Cerebral Artery Occlusion In Mice Research Articles

Platelets and lymphocytes drive progressive penumbral tissue loss during middle cerebral artery occlusion in mice

BackgroundIn acute ischemic stroke, cessation of blood flow causes immediate tissue necrosis within the center of the ischemic brain region accompanied by functional failure in the surrounding brain tissue designated the penumbra. The penumbra can be salvaged by timely thrombolysis/thrombectomy, the only available acute stroke treatment to date, but is progressively destroyed by the expansion of infarction. The underlying mechanisms of progressive infarction are not fully understood.MethodsTo address mechanisms, mice underwent filament occlusion of the middle cerebral artery (MCAO) for up to 4 h. Infarct development was compared between mice treated with antigen-binding fragments (Fab) against the platelet surface molecules GPIb (p0p/B Fab) or rat immunoglobulin G (IgG) Fab as control treatment. Moreover, Rag1−/− mice lacking T-cells underwent the same procedures. Infarct volumes as well as the local inflammatory response were determined during vessel occlusion.ResultsWe show that blocking of the platelet adhesion receptor, glycoprotein (GP) Ibα in mice, delays cerebral infarct progression already during occlusion and thus before recanalization/reperfusion. This therapeutic effect was accompanied by decreased T-cell infiltration, particularly at the infarct border zone, which during occlusion is supplied by collateral blood flow. Accordingly, mice lacking T-cells were likewise protected from infarct progression under occlusion.ConclusionsProgressive brain infarction can be delayed by blocking detrimental lymphocyte/platelet responses already during occlusion paving the way for ultra-early treatment strategies in hyper-acute stroke before recanalization.

Neuroprotective Effects Against Cerebral Ischemic Injury Exerted by Dexmedetomidine via the HDAC5/NPAS4/MDM2/PSD-95 Axis.

Numerous evidences have highlighted the efficient role of dexmedetomidine (DEX) in multi-organ protection. In the present study, the neuroprotective role of DEX on cerebral ischemic injury and the underlining signaling mechanisms were explored. In order to simulate cerebral ischemic injury, we performed middle cerebral artery occlusion in mice and oxygen-glucose deprivation in neurons. Immunohistochemistry, Western blot analysis, and RT-qPCR were used to examine expression of HDAC5, NPAS4, MDM2, and PSD-95 in hippocampus tissues of MCAO mice and OGD-treated neurons. MCAO mice received treatment with DEX and sh-PSD-95, followed by neurological function evaluation, behavioral test, infarct volume detection by TTC staining, and apoptosis by TUNEL staining. Additionally, gain- and loss-of-function approaches were conducted in OGD-treated neuron after DEX treatment. Cell viability and apoptosis were assessed with the application of CCK-8 and flow cytometry. The interaction between MDM2 and PSD-95 was evaluated using Co-IP assay, followed by ubiquitination of PSD-95 detection. As per the results, HDAC5 and MDM2 were abundantly expressed, while NPAS4 and PSD-95 were poorly expressed in hippocampus tissues of MCAO mice and OGD-treated neurons. DEX elevated viability, and reduced LDH leakage rate and apoptosis rate of OGD-treated neurons, which was reversed following the overexpression of HDAC5. Moreover, HDAC5 augmented MDM2 expression via NPAS4 inhibition. MDM2 induced PSD-95 ubiquitination and degradation. In MCAO mice, DEX improved neurological function and behaviors and decreased infarct volume and apoptosis, which was negated as a result of PSD-95 silencing. DEX plays a neuroprotective role against cerebral ischemic injury by disrupting MDM2-induced PSD-95 ubiquitination and degradation via HDAC5 and NPAS4.

Transcranial Focused Ultrasound Stimulation Improves Neurorehabilitation after Middle Cerebral Artery Occlusion in Mice.

Transcranial focused ultrasound stimulation (tFUS) regulates neural activity in different brain regions in humans and animals. However, the role of ultrasound stimulation in modulating neural activity and promoting neurorehabilitation in the ischemic brain is largely unknown. In the present study, we explored the effect of tFUS on neurological rehabilitation and the underlying mechanism. Adult male ICR mice (n=42) underwent transient middle cerebral artery occlusion. One week after brain ischemia, low frequency (0.5 MHz) tFUS was applied to stimulate the ischemic hemisphere of mice for 7 consecutive days (10 minutes daily). Brain infarct volume, neurobehavioral tests, microglia activation, IL-10 and IL-10R levels were further assessed for up to 14 days. We found that the brain infarct volume was significantly reduced in the tFUS treated mice compared to that in the non-treated mice (p<0.05). Similarly, neurological severity scores, elevated body swing test, and corner test improved in the tFUS treated mice (p<0.05). We also demonstrated that tFUS resulted in increased M2 microglia in the ischemic brain region. The expression of IL-10R and IL-10 levels were also substantially upregulated (p<0.05). We concluded that tFUS served as a unique technique to promote neurorehabilitation after brain ischemia by promoting microglia polarization and further regulating IL-10 signaling in the ischemic brain.

Inhibition of Long Noncoding RNA SNHG15 Ameliorates Hypoxia/Ischemia-Induced Neuronal Damage by Regulating miR-302a-3p/STAT1/NF-κB Axis.

PurposeIschemic brain injury results in high mortality and serious neurologic morbidity. Here, we explored the role of SNHG15 in modulating neuronal damage and microglial inflammation after ischemia stroke.Materials and MethodsThe hypoxia/ischemia models were induced by middle cerebral artery occlusion in mice and oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro. Quantitative real-time PCR (qRT-PCR) and Western blot were conducted to determine the levels of SNHG15, miR-302a-3p, and STAT1/NF-κB. Moreover, gain- or loss-of functional assays of SNHG15 and miR-302a-3p were conducted. MTT assay was used to evaluate the viability of HT22 cells, and the apoptotic level was determined by flow cytometry. Furthermore, enzyme-linked immunosorbent assay was performed to detect oxidative stress and inflammatory mediators in the ischemia cortex and OGD/R-treated BV2 microglia.ResultsThe SNHG15 and STAT1/NF-κB pathways were both distinctly up-regulated, while miR-302a-3p was notably down-regulated in the ischemia cortex. Additionally, overexpressing SNHG15 dramatically enhanced OGD/R-mediated neuronal apoptosis as well as the expression of oxidative stress and inflammation factors from microglia. In contrast, knocking down SNHG15 or overexpressing miR-302a-3p relieved OGD/R-mediated neuronal apoptosis and microglial activation. Moreover, the rescue experiment testified that overexpressing miR-302a-3p also attenuated SNHG15 up-regulation-induced effects. In terms of the mechanisms, SNHG15 sponged miR-302a-3p and activated STAT1/NF-κB as a competitive endogenous RNA, while miR-302a-3p targeted STAT1 and negatively regulated the STAT1/NF-κB pathway.ConclusionSNHG15 was up-regulated in the hypoxia/ischemia mouse or cell model. The inhibition of SNHG15 ameliorates ischemia/hypoxia-induced neuronal damage and microglial inflammation by regulating the miR-302a-3p/STAT1/NF-κB pathway.

The NLRP3 inflammasome drives inflammation in ischemia/reperfusion injury after transient middle cerebral artery occlusion in mice

PurposeCerebral ischemia induces a profound neuro-inflammatory response, but the underlying molecular mechanisms are poorly understood. Inflammasomes (NLRP1, NLRP3, NLRC4, AIM2) are intracellular multi-protein complexes which can induce sets of pro-inflammatory cyto- and chemokines, and thereby guide inflammation. We, here, assessed the functional role of NLRP3 in ischemia/reperfusion (I/R) injury in a mouse model of transient cerebral ischemia. MethodsIschemic stroke was induced in C57Bl/6 mice by 60 min transient middle cerebral artery occlusion (tMCAO) and 3, 7 or 23 h of reperfusion, a paradigm of I/R injury. The expression patterns of inflammasomes in the ischemic hemispheres were evaluated by semiquantitative real-time PCR and Western Blot analysis accompanied by protein localization using immunocytochemistry. Finally, animals were treated with the inflammasome inhibitors Sulforaphane, Genipin, MCC950 or vehicle, directly before or upon recanalization after tMCAO. Stroke outcome was assessed, including infarct size and functional deficits, local inflammatory response, neuronal survival as well as blood–brain barrier function on day 1 after tMCAO. ResultsAfter tMCAO the relative gene expression levels of NLRP3 increased 20-30x within 1 day in the ischemic hemisphere which translated into an increased expression of NLRP3 in neurons. Accordingly, the gene expression levels of the NLRP3-modulator, Bruton’s Tyrosine Kinase (BTK), and the NLRP3-inducible cytokine IL-1β significantly rose. Lesser or non-significant changes were seen for the other inflammasomes. Application of inflammasome inhibitors covering all inflammasomes or specifically NLRP3 significantly reduced infarct volumes when given before or after tMCAO and was accompanied by clear evidence for reduced activation of caspase 1. This stroke attenuating effect coincided with less immune cell infiltration in the ischemic hemisphere and preservation of the blood–brain barrier integrity. ConclusionsOur data show that induction of the NLRP3 inflammasome in neurons drives neuroinflammation in acute ischemic stroke. Early blockade of NLRP3 protects from I/R injury by mitigating inflammation and stabilizing the blood–brain barrier.

Targeting chronic and evolving neuroinflammation following traumatic brain injury to improve long-term outcomes: insights from microglial-depletion models.

Targeting chronic and evolving neuroinflammation following traumatic brain injury to improve long-term outcomes: insights from microglial-depletion models.

4035 Astrocyte LDLR-Related Protein 1 Increases Cytokine Sensitivity – The Role of Glia in Recovery after Brain Damage

OBJECTIVES/GOALS: The limited treatment options for ischemic stroke patients have resulted in stroke being a leading cause of death and the primary cause of long-term disability in the U.S. Finding effective treatment options requires a better fundamental understanding of the ongoing processes that contribute to poor long-term outcome. METHODS/STUDY POPULATION: Expression of Apolipoprotein E4 predisposes stroke patients to poor long-term outcome. This study aims to test one possible mechanism by which ApoE4 contributes to cognitive decline after stroke. Here, we examine the effect of a major ApoE4 receptor, low-density lipoprotein receptor related protein 1 (LRP1) on sensitivity to stress in astrocytes. LRP1 binds and moves extracellular ligands and plasma membrane proteins into the endocytic system. Others have shown that LRP1 regulates cell-surface TNF receptor (TNFR1) in non-astrocytic cells. We propose That LRP1 similarly regulates TNFR1 in the central nervous system to attenuate inflammatory response after stroke. Studies have shown that ApoE4 slows the recycling of endocytic LDL receptors. We hypothesize that ApoE4 inhibits the ability of LRP1 to remove TNFR1 from the plasma membrane. This is expected to increase cytokine sensitivity, resulting in worse outcome after stroke. We investigated the effect of LRP1 on astrocyte TNFα signaling and response in immortalized ApoE null mouse astrocytes subjected to lentiviral-mediated knockdown of LRP1. The astrocyte response to TNFα stimulation was tested in a time dependent manner using Western blotting of NFkB pathway components, which are the downstream mediators of TNFα signaling. We also tested astrocyte viability after prolonged TNFα stimulation using Alamar Blue reagent. We found that LRP1 deficient cells have increased phosphorylation of NFkB upon TNFα stimulation, and that loss of LRP1 resulted in significant loss of astrocyte viability after prolonged stimulation. RESULTS/ANTICIPATED RESULTS: Altogether, our results indicate that loss of LRP1 renders astrocytes more sensitive to TNFα. Future experiments will focus on testing the influence of LRP1 on recovery after middle cerebral artery occlusion in mice. DISCUSSION/SIGNIFICANCE OF IMPACT: These studies will elucidate how astrocyte-LRP1 contributes to outcome after stroke, and helps us to understand one potential way that ApoE4 exerts pathological effects. A better understanding of the long-term processes after stroke will allow identification of therapies which improve the morbidity and mortality associated with stroke. CONFLICT OF INTEREST DESCRIPTION: NA.

Up-regulation of sphingosine-1-phosphate receptors and sphingosine kinase 1 in the peri-ischemic area after transient middle cerebral artery occlusion in mice

There is thought to be a strong relationship between sphingosine-1-phosphate (S1P) signaling and pathophysiolosy of cerebral ischemia. We examined the change of expression and distribution of S1P receptors (S1PRs) and sphingosine kinases (SphKs) after cerebral ischemia in male C57BL6/J mice using immunohistochemical analysis at 1, 5, 14, and 28 days after 30 min of transient middle cerebral artery occlusion (tMCAO). S1PR1, 3, and 5 were transiently induced in the cells, which were morphologically similar to neurons in the peri-infarct lesion with a peak seen at 1 day after tMCAO (p < 0.01 vs. sham control). S1PR2 appeared in the inner layer of vessels in the ischemic core (p < 0.01 vs. sham control) and the peri-infarct lesion (p < 0.01 vs. sham control) at the acute phase after tMCAO. However, SphK1 was strongly induced at 1 and 5 days after tMCAO (p < 0.01 vs. sham control) in the peri-infarct lesion, whereas SphK2 expression did not change. Western blot analysis at 1 and 5 days after 30 min of tMCAO revealed that the expression of S1PRs were transiently enhanced at the acute phase, which was consistent with the immunohistochemical results. Double immunofluorescent analysis revealed S1PR2/NG2- and S1PR2/CD31-, S1PR3/CD31-, and S1PR5/CD31-double positive cells in the peri-infarct lesion 1 day after tMCAO. The present results suggest that S1PRs and SphK1 may be important therapeutic targets for rescuing the peri-infarct lesion.

Abstract TMP30: M2 Microglia Derived Exosomes Promote White Matter Repair and Long-Term Functional Recovery After Focal Cerebral Ischemia in Mice

Objectives: White matter injury aggravates neurological and cognitive impairment in experimental ischemic stroke. M2 microglia promote oligodendrocyte precursor cells survival and differentiation, and further enhance white matter repair. However, the molecular mechanism is unclear. Here, we explored the effect and mechanism of M2 microglia-derived exosomes on white matter repair after focal cerebral ischemia in mice. Methods: Microglia BV2 cells were polarized to M2 phenotype by IL-4 stimulation. Exosomes were isolated from M2 microglia (M2-Exo) and unstimulated microglia as a control (M0-Exo). M2-Exo and M0-Exo (100 μg) were intravenously injected after 90-minute middle cerebral artery occlusion in mice (n=72). Brain atrophy volume and neuro behavioral outcomes were examined in 28 days following focal cerebral ischemia. Oligodendrocyte precursor cells survival, differentiation and white matter integrity were evaluated. Exosomal miRNA and target gene were further examined to explore molecular mechanism. Results: M2-Exo treatment promoted sensorimotor and memory function recovery ( p &lt;0.05), and further reduced brain atrophy compared to the M0-Exo control group ( p &lt;0.001). Immunostaining showed that M2-Exo increased the number of BrdU + /Pdgfr-α + and BrdU + /adenomatous polyposis coli + cells, enhanced myelin basic protein fluorescence-intensity compared to the control ( p &lt;0.05). M2-Exo increased oligodendrocyte precursor cell survival under OGD in vi tro , ( p &lt;0.05) and differentiation ( p &lt;0.05). Exosomal miRNA sequencing and PCR identified that miR-23a-5p was enriched in M2-Exo. Conclusion: Our results showed that M2-Exo treatment enhanced oligodendrocyte precursor cell survival and differentiation, further promoted white matter repair and long-term functional recovery, suggesting that M2-Exo is a novel therapeutic strategy for the white matter repair after ischemic brain injury.

Dl-NBP (Dl-3-N-Butylphthalide) Treatment Promotes Neurological Functional Recovery Accompanied by the Upregulation of White Matter Integrity and HIF-1α/VEGF/Notch/Dll4 Expression.

Dl-3-n-butylphthalide (dl-NBP) was approved by the FDA of China for the treatment of acute ischemic stroke. Dl-NBP has been shown to promote neurological functional recovery and enhance white matter integrity using an endothelin-1-induced focal permanent cerebral ischemia model, which could mimic those patients who have no opportunity to receive either tissue plasminogen activator (tPA) thrombolysis or endovascular therapy. However, it is not clear whether dl-NBP could promote neurological functional recovery in a focal transient cerebral ischemia model, which could mimic those patients who have the opportunity to receive either tPA thrombolysis or endovascular therapy. In this study, using a model of middle cerebral artery occlusion in mice, we aim to explore the effect of two-week dl-NBP treatment on neurological functional recovery after ischemic stroke as well as its underlying mechanism. Our results showed that dl-NBP treatment promoted functional recovery assessed by neurological scores and an adhesive remove test, and this improved the integrity of white matter after 60-min ischemia and 14-day reperfusion. In addition, dl-NBP increased the number of RECA-1 positive vessels and enhanced the expression of the tight junction protein occludin. More importantly, dl-NBP also promoted the expression of hypoxia-induced factor-1α, the vascular endothelial growth factor, Notch, and delta-like ligand 4. In conclusion, our study provides evidence that dl-NBP treatment could also promote functional recovery after focal transient ischemia stroke, and this recovery is associated with upregulated white matter integrity, microvessels, and the tight junction protein occludin. Our results suggested that, in future, dl-NBP may also be applied in clinic to promote functional recovery during the later phase of focal transient ischemic stroke.

Stroke Induces Prolonged Changes in Lipid Metabolism, the Liver and Body Composition in Mice

During recovery, stroke patients are at risk of developing long-term complications that impact quality of life, including changes in body weight and composition, depression and anxiety, as well as an increased risk of subsequent vascular events. The aetiologies and time-course of these post-stroke complications have not been extensively studied and are poorly understood. Therefore, we assessed long-term changes in body composition, metabolic markers and behaviour after middle cerebral artery occlusion in mice. These outcomes were also studied in the context of obesity, a common stroke co-morbidity proposed to protect against post-stroke weight loss in patients. We found that stroke induced long-term changes in body composition, characterised by a sustained loss of fat mass with a recovery of lean weight loss. These global changes in response to stroke were accompanied by an altered lipid profile (increased plasma free fatty acids and triglycerides) and increased adipokine release at 60 days. After stroke, the liver also showed histological changes indicative of liver damage and a decrease in plasma alanine aminotransferase (ALT) was observed. Stroke induced depression and anxiety-like behaviours in mice, illustrated by deficits in exploration, nest building and burrowing behaviours. When initial infarct volumes were matched between mice with and without comorbid obesity, these outcomes were not drastically altered. Overall, we found that stroke induced long-term changes in depressive/anxiety-like behaviours, and changes in plasma lipids, adipokines and the liver that may impact negatively on future vascular health.

Pharmacological hypothermia induced neurovascular protection after severe stroke of transient middle cerebral artery occlusion in mice

Therapeutic hypothermia is a potential protective strategy after stroke. The present study evaluated the neurovascular protective potential of pharmacological hypothermia induced by the neurotensin receptor 1 agonist HPI-201 after severe ischemic stroke. Adult C57BL/6 mice were subjected to filament insertion-induced occlusion of the middle cerebral artery (60 min MCAO). HPI-201 was i.p. injected 120 min after the onset of MCAO to initiate and maintain the body temperature at 32-33°C for 6 hrs. The infarct volume, cell death, integrity of the blood brain barrier (BBB) and neurovascular unit (NVU), inflammation, and functional outcomes were evaluated. The hypothermic treatment significantly suppressed the infarct volume and neuronal cell death, accompanied with reduced caspase-3 activation and BAX expression while Bcl-2 increased in the peri-infarct region. The cellular integrity of the BBB and NVU was significantly improved and brain edema was attenuated in HPI-201-treated mice compared to stroke controls. The hypothermic treatment decreased the expression of inflammatory factors including tumor necrosis factor-α (TNF-α), MMP-9, interleukin-1β (IL-1β), the M1 microglia markers IL-12 and inducible nitric oxide synthase (iNOS), while increased the M2 marker arginase-1 (Arg-1). Stroke mice received the hypothermic treatment showed lower neurological severity score (NSS), performed significantly better in functional tests, the mortality rate in the hypothermic group was noticeably lower compared with stroke controls. Taken together, HPI-201 induced pharmacological hypothermia is protective for different neurovascular cells after a severely injured brain, mediated by multiple mechanisms.

Abstract 749: Circulating miRNAs and Apoptosis Genes With Hyperglycemia in Ischemic Stroke Patient

Stroke is the third cause of death and the leading cause of adult disability in Taiwan. More than half of acute ischemic stroke patients had hyperglycemia, which is one of the major reason for unfavorable outcomes. However, the physiopathology is remain unclear and better knowledge of underlying genetic mechanisms is needed to improve prognosis. This study aimed to identify possible genes and its regulated miRNA involving in hyperglycemia-induced unfavorable stroke outcomes. This is a two-stage study design. In the first screening stage, 3 hyperglycemia ischemic stroke patients and 3 normal-glycemia cases were recruited. MiRNA sequencing and PCR profile arrays were conducted. Messenger RNA with significant differences and miRNA were analyzed using miRSystem database and Cytoscape. The results showed that apoptosis regulatory pathway genes (FAS and BCL2L1) and its related microRNAs (has-let-7i-5p,has-let-7b-5p,has-let-7a-5p,has-let-7f-5p,has-let-7g-5p,has-let-7e-5p,has-miR-98-5p) were highly associated with acute hyperglycemia ischemic stroke. In the second validation stage, we then verified the above mentioned apoptosis regulatory pathway genes and its related miRNAs using middle cerebral artery occlusion (MCAO) mice model and validate in 143 ischemic stroke patients. From the animal model, the results showed that the FAS gene of hyperglycemic rats exhibited a higher performance than that of normal glycemic rats. The results of miRNAs indicated that the expression of has-let-7a-5p, has-let-7b-5p, has-let-7e-5p, and has-let-7i-5p was lower in hyperglycemia rats than those belonged to normal glycemic rats. For ischemic stroke patients, unfavorable outcome patients had higher FAS expression. Furthermore, there is a significant group-by-time interactions in FAS expression, with unfavorable outcome patients having increasing expression in FAS gene (p=0.0208). A remarkable group-by-time interaction in has-let-7e-5p expression was also observed, with unfavorable outcome patients having decreasing expression in has-let-7e-5p (p=0.0128). Our study uncovers FAS gene play an important role in hyperglycemia resulting in poor outcomes in ischemic stroke patients, and might provide new direction for treatment in the future.

Inactivation of endothelial adenosine A2A receptors protects mice from cerebral ischaemia-induced brain injury.

Inactivation of the gene for adenosine A2A receptors (ADORA2A for humans and Adora2a for rodents) protects against brain injury in experimental stroke. However, the cell-specific pathogenic effects of A2A receptors in thromboembolic stroke and the underlying mechanisms remain undefined. Here, we tested the hypothesis that inhibition of endothelial A2A receptors after thromboembolic stroke improves post-stroke outcomes via down-regulation of inflammation. Thromboembolic stroke was induced by embolic middle cerebral artery occlusion in mice. Post-stroke outcomes were determined with neurological deficit scoring, infarct volume, inflammatory marker expression, brain leukocyte infiltration, blood-brain barrier (BBB) leakage, and oedema assessment. Anti-inflammatory effects of silencing the gene for A2A receptors or pharmacological antagonism of these receptors were assessed in vitro. Thromboembolic stroke induced Adora2a expression in the brain. Mice globally deficient in Adora2a (Adora2a-/- ) were resistant to stroke injury. Mice specifically deficient in endothelial Adora2a (Adora2aΔVEC ) showed reduced leukocyte infiltration, BBB leakage, and oedema after stroke, along with attenuated downstream proinflammatory markers, both in vivo and in vitro. The A2A receptor antagonist, KW 6002, also reduced brain injury and inflammation after stroke. Inactivation of ADORA2A inhibited endothelial inflammation via suppression of the NLRP3 inflammasome, down-regulating cleaved caspase 1 and IL-1β expression. Specific inactivation of endothelial A2A receptors mitigated ischaemic brain injury and improved post-stroke outcomes, at least partly, through anti-inflammatory effects via blockade of NLRP3 inflammasome activity. Our findings may open new approaches to vascular protection after ischaemic stroke.

Temporal and spatial profile of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in ischemic stroke in mice.

Although T cells play important roles in the pathophysiology of ischemic stroke, the dynamics of T cells remains unclear. In cancer, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) contribute to the maintenance of the tumor microenvironment by suppressing T cells. However, the presence of these cells has never been examined in ischemic brain. Therefore, we examined the temporal and spatial profiles of PMN-MDSCs, which are defined as the CD11b+Ly6ClowLy6G+ cells with higher expression levels of Nox2 and C/EBP Homologous Protein (CHOP) mRNA than normal neutrophil. Fluorescence-activated cell sorter (FACS) analysis showed that the count of CD11b+Ly6ClowLy6G+ cells was increased in the ischemic hemisphere and bone marrow at 72 hours, as well as in the spleen 24 hours after transient middle cerebral artery occlusion in mice. In contrast, the contralateral hemisphere, normal bone marrow, and normal spleen contained few CD11b+Ly6ClowLy6G+ cells. Real-time reverse transcription polymerase chain reaction revealed that CD11b+Ly6ClowLy6G+ cells sorted from brain and spleen 72 hours after ischemia had greater expression of Nox2 and CHOP mRNA than neutrophils in bone marrow, suggesting that these cells constitute PMN-MDSCs. Immunohistochemistry showed that CD11b+Ly6G+ cells were located in the ischemic core and border zone, indicating that PMN-MDSCs might be endemic to these regions. Although neutrophils are believed to invade infarct regions 48–72 hours after ischemia, the present study suggested that some of these cells are in fact PMN-MDSCs. Further studies on the function of PMN-MDSCs might unveil the unknown mechanisms of T cell activation and recruitment in ischemic stroke.

Glucocorticoids Regulate Bone Marrow B Lymphopoiesis After Stroke.

After a stroke, patients frequently experience altered systemic immunity resulting in peripheral immunosuppression and higher susceptibility to infections, which is at least partly attributed to lymphopenia. The mechanisms that profoundly change the systemic leukocyte repertoire after stroke are incompletely understood. Emerging evidence indicates that stroke alters hematopoietic output of the bone marrow. To explore the mechanisms that lead to defects of B lymphopoiesis after ischemic stroke. We here report that ischemic stroke triggers brain-bone marrow communication via hormonal long-range signals that regulate hematopoietic B lineage decisions. Bone marrow fluorescence-activated cell sorter analyses and serial intravital microscopy indicate that transient middle cerebral artery occlusion in mice arrests B-cell development beginning at the pro-B-cell stage. This phenotype was not rescued in Myd88-/- and TLR4-/- mice with disrupted TLR (Toll-like receptor) signaling or after blockage of peripheral sympathetic nerves. Mechanistically, we identified stroke-induced glucocorticoid release as the main instigator of B lymphopoiesis defects. B-cell lineage-specific deletion of the GR (glucocorticoid receptor) in CD19-Cre loxP Nr3c1 mice attenuated lymphocytopenia after transient middle cerebral artery. In 20 patients with acute stroke, increased cortisol levels inversely correlated with blood lymphocyte numbers. Our data demonstrate that the hypothalamic-pituitary-adrenal axis mediates B lymphopoiesis defects after ischemic stroke.

Loss of lipoprotein receptor LRP1 ablates neural stem cell migration to ischemic lesions

Brain damage due to stroke is a leading cause of death and long‐term disability in the US. Treatment options are limited, necessitating a better understanding of reparative and pathological processes after stroke. Ischemia stimulates the proliferation and migration of neural stem cells (NSCs) from the subventricular zone toward the ischemic lesion in mice. During this time, the migrating NSCs differentiate into GFAP positive cells and pro‐reparative glia. While the importance of this process is not fully elucidated, it is clear that limiting NSC response increases lesion size and worsens behavioral outcomes after ischemic stroke. We tested the effect of low‐density lipoprotein receptor related protein 1 (LRP1) on modulating NSC response and differentiation, and found that virtually every aspect of NSC physiology we tested was altered after deletion of LRP1; increased glial differentiation, proliferation, and self‐renewal were measured. After middle cerebral artery occlusion in mice, we observed a marked reduction in the migration of neuroblasts toward the site of the lesion, which correlated with increased gliosis and worsened behavioral outcomes. Loss of LRP1 also caused reduction in expression of the chemokine receptor CXCR4, which could account for the loss of migration. Ongoing studies involve elucidating the mechanism by which LRP1 regulates CXCR4 expression as well as understanding the effect on pro‐reparative processes after stroke.Support or Funding InformationThis research is supported by a pilot grant from the William and Ella Owen's Medical Research Foundation, and a Veteran's Health Administration Career Development Award K2BX003240.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Long noncoding RNA-regulator of reprogramming alleviates hypoxia-induced cerebral injury in mice model and human via modulating apoptosis complexes.

Ischemic stroke causes cerebral hypoxia, while long non-coding RNA regulator of reprogramming is associated with hypoxia. To find a new intervention target to protect hypoxic cerebral tissue and a potential biomarker from reflecting the severity of hypoxia after ischemic stroke, our research explored the expression pattern and function of the regulator of reprogramming in cerebral hypoxia-induced injury. The expression pattern and the function of the regulator of reprogramming were explored in mice with middle cerebral artery occlusion, and human brain microvascular endothelial cells underwent oxygen-glucose deprivation treatment. A case-control study, including 223 ischemic stroke patients and 155 controls were also conducted to investigate its correlation with ischemic stroke clinical characteristics. Results showed that the regulator of reprogramming increased significantly in middle cerebral artery occlusion in mice (P < 0.05), and its level remained stable within 2 to 48 h after the implement of middle cerebral artery occlusion. Oxygen-glucose deprivation up-regulated the expression of regulator of reprogramming, and regulator of reprogramming promoted ASK-1/STRAP/14-3-3 complex formation to inhibit the activation of TNF-α/ASK-1-mediated apoptosis of human brain microvascular endothelial cells, while small interfering ribonucleic acid (RNA) targeting regulator of reprogramming amplified these effects. Regulator of reprogramming increased and maintained stable within 3 to 48 h of ischemic stroke onset in patients, and was negatively associated with the National Institutes of Health Stroke Scale (NIHSS) (r = -0.708, P < 0.001), high sensitivity C-reactive protein (Hs-CRP) (r = -0.683, P < 0.001) level, infarct volume (r = -0.579, P < 0.001), and modified Rankin scale (mRS) (r = -0.728, P < 0.001). These results indicate that the regulator of reprogramming can alleviate cerebral hypoxia-induced injury by suppressing TNFα-induced apoptosis of vascular endothelial cells.

An enriched environment promotes synaptic plasticity and cognitive recovery after permanent middle cerebral artery occlusion in mice.

Cerebral ischemia activates an endogenous repair program that induces plastic changes in neurons. In this study, we investigated the effects of environmental enrichment on spatial learning and memory as well as on synaptic remodeling in a mouse model of chronic cerebral ischemia, produced by subjecting adult male C57BL/6 mice to permanent left middle cerebral artery occlusion. Three days postoperatively, mice were randomly assigned to the environmental enrichment and standard housing groups. Mice in the standard housing group were housed and fed a standard diet. Mice in the environmental enrichment group were housed in a cage with various toys and fed a standard diet. Then, 28 days postoperatively, spatial learning and memory were tested using the Morris water maze. The expression levels of growth-associated protein 43, synaptophysin and postsynaptic density protein 95 in the hippocampus were analyzed by western blot assay. The number of synapses was evaluated by electron microscopy. In the water maze test, mice in the environmental enrichment group had a shorter escape latency, traveled markedly longer distances, spent more time in the correct quadrant (northeast zone), and had a higher frequency of crossings compared with the standard housing group. The expression levels of growth-associated protein 43, synaptophysin and postsynaptic density protein 95 were substantially upregulated in the hippocampus in the environmental enrichment group compared with the standard housing group. Furthermore, electron microscopy revealed that environmental enrichment increased the number of synapses in the hippocampal CA1 region. Collectively, these findings suggest that environmental enrichment ameliorates the spatial learning and memory impairment induced by permanent middle cerebral artery occlusion. Environmental enrichment in mice with cerebral ischemia likely promotes cognitive recovery by inducing plastic changes in synapses.

Stroke target identification guided by astrocyte transcriptome analysis.

Astrocytes support normal brain function, but may also contribute to neurodegeneration when they become reactive under pathological conditions such as stroke. However, the molecular underpinnings of this context-dependent interplay between beneficial and detrimental properties in reactive astrogliosis have remained incompletely understood. Therefore, using the RiboTag technique, we immunopurified translating mRNAs specifically from astrocytes 72 hr after transient middle cerebral artery occlusion in mice (tMCAO), thereby generating a stroke-specific astroglial translatome database. We found that compared to control brains, reactive astrocytes after tMCAO show an enrichment of transcripts linked to the A2 phenotype, which has been associated with neuroprotection. However, we found that astrocytes also upregulate a large number of potentially neurotoxic genes. In total, we identified the differential expression of 1,003 genes and 38 transcription factors, of which Stat3, Sp1, and Spi1 were the most prominent. To further explore the effects of Stat3-mediated pathways on stroke pathogenesis, we subjected mice with an astrocyte-specific conditional deletion of Stat3 to tMCAO, and found that these mice have reduced stroke volume and improved motor outcome 72 hr after focal ischemia. Taken together, our study extends the emerging database of novel astrocyte-specific targets for stroke therapy, and supports the role of astrocytes as critical safeguards of brain function in health and disease.