followed by reperfusion to compare the in-vitro effects of JR14a and SB290157, using ELISA performed for TNF-α and IL-6 and immunofluorescence for ICAM-1 expression. We also compared JR14a and SB290157 in the middle cerebral artery occlusion (MCAO, C57BL/6) stroke model, evaluating infarct volume. Results: JR14a treated endothelial cells exhibited reduced LDH release (Fig.1) and more potent anti-inflammatory response following OGD/R compared to SB290157. We found that JR14a treatment significantly reduced endothelial TNF-α, IL-6 and ICAM-1 expression. JR14a also significantly reduced brain infarction and microglial activation relative to SB290157 following MCAO in C57BL/6 mice (Fig 2). Conclusion: We conclude that the novel C3aR inhibitor JR14a attenuates inflammation and endothelial dysfunction in stroke and may confer a more potent neuroprotective effect than SB290157. Further work is warranted to explore the mechanisms underlying JR14a-mediated neuroprotection

Post Stroke Safinamide Treatment Attenuates Neurological Damage by Modulating Autophagy and Apoptosis in Experimental Model of Stroke in Rats.

Exploring and repurposing a drug have become a lower risk alternative. Safinamide, approved for Parkinson's disease, has shown neuroprotection in various animal models of neurological disorders. The present study aimed to explore the potential of safinamide in cerebral ischemia/reperfusion (I/R) in rats. Sprague-Dawley rats were used in middle cerebral artery occlusion model of stroke. The effective dose of safinamide was selected based on the results of neurobehavioral parameters and reduction in infarct size assessed 24h post-reperfusion. For sub-acute study, the treatment with effective dose was extended for 3days and effects on neurobehavioral parameters, infarct size (TTC staining and MRI), oxidative stress parameters (MDA, GSH, SOD, NOX-2), inflammatory cytokines (TNF-α, IL-1β, IL-10), apoptosis (Bax, Bcl-2, cleaved caspase-3 expression, and TUNEL staining), and autophagy (pAMPK, Beclin-1, LC3-II expression) were studied. The results of dose selection study showed significant reduction (p < 0.05) in infarct size and improvement in neurobehavioral parameters with safinamide (80mg/kg). In sub-acute study, safinamide showed significant (p < 0.05) improvement in motor coordination and infarct size reduction. Additionally, safinamide treatment significantly normalized altered redox homeostasis and inflammatory cytokine levels. However, no change was observed in expression of NOX-2 in I/R or safinamide treatment group when compared with sham. I/R induced deranged expression of apoptotic markers and increased TUNEL positive cells in cortex were significantly normalized with safinamide treatment. Further, safinamide significantly (p < 0.05) induced the expressions of autophagic proteins (Beclin-1 and LC3-II) in cortex. Overall, the results demonstrated neuroprotective potential of safinamide via anti-oxidant, anti-inflammatory, anti-apoptotic, and autophagy inducing properties. Thus, safinamide can be explored for repurposing in ischemic stroke after further exploration.

Abstract P217: Direct Myosin-2 Inhibition Restores Cerebral Perfusion in Cortical and Subcortical Regions Resulting in Functional Improvement After Ischemic Stroke

Inefficient cerebral reperfusion is a major unresolved problem in ischemic stroke treatment because hypoxia-induced constriction of capillaries remains persistent even after recanalization by thrombolysis or thrombectomy. Capillary constriction is mediated by actomyosin contraction in precapillary smooth muscle cells (SMCs). We have developed and tested a formulation and administration technique to target the ischemic brain region and developed a promising small-molecule myosin-2 inhibitor (para-aminoblebbistatin (AmBleb) that mainly exerts its effect through direct inhibition of smooth muscle myosin-2 (SMM) in SMCs. The efficacy of SMM inhibition was tested in a rodent transient Middle Cerebral Artery Occlusion (tMCAO) stroke model. SMM was targeted by the direct administration of AmBleb into the ischemic region. AmBleb significantly accelerated the improvement of neurological deficits. Regional cerebral blood flow (rCBF) in the most important cortical and subcortical regions (e.g. motor- and somatosensory cortices, optic pathways, striatum, corpus callosum) showed drastic improvement in the AmBleb treated animals in line with the significant reduction of neurological deficits. We further optimized AmBleb and developed our lead compound MPH-222, which possesses significantly improved pharmacological properties than those of AmBleb. As observed with AmBleb, MPH-222 also fully relaxed isolated human and rat cerebral arterioles, and improved neurological functions of tMCAO operated rats characterized by significantly improved neurological deficits as well as enhanced locomotor symmetry. Moreover, as opposed to AmBleb, markedly lower MPH-222 dose was enough to achieve efficient concentrations in the ischemic region when catheter-based direct intra-arterial administration was applied. This result suggests that direct myosin inhibitor administration may be an optimal add-on therapy to thrombectomy. Funded by the Hungarian National Research, Development and Innovation Office (NVKP 16-1-2016-0051 and PIACI-KFI-2019-00488).

Semi-Automated Cell and Tissue Analyses Reveal Regionally Specific Morphological Alterations of Immune and Neural Cells in a Porcine Middle Cerebral Artery Occlusion Model of Stroke.

Histopathological analysis of cellular changes in the stroked brain provides critical information pertaining to inflammation, cell death, glial scarring, and other dynamic injury and recovery responses. However, commonly used manual approaches are hindered by limitations in speed, accuracy, bias, and the breadth of morphological information that can be obtained. Here, a semi-automated high-content imaging (HCI) and CellProfiler histological analysis method was developed and used in a Yucatan miniature pig permanent middle cerebral artery occlusion (pMCAO) model of ischemic stroke to overcome these limitations. Evaluation of 19 morphological parameters in IBA1+ microglia/macrophages, GFAP+ astrocytes, NeuN+ neuronal, FactorVIII+ vascular endothelial, and DCX+ neuroblast cell areas was conducted on porcine brain tissue 4 weeks post pMCAO. Out of 19 morphological parameters assessed in the stroke perilesional and ipsilateral hemisphere regions (38 parameters), a significant change in measured IBA1+ parameters, GFAP+ parameters, NeuN+ parameters, FactorVIII+ parameters, and DCX+ parameters were observed in stroked vs. non-stroked animals. Principal component analysis (PCA) and correlation analyses demonstrated that stroke-induced significant and predictable morphological changes that demonstrated strong relationships between IBA1+, GFAP+, and NeuN+ areas. Ultimately, this unbiased, semi-automated HCI and CellProfiler histopathological analysis approach revealed regional and cell specific morphological signatures of immune and neural cells after stroke in a highly translational porcine model. These identified features can provide information of disease pathogenesis and evolution with high resolution, as well as be used in therapeutic screening applications.

Edaravone-Loaded Macrophage-Derived Exosomes Enhance Neuroprotection in the Rat Permanent Middle Cerebral Artery Occlusion Model of Stroke.

Edaravone (Edv) can inhibit tissue damage, cause cerebral edema, and delay neuronal death caused by acute cerebral infarction. Exosomes are considered as cargo carriers for intercellular communication and serve as important regulators in many pathological processes. Here, we developed macrophage-derived exosomes (Exo) containing Edv (Exo + Edv) to improve the bioavailability of Edv and enhance the neuroprotective effects in a rat model of permanent middle cerebral artery occlusion (PMCAO). The results showed that Exo + Edv significantly improved the bioavailability of Edv and prolonged half-life (t1/2). At the same time, Exo + Edv made Edv more easily reach the ischemic side of rats with PMCAO and was localized with neuronal cells and microglia, thus reducing the death of neuronal cells and promoting the polarization of microglia from M1 to M2. Taken together, Exo + Edv may become a potential clinical treatment option for PMCAO.

Abstract TP266: Ischemic Stroke Induces Striking Heterogeneity in the Inflammatory Leukocyte Infiltrate Across the Core and Penumbra

Background: Current therapies for ischemic stroke focus on reperfusion but do not address the acute inflammatory response. Previous clinical trials aimed at modulating the inflammatory milieu by disrupting leukocyte infiltration failed to show clinical efficacy. One possible explanation for this unexpected shortcoming is an incomplete understanding of the precise spatio-temporal underpinnings of leukocyte extravasation and infiltration. Methods: Here we describe the evolution of the inflammatory response in a mouse transient middle cerebral artery occlusion (tMCAO) stroke model at 0, 1, 2 and 3 days post reperfusion. We used wide field and confocal immunofluorescence microscopy to examine the exact nature and location of the invading myelomonocytic populations, with close examination of the leukocyte position with regard to the brain vasculature and the perivascular space. Results: Our findings suggest that the vast majority of infiltrating myelomonocytic cells escape the perivascular compartment and enter the parenchyma. Interestingly, leukocyte extravasation and accumulation in the subcortex occurred over several days. Dramatic heterogeneity in the inflammatory infiltrate was observed across the infarcted tissue, but also in the surrounding penumbra and adjacent cortical surface. In addition, triphenyl tetrazolium chloride staining, a common indicator for infarcted tissue, did not correlate with the amount or location of leukocyte infiltration. Conclusion: Taken together our findings demonstrate that the infiltration of leukocytes dynamically evolves over several days following reperfusion. Furthermore, leukocytes infiltrate in a heterogeneous pattern that does not correlate well with traditional markers of cellular dysfunction. A better understating of the precise spatio-temporal infiltration of inflammatory cells could help inform the next generation of therapeutic interventions.

Abstract 176: Platelet Cyclophilin D Mediates Neutrophil Recruitment and Ischemic Stroke Outcomes in Mice

Rationale: Besides their role in thrombosis, platelets also mediate inflammation through platelet-neutrophil aggregates (PNA). Recently, cyclophilin D (CypD)-mediated platelet necrosis emerged as a potential mediator of detrimental PNA during thrombosis. However, the role of platelet CypD in ischemic stroke has never been examined. Objective: Here, we investigate the contribution of platelet CypD following ischemic stroke. Methods: We generated mice lacking CypD specifically in platelets (KO). Both wild-type (WT) and KO mice were subjected to a 1h transient middle cerebral artery occlusion (tMCAO) stroke model. Twenty-four hours after occlusion, neurological and motor function, and stroke infarct size were determined. We also examined if the CypD pathway was altered in human platelets after ischemic stroke. Results: Loss of platelet CypD significantly improved neurological (p&lt;0.001) and motor (p&lt;0.005) functions compared to WT mice after tMCAO. Furthermore, platelet CypD deficient significantly reduced ischemic stroke infarct volume (39.1±15.7mm 3 vs. 78.6±27.7mm 3 , n=15; p&lt;0.0001). Smaller infarcts in KO mice was not due to difference in blood flow during the ischemia stage. Twenty-four hours after stroke, a greater than 2-fold reduction in neutrophils was observed in the brains from KO mice (p&lt;0.0001). In addition, we observed significantly fewer circulating and cerebral PNA (p&lt;0.01). Depletion of neutrophils significantly (p&lt;0.05) reduced infarct size and neurological damage following ischemic stroke in WT mice, however, no additional protective effect was observed in KO mice, suggesting necrotic PNAs are critical during ischemic stroke. RNA-sequencing on platelets isolated from ischemic stroke patients (n=8) and healthy aged, gender matched controls (n=7) revealed significant increases in several targets involved in CypD-mediated necrosis, including MCUR1, TMEM16F and calpain2 (p&lt;0.005). Conclusion: Our results demonstrate necrotic platelets interact with neutrophils to exacerbate brain injury following ischemic stroke. As inhibiting platelet necrosis does not compromise hemostasis, targeting platelet CypD may be a potential therapeutic strategy to limit brain damage after ischemic stroke.

Human Milk Oligosaccharide 2'-Fucosyllactose Reduces Neurodegeneration in Stroke Brain.

2'-Fucosyllactose (2'-FL) is a major oligosaccharide in human milk and is present at trace levels in cow milk. 2'-FL reduces inflammation in the gastrointestinal tract. Its action in the central nervous system has not been well characterized. The purpose of this study is to determine 2'-FL-mediated neural protection and repair in culture and stroke brain. In rat primary cortical neuronal cultures, 2'-FL significantly antagonized N-methyl-D-aspartate (NMDA) or glutamate-mediated changes in ATP production, MAP2 immunoreactivity, and TUNEL. The influx of Ca++ (Ca++i) was examined in primary cortical neurons expressing GCaMP5, an endogenous calcium probe. NMDA increased Ca++i; 2'-FL significantly attenuated this reaction. In a rat middle cerebral artery occlusion model of stroke, we found that intracerebroventricular pretreatment or oral posttreatment with 2'-FL significantly reduced brain infarction, mitigated microglial activation, improved locomotor activity, and upregulated brain-derived neurotrophic factor (BDNF) expression. Post-stroke delivery of 2'-FL increased bromodeoxyuridine (BrdU) labeling in the perilesioned area. These BrdU cells co-expressed NeuN, or nestin, or GFAP. Using subventricular Matrigel cultures, we demonstrated that 2'-FL increased cell migration from subventricular zone explant. This response was reduced by anti-BDNF blocking antibody. In conclusion, our data suggest that 2'-FL has neuroprotective action through inhibition of Ca++i, inflammation, and apoptosis. Posttreatment with 2'-FL facilitates neural repair in stroke brain.

Paired associated magnetic stimulation promotes neural repair in the rat middle cerebral artery occlusion model of stroke.

Paired associative stimulation has been used in stroke patients as an innovative recovery treatment. However, the mechanisms underlying the therapeutic effectiveness of paired associative stimulation on neurological function remain unclear. In this study, rats were randomly divided into middle cerebral occlusion model (MCAO) and paired associated magnetic stimulation (PAMS) groups. The MCAO rat model was produced by middle cerebral artery embolization. The PAMS group received PAMS on days 3 to 20 post MCAO. The MCAO group received sham stimulation, three times every week. Within 18 days after ischemia, rats were subjected to behavioral experiments-the foot-fault test, the balance beam walking test, and the ladder walking test. Balance ability was improved on days 15 and 17, and the foot-fault rate was less in their affected limb on day 15 in the PAMS group compared with the MCAO group. Western blot assay showed that the expression levels of brain derived neurotrophic factor, glutamate receptor 2/3, postsynaptic density protein 95 and synapsin-1 were significantly increased in the PAMS group compared with the MCAO group in the ipsilateral sensorimotor cortex on day 21. Resting-state functional magnetic resonance imaging revealed that regional brain activities in the sensorimotor cortex were increased in the ipsilateral hemisphere, but decreased in the contralateral hemisphere on day 20. By finite element simulation, the electric field distribution showed a higher intensity, of approximately 0.4 A/m2, in the ischemic cortex compared with the contralateral cortex in the template. Together, our findings show that PAMS upregulates neuroplasticity-related proteins, increases regional brain activity, and promotes functional recovery in the affected sensorimotor cortex in the rat MCAO model. The experiments were approved by the Institutional Animal Care and Use Committee of Fudan University, China (approval No. 201802173S) on March 3, 2018.

Motor deficit in the mouse ferric chloride-induced distal middle cerebral artery occlusion model of stroke

Ferric chloride-induced distal middle cerebral artery occlusion (MCAO) model of stroke was described in mice several years ago, however it lacked in-depth evaluation of the post-stroke functional outcomes in the animals. In this study, we reproduced the recently developed model and expanded its characterization by thorough evaluation of blood supply, cerebral infarction, and motor function in adult male and female mice up to 14 days after stroke. Our observations indicate near complete interruption of blood flow in the distal MCA shortly after application of 20 % ferric chloride over the artery through a cranial window, which remained occluded for at least 4 h. As expected, infarction of the brain tissue, documented by TTC and hematoxylin stains, was restricted to the cerebral cortex. We also systematically evaluated motor impairment of the animals in this model. For this, a series of studies were carried out in male and female mice up to 14 days after stroke, and motor function was assessed in cylinder and grid-walking tests in blinded manner. Contrary to our expectations, the results of both motor tests indicated minor, transient motor deficit in mice after stroke. Based on these observations, we conclude that the mouse ferric chloride-induced distal MCAO model is likely not suitable for proof-of-concept and preclinical studies where motor function is an important outcome measure.

Abstract 132: The Mammalian Target of Rapamycin Regulates Platelet Integrin Activation, Aggregation, and Ischemic Stroke

Rationale: Platelets are critical cellular effectors in the development of ischemic stroke. Translational control pathways, such as the mammalian target of rapamycin (mTOR), are active in platelets, and can be targeted therapeutically, but have not been examined in stroke. Objective: To determine if activation of the mTOR pathway regulates platelet activation and the development of ischemic stroke following stroke in vivo . Methods: We generated mice lacking mTOR specifically in platelets (KO). We assessed mTOR activation, platelet aggregation, integrin activation, and clot retraction in KO and wild-type control mice (WT). In both KO and WT mice, we also performed a transient middle cerebral artery occlusion (tMCAO) stroke model (t=1h followed with reperfusion for 24hrs). Following tMACO, blood flow in the MCA and stroke infarct size were determined. We also examined mTOR activation in human platelets from individuals with ischemic stroke (n=5) and healthy controls (n=4). Results: We confirmed the absence of mTOR in KO platelets. In platelets from WT mice, the mTOR pathway is activatable, triggering platelet activation and clot retraction in vitro . In comparison, indices of mTOR activation, including pAKT and pS6K, were significantly (p&lt;0.05) reduced in platelets from KO mice. Platelets from KO mice also had significant reductions in platelet aggregation (50% less), integrin αIIbβ3 activation (40% less), and clot retraction compared to WT mice. These differences were not due to alterations in platelet count. In the tMCAO model, KO mice had significantly smaller infarct sizes compared to WT mice (36.8±9.4mm 3 , n=7 vs. 80.3±8.1mm 3 , n=8; p&lt;0.004). Smaller infarct sizes in KO mice were not due to differences in blood flow during the ischemia or reperfusion phase, which were similar to WT mice. In platelets from human ischemic stroke patients, the mTOR pathway was activated as p4EBP1, a downstream target of mTOR, was 1.72-fold higher compared to healthy controls. Conclusion: These data demonstrate that inhibition of the mTOR pathway in platelets significantly reduces platelet activation and protects from ischemic stroke . Therapeutic targeting of the mTOR pathway in platelets may provide clinical benefit in the setting of ischemic stroke.

Citalopram attenuated neurobehavioral, biochemical, and metabolic alterations in transient middle cerebral artery occlusion model of stroke in male Wistar rats.

Oxidative stress and inflammation are implicated as cardinal mechanisms of neuronal death following stroke. In the present study citalopram (Cit) was investigated in a 2 h middle cerebral artery occlusion (MCAo) model of stroke in male Wistar rats. Pretreatment, posttreatment (Post Cit) and pre plus posttreatment (Pre + Post Cit) with Cit were evaluated for its neuroprotective effect. In pretreatment protocol, effect of Cit at three doses (2, 4, and 8 mg/kg) administered i.p., 1 h prior to MCAo was evaluated using neurological deficit score (NDS), motor deficit paradigms, and cerebral infarction 24 h post-MCAo. In posttreatment and pre plus posttreatment protocol, the effective dose of Cit (4 mg/kg) was administered i.p., 0.5 h post-reperfusion (Post Cit) only, and 1 h prior to MCAo and again at 0.5 h post-reperfusion (Pre + Post Cit), respectively. These two groups were assessed for NDS and cerebral infarction. Though NDS was significantly reduced in both Post Cit and Pre + Post Cit groups, significant reduction in cerebral infarction was evident only in Pre + Post Cit group. Infarct volume assessed by magnetic resonance imaging was significantly attenuated in Pre + Post Cit group (10.6 ± 1.1%) compared to MCAo control group (18.5 ± 3.0%). Further, Pre + Post Cit treatment significantly altered 17 metabolites along with attenuation of malondialdehyde, reduced glutathione, matrix metalloproteinases, and apoptotic markers as compared to MCAo control. These results support the neuroprotective effect of Cit, mediated through amelioration of oxidative stress, inflammation, apoptosis, and altered metabolic profile.

Monoacylglycerol lipase inhibitor, JZL-184, confers neuroprotection in the mice middle cerebral artery occlusion model of stroke

IntroductionInvestigators are searching to find new therapeutic strategies to reduce stroke secondary injury. JZL-184 (JZL) is an inhibitory factor for production of arachidonic acid (AA). Thus, it suppresses production of AA metabolites which are the cause of inflammation and tissue edema. Therefore, JZL may be considered for suppression of stroke secondary injury in mice middle cerebral artery occlusion (MCAO) model. Additionally, Aspirin is a known anti-inflammatory factor which is used to reduce pro-inflammatory secondary injury. The aim of this study was to determine the effects of JZL on the reduction of stroke secondary injury and to compare them with Aspirin effects. Material and methodsMCAO model has been induced and accordingly 83 male MCAO induced mice have been introduced to the study. The animals were divided to seven groups including intact, controls, vehicle, Aspirin, JZL 4, 8 and 16 mg/kg administrated groups. Brain edema and infarction, behavioral functions and brain levels of IL-10, TNF-α and matrix metaloperoteinase-9 (MMP9) have been examined in the evaluated groups. ResultsThe results revealed that JZL reduced brain edema, infarction, brain levels of TNF-α and MMP9 and also increased brain levels of IL-10 as well as improved behavioral functions in all three concentrations. The therapeutic effects of JZL were observed as well as Aspirin. DiscussionBased on the results, it seems that JZL can be considered as a good candidate for inhibition of stroke secondary injury in the case of delayed treatment.

Abstract WMP78: Brain Ischemia Induces Diversified Neuroantigen-Specific T Cell Responses That Exacerbate Brain Injury

Background: Autoimmune responses can occur when antigens from the central nervous system (CNS) are presented to lymphocytes in the periphery or CNS. However, whether autoimmune responses emerge after brain ischemia remains controversial. Similarly, it is debated whether genesis of autoimmune responses can impact outcome of ischemic stroke. We hypothesized that that brain ischemia can diversify T cell responses against CNS antigens, and that expanded CNS antigen-specific T cells can promote ischemic brain injury. Methods: We quantified the presence and status of T cells from brain slices of ischemic patients. Using a mouse strain that harbor a transgenic T cell receptor (TCR) to a CNS antigen, the myelin oligodendrocyte glycoprotein (MOG 35-55 ) epitope (2D2), we determined the anatomic location and involvement of antigen presentation cells in the development of T cell reactivity after brain ischemia and how T cell reactivity impacts stroke outcome. Transient middle cerebral artery occlusion (MCAO) and photothrombotic stroke models were used in this study. Results: By coupling transfer of labeled myelin oligodendrocyte glycoprotein (MOG) 35-55 -specific (2D2) T cells with tetramer tracking, we show an expansion in reactivity of 2D2 T cells to MOG 97-108 and MOG 103-125 in transient MCAO and photothrombotic stroke models. This reactivity and T cell activation occur in the brain and not in the peripheral lymphoid organs after ischemia. Also, microglia act as antigen-presenting cells (APCs) that effectively present MOG antigen, and depletion of microglia ablates expansion of 2D2 reactive T cells. Notably, the adoptive transfer of neuroantigen-experienced 2D2 T cells exacerbates Th1/Th17 responses and brain injury. Finally, T cell activation and MOG-specific T cells can be seen in the brain of ischemic stroke patients. Conclusion: Our findings suggest that brain ischemia activates and diversifies T cell responses locally, which exacerbates ischemic brain injury.

Catgut implantation at acupoints increases the expression of glutamate aspartate transporter and glial glutamate transporter-1 in the brain of rats with spasticity after stroke

Catgut implantation at acupoints has been shown to alleviate spasticity after stroke in rats. However, the underlying mechanisms are poorly understood. In this study, we used the rat middle cerebral artery occlusion model of stroke. Three days after surgery, absorbable surgical catgut sutures were implanted at Dazhui (GV14), Jizhong (GV6), Houhui, Guanyuan (CV4) and Zhongwan (CV12). The Zea Longa score was used to assess neurological function. The Modified Ashworth Scale was used to evaluate muscle tension. The 2,3,5-triphenyl-tetrazolium chloride assay was used to measure infarct volume. Immunohistochemical staining was performed for glutamate aspartate transporter (GLAST) and glial glutamate transporter-1 (GLT-1) expression. Western blot assay was used to analyze the expression of GLAST and GLT-1. Reverse transcription and polymerase chain reaction were carried out to assess the expression of GLAST and GLT-1 mRNAs. After catgut implantation at the acupoints, neurological function was substantially improved, muscle tension was decreased, and infarct volume was reduced in rats with spasticity after stroke. Furthermore, the expression of GLAST and GLT-1 mRNAs was increased on the injured (left) side. Our findings demonstrate that catgut implantation at acupoints alleviates spasticity after stroke, likely by increasing the expression of GLAST and GLT-1.

Gemst: a taylor-made combination that reverts neuroanatomical changes in stroke.

In a single transient middle cerebral artery occlusion model of stroke and using immunohistochemical techniques, the effects of a new therapeutic approach named Gemst (a member of the Poly-LLysine innovative therapies) have been studied in the rat brain. The expression of inflammatory (CD45, CD11b), oxidative (NO-tryptophan, NO2-tyrosine) and indoleamine 2, 3-dioxygenase pathway (kynurenic acid, 3-hydroxy anthranilic acid) markers has been evaluated in early and late phases of stroke. For this purpose, we have developed eight highly specific monoclonal antibodies directed against some of these markers. In the early phase (3 and 5 days of the stroke, we observed no effect of Gemst treatment (7.5 mg/day, subcutaneously for 3, 5 days). In the late phase (21 days) of stroke and exclusively in the ipsilateral side of non-treated animals an overexpression of kynurenic acid, 3- hydroxy anthranilic acid, CD45, CD11b, GFAP and ionized calcium-binding adapter molecule 1 (IBA-1) was found. In treated animals, the overexpression of the four former markers was completely abolished whereas the overexpression of the two latter ones was decreased down to normal levels. Gemst reversed the pathological conditions of stroke to normal situations. Gemst exerts a multifunctional action: down-regulates the indoleamine 2, 3-dioxygenase pathway and abolishes brain infiltration, microglial activation and gliosis. Moreover, Gemst has no effect on the expression of doublecortin, a protein involved in neuronal migration. Gemst could be a new drug for the treatment of stroke since it reverses the pathological findings of stroke and normalizes brain tissue conditions following the ischemic insult.

Regulatory T cells ameliorate tissue plasminogen activator-induced brain haemorrhage after stroke.

Delayed thrombolytic treatment with recombinant tissue plasminogen activator (tPA) may exacerbate blood-brain barrier breakdown after ischaemic stroke and lead to lethal haemorrhagic transformation. The immune system is a dynamic modulator of stroke response, and excessive immune cell accumulation in the cerebral vasculature is associated with compromised integrity of the blood-brain barrier. We previously reported that regulatory T cells, which function to suppress excessive immune responses, ameliorated blood-brain barrier damage after cerebral ischaemia. This study assessed the impact of regulatory T cells in the context of tPA-induced brain haemorrhage and investigated the underlying mechanisms of action. The number of circulating regulatory T cells in stroke patients was dramatically reduced soon after stroke onset (84 acute ischaemic stroke patients with or without intravenous tPA treatment, compared to 115 age and gender-matched healthy controls). Although stroke patients without tPA treatment gradually repopulated the numbers of circulating regulatory T cells within the first 7 days after stroke, post-ischaemic tPA treatment led to sustained suppression of regulatory T cells in the blood. We then used the murine suture and embolic middle cerebral artery occlusion models of stroke to investigate the therapeutic potential of adoptive regulatory T cell transfer against tPA-induced haemorrhagic transformation. Delayed administration of tPA (10 mg/kg) resulted in haemorrhagic transformation in the ischaemic territory 1 day after ischaemia. When regulatory T cells (2 × 106/mouse) were intravenously administered immediately after delayed tPA treatment in ischaemic mice, haemorrhagic transformation was significantly decreased, and this was associated with improved sensorimotor functions. Blood-brain barrier disruption and tight junction damages were observed in the presence of delayed tPA after stroke, but were mitigated by regulatory T cell transfer. Mechanistic studies demonstrated that regulatory T cells completely abolished the tPA-induced elevation of MMP9 and CCL2 after stroke. Using MMP9 and CCL2 knockout mice, we discovered that both molecules partially contributed to the protective actions of regulatory T cells. In an in vitro endothelial cell-based model of the blood-brain barrier, we confirmed that regulatory T cells inhibited tPA-induced endothelial expression of CCL2 and preserved blood-brain barrier integrity after an ischaemic challenge. Lentivirus-mediated CCL2 knockdown in endothelial cells completely abolished the blood-brain barrier protective effect of regulatory T cells in vitro. Altogether, our studies suggest that regulatory T cell adoptive transfer may alleviate thrombolytic treatment-induced haemorrhage in stroke victims. Furthermore, regulatory T cell-afforded protection in the tPA-treated stroke model is mediated by two inhibitory mechanisms involving CCL2 and MMP9. Thus, regulatory T cell adoptive transfer may be useful as a cell-based therapy to improve the efficacy and safety of thrombolytic treatment for ischaemic stroke.