Middle Cerebral Artery Occlusion Stroke Research Articles

Nt5e deficiency does not affect post-stroke inflammation and lesion size in a murine ischemia/reperfusion stroke model

SummaryExtracellular ATP released to the ischemic brain parenchyma is quickly metabolized by ectonucleotidases. Among them, the ecto-5′-nucleotidase CD73 encoded by Nt5e generates immunosuppressive adenosine. Genetic deletion of Nt5e led to increased infarct size in the murine photothrombotic stroke model. We aimed at validating this result using the transient middle cerebral artery occlusion (tMCAO) stroke model that represents pathophysiological aspects of penumbra and reperfusion. Three days after tMACO, we did not detect a difference in stroke size between CD73-deficient (CD73−/−) and control mice. Consistent with this finding, CD73−/− and control mice showed comparable numbers and composition of brain-infiltrating leukocytes measured by flow cytometry. Using NanoString technology, we further demonstrated that CD73−/− and control mice do not differ regarding glia cell gene expression profiles. Our findings highlight the potential impact of stroke models on study outcome and the need for cross-validation of originally promising immunomodulatory candidates.

Inhibition of PECAM-1 Significantly Delays Leukocyte Extravasation into the Subcortex Post-Stroke.

Current therapies for ischemic stroke focus on reperfusion but do not address the acute inflammatory response that results in significant reperfusion injury. To advance future therapies, a thorough understanding of the precise spatiotemporal underpinnings of leukocyte extravasation and infiltration is necessary. We describe the evolution of the inflammatory response in a mouse transient middle cerebral artery occlusion (tMCAO) stroke model at several time points after reperfusion and the modulation of this response with PECAM blockade. The transient Middle Cerebral Artery Occlusion model (90 minutes of ischemia followed by reperfusion) was used to simulate large vessel occlusion stroke and recanalization. We used wide field and confocal immunofluorescence microscopy to examine the exact distribution of neutrophils with close examination of the leukocyte position with regard to the brain vasculature and the perivascular space. Flow cytometry of single cell suspensions was used to confirm cell identity at different time points post-stroke. Large ischemic strokes involving both the subcortex and cortex (over 20% of the ischemic hemisphere) were induced in mice. At 12 and 24 hours, leukocyte recruitment and extravasation was primarily localized to the cortical surface. This contrasts with other organs where there is considerable migration of neutrophils deep into the inflamed tissue by 24 hours. Flow cytometry showed at 24 hours a majority of leukocytes were neutrophils. Over 48 to 72 hours, leukocytes were increasingly found deeper into the subcortex. Throughout the infarct (determined with triphenyl tetrazolium chloride staining), leukocyte recruitment was not uniform but rather organized in clusters. Disrupting leukocyte diapedesis with PECAM function-blocking monoclonal antibody restricted leukocytes to within 500 microns of the surface when compared to control; and this was still evident at 72 hours (n=3 mice per group, p<0.01, Control 46% ± 4.0 %; PECAM-1 Ab 62% ± 5.0%). High-resolution wide-field microscopy confirmed inhibition of TEM by PECAM-1 blockade at 24 hours. Flow cytometry showed approximately equal numbers of monocytes and neutrophils at 72 hours. Our findings demonstrate that leukocyte infiltration into a stroke evolves over several days following reperfusion. The use of PECAM blockade modulates the natural progression of leukocytes into the infarcted stroke bed. A better understanding of leukocyte spatiotemporal infiltration and its regulators could help inform the next generation of therapeutic interventions.

3,6'- and 1,6'-Dithiopomalidomide Mitigate Ischemic Stroke in Rats and Blunt Inflammation.

(1) Background: An important concomitant of stroke is neuroinflammation. Pomalidomide, a clinically available immunomodulatory imide drug (IMiD) used in cancer therapy, lowers TNF-α generation and thus has potent anti-inflammatory actions. Well-tolerated analogs may provide a stroke treatment and allow evaluation of the role of neuroinflammation in the ischemic brain. (2) Methods: Two novel pomalidomide derivatives, 3,6′-dithiopomalidomide (3,6′-DP) and 1,6′-dithiopomalidomide (1,6′-DP), were evaluated alongside pomalidomide in a rat middle cerebral artery occlusion (MCAo) stroke model, and their anti-inflammatory actions were characterized. (3) Results: Post-MCAo administration of all drugs lowered pro-inflammatory TNF-α and IL1-β levels, and reduced stroke-induced postural asymmetry and infarct size. Whereas 3,6′- and 1,6′-DP, like pomalidomide, potently bound to cereblon in cellular studies, 3,6′-DP did not lower Ikaros, Aiolos or SALL4 levels—critical intermediates mediating the anticancer/teratogenic actions of pomalidomide and IMiDs. 3,6′-DP and 1,6′-DP lacked activity in mammalian chromosome aberration, AMES and hERG channel assays –critical FDA regulatory tests. Finally, 3,6′- and 1,6′-DP mitigated inflammation across rat primary dopaminergic neuron and microglia mixed cultures challenged with α-synuclein and mouse LPS-challenged RAW 264.7 cells. (4) Conclusion: Neuroinflammation mediated via TNF-α plays a key role in stroke outcome, and 3,6′-DP and 1,6′-DP may prove valuable as stroke therapies and thus warrant further preclinical development.

Loss of Kir6.1 facilitates peri-infarct depolarizations in focal cerebral ischemia

ABSTRACT Objective Peri-infarct depolarizations (PIDs) are spontaneous waves that propagate slowly across the penumbra region following stroke, contributing to secondary infarct growth and negatively affecting stroke outcomes. KATP channels are generally spread in the brain. Under conditions of ischemia and/or hypoxia, KATP channels play a cytoprotective role in neurons. However, it is still unknown whether KATP channels are involved in the initiation and propagation of PIDs. Methods The Kir6.1 knockout (Kir6.1−/−) mice, Kir6.2 knockout (Kir6.2−/−) mice, and wild-type C57Bl6 mice (n = 8) were used. The middle cerebral artery occlusion (MCAO) stroke model was made and PIDs were detected by an optical intrinsic signal (OIS) imaging system. Results Much more PIDs appeared in Kir6.1−/−mice than that in Kir6.2−/− and WT mice in both the first hour and 4 hours following MCAO (3.9 ± 0.7 vs. 1.5 ± 0.3, p < 0,05; 3.9 ± 0.7 vs. 1.9 ± 0.3, p < 0.05; 20.0 ± 2.5 vs. 10.4 ± 2.4, p < 0.05; 20.0 ± 2.5 vs. 11.3 ± 1.4, p < 0.05). Furthermore, the first PID occurred much earlier in Kir6.1−/− mice than that in Kir6.2−/− mice and WT mice (21.3 ± 2.1 min vs. 34.1 ± 4.8 min, p < 0.05; 21.3 ± 2.1 min vs. 38.8 ± 3.4 min, p < 0.01). No significant differences in other characteristics of PIDs including originating sites, duration time, propagation patterns, and velocity were detected. Additionally, the migration of originating sites was observed. Conclusion This study shows that loss of Kir6.1, not Kir6.2 facilitates the induction of PIDs in focal cerebral ischemia, indicating that Kir6.1-forming channels in the brain may provide protection against PIDs.

The neuroprotective mechanism of lithium after ischaemic stroke

Stroke causes degeneration and death of neurones leading to the loss of motor function and frequent occurrence of cognitive impairment and depression. Lithium (Li+), the archetypal mood stabiliser, is neuroprotective in animal models of stroke, albeit underlying mechanisms remain unknown. We discover that Li+ inhibits activation of nucleotide-binding oligomerisation domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasomes in the middle cerebral artery occlusion (MCAO) stroke model in mice. This action of Li+ is mediated by two signalling pathways of AKT/GSK3β/β-catenin and AKT/FoxO3a/β-catenin which converge in suppressing the production of reactive oxygen species (ROS). Using immunocytochemstry, MRI imaging, and cell sorting with subsequent mRNA and protein quantification, we demonstrate that Li+ decreases the infarct volume, improves motor function, and alleviates associated cognitive and depressive impairments. In conclusion, this study reveals molecular mechanisms of Li+ neuroprotection during brain ischaemia, thus providing the theoretical background to extend clinical applications of Li+ for 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

Benefits of a Skull-Interfaced Flexible and Implantable Multilight Emitting Diode Array for Photobiomodulation in Ischemic Stroke.

Photobiomodulation (PBM) has received attention due to its potential for improving tissue function and enhancing regeneration in stroke. A lightweight, compact, and simple system of miniaturized electronic devices consisting of packaged light‐emitting diodes (LEDs) that incorporates a flexible substrate for in vivo brain PBM in a mouse model is developed. Using this device platform, the preventive and therapeutic effects of PBM affixed to the exposed skull of mice in the photothrombosis and middle cerebral artery occlusion stroke model are evaluated. Among the wavelength range of 630, 850, and 940 nm LED array, the PBM with 630‐nm LED array is proved to be the most effective for reducing the infarction volume and neurological impairment after ischemic stroke. Moreover, the PBM with 630 nm LED array remarkably improves the capability of spatial learning and memory in the chronic poststroke phase, attenuates AIM2 inflammasome activation and inflammasome‐mediated pyroptosis, and modulates microglial polarization in the hippocampus and cortex 7 days following ischemic stroke. Thus, PBM may prevent tissue and functional damage in acute ischemic injury, thereby attenuating the development of cognitive impairment after stroke.

Clenbuterol, a Selective β2-Adrenergic Receptor Agonist, Inhibits or Limits Post-Stroke Pneumonia, but Increases Infarct Volume in MCAO Mice.

BackgroundLarge ischemic stroke provokes an inflammatory response, promoting the release of norepinephrine (NE) by intensifying the sympathetic nervous system. This augmented sympathetic outflow was deemed to act on β2-adrenergic receptors (β2-ARs) expressed by immune cells, rendering organisms to post-stroke infections, like pneumonia. To clarify this issue, we introduced selective β2-ARs agonist clenbuterol (CLEN) to stroke mice to investigate how β2-adrenergic signaling augmentation after stroke affects immune response and post-stroke outcomes, including central and peripheral.MethodsWe developed a middle cerebral artery occlusion (MCAO) stroke model in mice to induce large ischemic stroke and administered CLEN 24 h after the onset of MCAO stroke. First, we assessed infarct volume and NE levels in plasma and spleen 3ds later. Next, the immune state was identified by analyzing the spleen index, immune cell populations, and immune cytokines. Finally, peripheral outcomes were assessed by measuring signs of pneumonia, such as pathology, bacterial burden, and lung cytokines.ResultsWe report that CLEN treatment 24 h after MCAO stroke causes an enlarged infarct volume and a decrease in NE levels at 3ds after stroke. Consistent with a reduction of total T cells, T helper cells, and increase of cytotoxic T cells, the immune milieu after CLEN treatment presents an anti-inflammatory landscape, showing raised expression of anti-inflammatory cytokines: IL-4, IL-10, and TGF-β1, and decreased expression of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IFN-γ, with a dramatically reduced percentage of Gr-1+ neutrophils and B cells but an increased percentage of NK cells. In our study, CLEN treatment results in no higher risk of pneumonia but relieves bacterial burden, inhibits or limits pneumonia, and diminishes TNF-α expression in lung tissues after MCAO.ConclusionWe identified increased β2-adrenergic signaling after MCAO stroke, inhibits or limits post-stroke pneumonia but enlarges stroke volume in MCAO mice. Thus, careful consideration must be taken to improve post-stroke outcomes by manipulation over β2-adrenergic receptors.

Melatonin and calpeptin synergistically protect against post-reperfusion injury in a rat middle cerebral artery occlusion stroke model

Cerebral ischemia induces oxidative injury and increases the intracellular calcium ion concentration to activate several calcium-dependent proteases such as calpains. Calpain activation leads to various necrotic and apoptotic processes. Calpeptin is a potent, cell-permeable calpain inhibitor. As a strong antioxidant and free radical scavenger, melatonin shows beneficial effect in rodent models of focal cerebral ischemia when given prior to ischemia or reperfusion. This study was focused on the neuroprotective effects of melatonin and/or calpeptin given after onset of reperfusion. For this purpose, right-sided middle cerebral artery occlusion (MCAO) for 90 minutes followed by 24 or 72 hours of reperfusion was performed in male Sprague Dawley rats, then, melatonin 50 or 150 µg/kg, calpeptin 10, 15 or 50 µg/kg or a combination of melatonin 50 µg/kg plus calpeptin 15 or 50 µg/kg were injected via an intracerebroventricular route at 15 minutes after onset of reperfusion. Melatonin or calpeptin tended to reduce the relative infarct volume and significantly decreased the neurological deficit at 24 hours. The combination achieved a greater protection than each of them alone. Melatonin, calpeptin or the combination all decreased Fluoro-Jade B (FJB)+ degenerative neurons and cleaved/total caspase-3 ratio at 24 hours. These treatments did not significantly impact the density of surviving neurons and ED-1+ macrophage/activated microglia. At the 72-hour-reperfusion, melatonin or the combination decreased the relative infarct volume and neurological deficit. Nevertheless, only the combination reduced FJB+ degenerating neurons at 72 hours. In conclusion, a combination of melatonin and calpeptin exerted synergistic protection against post-reperfusion injury in a rat MCAO stroke model.

Histone Methyltransferase Dot1L Contributes to RIPK1 Kinase-Dependent Apoptosis in Cerebral Ischemia/Reperfusion.

BackgroundNeuron apoptosis is a pivotal process for brain damage in cerebral ischemia. Dot1L (disruptor of telomeric silencing 1‐like) is only known histone H3K79 methyltransferase. It is not clear whether the role and mechanism of Dot1L on cerebral ischemia is related to regulate neuron apoptosis.Methods and ResultsWe use a combination of mice middle cerebral artery occlusion stroke and neurons exposed to oxygen‐glucose deprivation followed by reoxygenation to investigate the role and mechanism of Dot1L on cerebral ischemia. We find knockdown or inhibition of Dot1L reversed ischemia‐induced neuronal apoptosis and attenuated the neurons injury treated by oxygen‐glucose deprivation followed by reoxygenation. Further, blockade of Dot1L prevents RIPK1 (receptor‐interacting protein kinase 1)‐dependent apoptosis through increased RIPK1 K63‐ubiquitylation and decreased formation of RIPK1/Caspase 8 complexes. In line with this, H3K79me3 enrichment in the promoter region of deubiquitin‐modifying enzyme A20 and deubiquitinase cylindromatosis gene promotes the increasing expression in oxygen‐glucose deprivation followed by reoxygenation ‐induced neuronal cells, on the contrary, oxygen‐glucose deprivation followed by reoxygenation decreases H3K79me3 level in the promoter region of ubiquitin‐modifying enzyme cIAP1 (cellular inhibitors of apoptosis proteins), and both these factors ultimately cause K63‐deubiquitination of RIPK1. Importantly, knockdown or inhibition of Dot1L in vivo attenuates apoptosis in middle cerebral artery occlusion mice and reduces the extent of middle cerebral artery occlusion ‐induced brain injury.ConclusionsThese data support for the first time, to our knowledge, that Dot1L regulating RIPK1 to the apoptotic death trigger contributes to cerebral ischemia injury. Therefore, targeting Dot1L serves as a new therapeutic strategy for ischemia stroke.

A Co-Doped Fe3O4 Nanozyme Shows Enhanced Reactive Oxygen and Nitrogen Species Scavenging Activity and Ameliorates the Deleterious Effects of Ischemic Stroke.

Acute ischemic stroke has become the major cause of mortality and disability worldwide. Following ischemic stroke, the reperfusion injury is mainly mediated by the burst of reactive oxygen and nitrogen species (RONS). Therefore, blocking the excessive production or removing RONS holds great promise as a potential therapeutic strategy. Herein, we developed a Co-doped Fe3O4 nanozyme that is capable of scavenging H2O2, O2•-, •NO, and ONOO- in vitro and in vivo and provides neuroprotection against ischemic stroke. In vitro experiments showed that pre-incubation with the Co-Fe3O4 nanozyme could prevent neurotoxicity and neuroinflammation induced by H2O2 or lipopolysaccharide, respectively, in HT22 cells. After intravenous administration, the Co-Fe3O4 nanozyme showed no signs of toxicity in peripheral organs of C57BL/6J mice, even after prolonged delivery for 4 weeks. In permanent photothrombotic stroke model and transient middle cerebral artery occlusion stroke model, the Co-Fe3O4 nanozyme specifically accumulated in the infarct rim at 72 h post-stroke and was endocytosed by neurons, astrocytes, microglia, and endothelial cells. Importantly, the Co-Fe3O4 nanozyme delivery reduced the infarct volume in both stroke models. The observation that the Co-Fe3O4 nanozyme was efficacious in two well-characterized ischemic stroke models provides strong evidence that it represents a powerful tool for targeting oxidative and nitrosative stress in the ischemic brain.

A low-cost mouse cage warming system provides improved intra-ischemic and post-ischemic body temperature control – Application for reducing variability in experimental stroke studies

BackgroundBrain temperature is a strong determinant of ischemic stroke injury. For this reason, tight management of brain or body temperature (Tcore) in experimental rodent stroke models is recommended to improve the rigor and reproducibility of outcomes. However, methods for managing Tcore during and after stroke vary widely in approach and effectiveness. New MethodWe developed a low-cost warm ambient air cage (WAAC) system to provide improved temperature control during the intra-ischemic and post-ischemic recovery periods. The system is incorporated into standard holding cages for maintaining Tcore during the intra-ischemic period as well as for several hours into the recovery period. Results and Comparison with Existing MethodsWe compared the WAAC system with a commonly used heat support method, consisting of a cage on a heating pad. Both heat support systems were evaluated for the middle cerebral artery occlusion (MCAo) stroke model in mice. The WAAC system provided improved temperature control (more normothermic Tcore and less Tcore variation) during the intra- ischemic period (60 min) and post-ischemic period (3 h). Mean infarct volume was not statistically different by heat support system, however, standard deviation was 54 % lower in the WAAC system group. ConclusionsMice and other small rodents are highly vulnerable to heat loss during and after the MCAo procedure. The WAAC system provides more precise and controlled Tcore maintenance compared with frequently used induction heating methods in mice undergoing the MCAo stroke model. The improved temperature control should enhance experimental rigor and reduce the number of experimental animals needed.

Neutrophil Distribution in the Post Stroke Brain Evolves Dramatically Over Time and can be Modulated by Disrupting PECAM Function

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. Here we describe the pattern of the acute inflammatory response in a mouse transient middle cerebral artery occlusion (tMCAO) stroke model at several time points after reperfusion. We used widefield and confocal immunofluorescence microscopy to examine the distribution of neutrophils with precise examination of the leukocyte position relative to the cerebrovasculature and adjacent perivascular space. The recruitment of neutrophils varied dramatically across the infarcted tissue and surrounding penumbra, especially at early time points. At 12 and 24 hours, neutrophil recruitment and extravasation are predominantly observed at the cortical surface. Over the next few days (48 and 72 hours), neutrophils are increasingly found deeper into the subcortex. Disrupting leukocyte transendothelial migration with PECAM function-blocking antibodies caused a marked redistribution of neutrophils away from the subcortex with a corresponding increase at the cortical surface at 72 hours. Our findings suggest that the vast majority of infiltrating neutrophils are initially recruited to cortical venules. Furthermore, neutrophils rapidly escape the perivascular compartment and enter the parenchyma. In addition, this initial recruitment to the cortical surface was highly regionalized. In spite of this, over the course of several days neutrophils did redistribute more evenly across the entire infarcted hemisphere. Taken together our findings demonstrate that the infiltration of neutrophils dynamically evolves over several days following reperfusion. The initial heterogeneous pattern of neutrophil recruitment does not correlate well with traditional markers of cellular dysfunction but does become more homogenous as the pathology evolves. A better understanding of the precise spatio-temporal infiltration of inflammatory cells could help inform the next generation of therapeutic interventions.

Abstract P766: Ischemic Injury in a Dmcao Model of Stroke Demonstrates Long-Term Immune Cell Diapedesis Into the Brain Parenchyma

Background: Stroke injury following a middle cerebral artery occlusion (MCAo) induces a rapid migration of leukocytes into the injured brain that lasts for weeks. The current study focuses on whether a focal cortical stroke using the distal MCAo (dMCAo) model induces similar long-term immune cell diapedesis into the brain parenchyma as seen following transient (t)MCAo stroke. Methods: Cells were isolated from spleens and brain hemispheres of adult 1-year old male C57BL/6J (B6; Jackson Labs) mice 30 days after a dMCAo (n=10). Sham animals (n=5) received a craniotomy without the distal MCA ligation. Peripheral migration was assessed in the spleen and brain using flow cytometry (FACSymphony) to identify viable (Ghost dye 780) CD3, CD4, CD8b, CD11b, Ly6C/Ly6G, CD19, CD45, and NK1.1 leukocytes. Populations were analyzed with FlowJo and assessed via repeated measures two-way ANOVA, Sidak post-hoc test (Graphpad Prism). Significance was p&lt;0.05. Results: CD45 + leukocytes were elevated in the ipsilesional (ipsi) hemisphere compared to the contralesional hemisphere (p=0.01) after dMCAo, though a group hemispheric effect (F(1,13)=5.4; p=0.04) suggests long-term inflammation in sham-treated mice. Hemispheric effects also occurred for CD8 + T cells (p=0.046), B cells (p=0.03), monocytes (p=0.01), and macrophages (p=0.06), with elevations in both dMCAo and sham-treated mice in the ipsi vs. contralesional hemispheres. Only monocyte populations were significantly elevated (p=0.03) in dMCAo vs. sham mice. Conclusions: Our study shows that immune cells remain elevated in the injured hemisphere at 30 days after a focal stroke confined to the neocortex, but inflammation occurred in both sham and dMCAo-treated animals. Only monocytes were differentially affected by dMCAo, and infiltrating cell numbers are not as robust as after tMCAo. This demonstrates a long-term injury response from the craniotomy in the dMCAo model that should be considered for long-term studies using this model.

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).

Autoantibodies to Annexin A2 and cerebral thrombosis: Insights from a mouse model.

IntroductionAntiphospholipid syndrome (APS) is an autoimmune disorder manifested by thromboembolic events, recurrent spontaneous abortions and elevated titers of circulating antiphospholipid antibodies. In addition, the presence of antiphospholipid antibodies seems to confer a fivefold higher risk for stroke or transient ischemic attack. Although the major antigen of APS is β2 glycoprotein I, it is now well established that antiphospholipid antibodies are heterogeneous and bind to various targets. Recently, antibodies to Annexin A2 (ANXA2) have been reported in APS. This is of special interest since data indicated ANXA2 as a key player in fibrinolysis. Therefore, in the present study we assessed whether anti-ANXA2 antibodies play a pathological role in thrombosis associated disease.Materials and MethodsMice were induced to produce anti-ANXA2 antibodies by immunization with ANXA2 (iANXA2) and control mice were immunized with adjuvant only. A middle cerebral artery occlusion stroke model was applied to the mice. The outcome of stroke severity was assessed and compared between the two groups.ResultsOur results indicate that antibodies to ANXA2 lead to a more severe stroke as demonstrated by a significant larger stroke infarct volume (iANXA2 133.9 ± 3.3 mm3 and control 113.7 ± 7.4 mm3; p = 0.017) and a more severe neurological outcome (iANXA2 2.2 ± 0.2, and control 1.5 ± 0.18; p = 0.03).ConclusionsThis study supports the hypothesis that auto-antibodies to ANXA2 are an independent risk factor for cerebral thrombosis. Consequently, we propose screening for anti-ANXA2 antibodies should be more widely used and patients that exhibit the manifestations of APS should be closely monitored by physicians.

Abstract 14278: Microglial Calcium Activity During Ischemic Stroke

Introduction: Ischemic stroke triggers waves of propagating action potentials followed by a loss of homeostatic ion gradients, known as cortical spreading depolarizations (SD). Our data indicate that microglia respond to SD by raising intracellular Ca 2+ , triggering a release of proinflammatory cytokines that may exacerbate post-stroke morbidity. Hypothesis: Inhibiting calcium release-activated calcium (CRAC) channels may block microglial Ca2+ influx and activation to potentially provide therapeutic benefit in ischemic stroke. Methods: We generated a mouse line expressing the Ca2+ indicator GCaMP5 in cortical microglia. Using two-photon microscopy, we imaged microglia in vivo following physical stroke via middle cerebral artery occlusion (MCAo) or chemical stroke via 1M KCl solution application. Controls were compared to mice treated with lipopolysaccharide (LPS) to mimic the inflammation of ischemic stroke. To study CRAC channels as a therapeutic target, we administered CM-EX-137, a CRAC channel blocker, and compared Ca2+ activity to controls. Results: We identified periodical Ca2+ waves in cortical microglia following MCAo (Fig. 1A), or localized KCl application (Fig. 1B-E). Further, when compared to controls, LPS-exposed mice expressed significantly greater microglial Ca2+ activity during KCl-triggered SD (Fig. 1C). Additionally, administration of CM-EX-137 effectively abolished the Ca2+ signals in the microglia and propagation of SD upon application of KCl (Fig. 1E). Conclusions: Blocking the Ca2+ influx into microglia after ischemic stroke may decrease the frequency of SD and reduce microglial activation, potentially leading to smaller stroke volumes and improved clinical outcomes. Figure 1: Microglial Ca 2+ after SD. (A) Ca 2+ transients in microglia after MCAo. (B) KCl-induced Ca 2+ activity in naïve microglia. (C) KCl-induced Ca 2+ activity after LPS. (D, E) KCl-induced Ca 2+ wave after (D) vehicle or (E) CM-EX-137 administration.

Coicis semen protects against focal cerebral ischemia-reperfusion injury by inhibiting oxidative stress and promoting angiogenesis via the TGFβ/ALK1/Smad1/5 signaling pathway.

Background: Ischemic stroke is a devastating disease that causes long-term disability. However, its pathogenesis is unclear, and treatments for ischemic stroke are limited. Recent studies indicate that oxidative stress is involved in the pathological progression of ischemic stroke and that angiogenesis participates in recovery from ischemic stroke. Furthermore, previous studies have shown that Coicis Semen has antioxidative and anti-inflammatory effects in a variety of diseases. In the present study, we investigated whether Coicis Semen has a protective effect against ischemic stroke and the mechanism of this protective effect.Results: Coicis Semen administration significantly decreased the infarct volume and mortality and alleviated neurological deficits at 3, 7 and 14 days after MCAO. In addition, cerebral edema at 3 days poststroke was ameliorated by Coicis Semen treatment. DHE staining showed that ROS levels in the vehicle group were increased at 3 days after reperfusion and then gradually declined, but Coicis Semen treatment reduced ROS levels. The levels of GSH and SOD in the brain were increased by Coicis Semen treatment, while MDA levels were reduced. Furthermore, Coicis Semen treatment decreased the extravasation of EB dye in MCAO mouse brains and elevated expression of the tight junction proteins ZO-1 and Occludin. Double immunofluorescence staining and western blot analysis showed that the expression of angiogenesis markers and TGFβ pathway-related proteins was increased by Coicis Semen administration. Consistent with the in vivo results, cytotoxicity assays showed that Coicis Semen substantially promoted HUVEC survival following OGD/RX in vitro. Additionally, though LY2109761 inhibited the activation of TGFβ signaling in OGD/RX model animals, Coicis Semen cotreatment markedly reversed the downregulation of TGFβ pathway-related proteins and increased VEGF levels.Methods: Adult male wild-type C57BL/6J mice were used to develop a middle cerebral artery occlusion (MCAO) stroke model. Infarct size, neurological deficits and behavior were evaluated on days 3, 7 and 14 after staining. In addition, changes in superoxide dismutase (SOD), GSH and malondialdehyde (MDA) levels were detected with a commercial kit. Blood-brain barrier (BBB) permeability was assessed with Evans blue (EB) dye. Western blotting was also performed to measure the levels of tight junction proteins of the BBB. Additionally, ELISA was performed to measure the level of VEGF in the brain. The colocalization of CD31, angiogenesis markers, and Smad1/5 was assessed by double immunofluorescent staining. TGFβ pathway-related proteins were measured by western blotting. Furthermore, the cell viability of human umbilical vein endothelial cells (HUVECs) following oxygen-glucose deprivation/reoxygenation (OGD/RX) was measured by Cell Counting Kit (CCK)-8 assay.Conclusions: Coicis Semen treatment alleviates brain damage induced by ischemic stroke through inhibiting oxidative stress and promoting angiogenesis by activating the TGFβ/ALK1 signaling pathway.

Blood substitution therapy rescues the brain of mice from ischemic damage

Acute stroke causes complex, pathological, and systemic responses that have not been treatable by any single medication. In this study, using a murine transient middle cerebral artery occlusion stroke model, a novel therapeutic strategy is proposed, where blood replacement (BR) robustly reduces infarctions and improves neurological deficits in mice. Our analyses of immune cell subsets suggest that BR therapy substantially decreases neutrophils in blood following a stroke. Electrochemiluminescence detection demonstrates that BR therapy reduces cytokine storm in plasma and ELISA demonstrates reduced levels of matrix metalloproteinase-9 (MMP-9) in the plasma and brains at different time points post-stroke. Further, we have demonstrated that the addition of MMP-9 to the blood diminishes the protective effect of the BR therapy. Our study is the first to show that BR therapy leads to profoundly improved stroke outcomes in mice and that the improved outcomes are mediated via MMP-9. These results offer new insights into the mechanisms of stroke damage.

Effects of Aerobic Exercise on Tau and Related Proteins in Rats with the Middle Cerebral Artery Occlusion.

Although Alzheimer’s disease (AD)-like pathology is frequently found in patients with post-stroke dementia, little is known about the effects of aerobic exercise on the modifications of tau and related proteins. Therefore, we evaluated the effects of aerobic exercise on the phosphorylation and acetylation of tau and the expressions of tau-related proteins, after middle cerebral artery occlusion (MCAO) stroke. Twenty-four Sprague–Dawley rats with MCAO infarction were used in this study. The rehabilitation group (RG) received treadmill training 40 min/day for 12 weeks, whereas the sedentary group (SG) did not receive any type of training. Functional tests, such as the single pellet reaching task, rotarod, and radial arm maze tests, were performed monthly for 3 months. In ipsilateral cortices in the RG and SG groups, level of Ac-tau was lower in the RG, whereas levels of p-tauS396, p-tauS262, and p-tauS202/T205 were not significantly lower in the RG. Level of phosphorylated glycogen synthase kinase 3-beta Tyr 216 (p-GSK3βY216) was lower in the RG, but levels of p-AMPK and phosphorylated glycogen synthase kinase 3-beta Ser 9 (p-GSK3βS9) were not significantly lower. Levels of COX-2 and BDNF were not significantly different between the two groups, while SIRT1 significantly decreased in ipsilateral cortices in RG. In addition, aerobic training also improved motor, balance, and memory functions. Rehabilitation with aerobic exercise inhibited tau modification, especially tau acetylation, following infarction in the rat MCAO model, which was accompanied with the improvement of motor and cognitive functions.