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Introduction: There is increasing interest in endovascular therapy (EVT) for patients with large vessel occlusion (LVO) acute ischemic stroke (AIS) presenting with low NIH Stroke Scale. Here, we assess the prevalence of LVO with low NIHSS and real-world trends in utilization of EVT in this population. Methods: We performed a cross-sectional study using the Texas Public Use Data File, which captures all inpatient admissions in the state from non-federal hospitals (10/01/2016-06/30/2020). AIS cases were defined as hospitalizations to acute care hospitals with a primary diagnosis of AIS using ICD-10-CM codes I63.x and NIHSS was determined using ICD-10-CM codes R29.700- R29.742. Low NIHSS was defined as Results: Among 61,524 AIS cases, patients with low NIHSS accounted for 63% and were more likely to be younger, male and white than those with high NIHSS. LVO was twice as prevalent among high NIHSS AIS (52% vs 24% p Conclusions: In this large state-level cohort, nearly one-quarter of low NIHSS patients were found to have LVO, and increasing trends in EVT use were observed in this group. The effectiveness and safety of EVT in low NIHSS LVOs need to be further evaluated in real world clinical settings.

CD11b expression is essential for immunity due to its role in cell motility and phagocytosis. Leukocytes, including monocytes, neutrophils and macrophages, express CD11b. B lymphocytes, which are both adaptive immune cells and antigen presenting cells (APCs), also express CD11b at varying levels. The role of B lymphocytes after stroke is multifaceted, and recent studies have highlighted their deleterious contributions to post-stroke recovery and cognitive decline. CD11b high B cells have not been studied in the context of aging and stroke. We hypothesized that CD11b high B cells increase in the brain after stroke and regulate microglia function. We used a middle cerebral artery occlusion (MCAO) model of stroke in young (2mo) and aged (18mo) WT male mice to investigate the role of CD11b high B cells in post-ischemic immune responses. In the acute phase following stroke, resident immune cells, primarily microglia (MG), are rapidly activated. Thus, we studied the ability of CD11b high B cells to influence MG phenotype in young and aged brain. We found that CD11b high B cells are a heterogeneous subpopulation of B lymphocytes and predominantly found in aged mice (n=4/gp, p=0.0089 ) and after stroke (n=5/gp, p=0.0078 ). This subset is characterized by their higher expression of CD80 and CD73 (n=8/gp, p ). The frequency of CD11b high B cells increases in the brain after stroke in both young and aged mice. These cells are capable of changing MG phenotype sorted from either young or aged brain, as assessed by decreased expression of CD45, CD11b, and MHC-II by MG (n=4/gp, p=0.0013, p=0.0033, p=0.0310 , respectively). When co-incubated with ex vivo sorted MG, CD11b high B cells increased phagocytic function MG more than their CD11b low B cell counterparts (n=4/gp, p=0.0486 ). Lastly, the regulatory influence of CD11b high B cells is likely through production of inflammatory cytokines including TNF-α (n=5/gp, p ). As both APCs and adaptive immune cells with memory function, B lymphocytes are uniquely positioned to regulate both acute and chronic phases of the post-stroke immune response, and their influence is subset-specific. Future studies are warranted to better understand the function of these age-associated CD11b high B cells in neuroinflammation.

Senescence in the cerebral endothelium has been proposed as a mechanism that can drive dysfunction of the cerebral vasculature, which precedes vascular dementia. Cysteine-rich angiogenic inducer 61 (Cyr61/CCN1) is a matricellular protein secreted by cerebral endothelial cells (CEC). CCN1 induces senescence in fibroblasts. Whether CCN1 contributes to senescence of CEC and how it is regulated require further study. Aging has been associated with the formation of four-stranded Guanine-quadruplexes (G4s) in G-rich motifs of DNA and RNA. G4 stabilization regulates transcription and translation either by upregulation or downregulation depending on the gene target. Previously, we showed that aged mice treated with a G4-stabilizing compound had enhanced senescence-associated (SA) phenotypes in their brains, and exhibited cognitive deficits. The expression of human CCN1 can be upregulated via the stabilization of G4s. A sequence in the 3’- untranslated region (UTR) of the CCN1 mRNA has the ability to fold in to G4 in vitro . Thus, we hypothesize that CCN1 is upregulated with aging and induces senescence, and that G4 stabilization upregulates CCN1 in cultured primary CEC. We used whole brain lysates, cerebral vessel fractions, and cultured primary CEC from young (4-months old, mo) and aged (18-mo) female mice to determine the CCN1 levels. SA phenotypes were determined by high-resolution fluorescence microscopy in cultured primary CEC, and we used Thioflavin T to recognize RNA G4s for fluorescence spectra. We found that CCN1 levels were increased in the brains of aging mice (p=0.0314, n=5), in cerebral vessel fractions (p=0.001, n=5), and in cultured primary CEC derived from aged mice (p=0.012, n=3), compared with those derived from young mice. In cultured CEC, a recombinant CCN1 protein induced SA phenotypes, such as SA beta-galactosidase activity (p=0.003, n=3), and double-strand DNA damage (p=0.016, n=3). Furthermore, CCN1 levels were upregulated by a G4 ligand (p=0.0103, n=3), and a G-rich motif in the 3’-UTR of the Ccn1 mRNA folded into a G4 (p=0.008, n=4). In conclusion , we demonstrate that CCN1 induces senescence in cultured primary CEC, and we propose G4 stabilization as a novel mechanism that regulates the SASP component CCN1.

Introduction: The alpha7 nicotinic acetylcholine receptor (α7nAChR) is the principal mediator of the cholinergic anti-inflammatory pathway (CAP). CAP is mediated through several signaling pathways, including the JAK2/ STAT3 pathway. Studies have shown decrease in brain α7nAChR and increase in phosphorylation (p) of JAK2 and STAT3 expression after stroke. Increase JAK2/STAT3 signaling induces expression of high mobility group box 1 (HMGB1) and receptor for advanced glycation end products (RAGE), which promotes release of cytokines. Pharmacological activation of α7nAChR reduced microglial activation and inflammatory cytokines and improved functional outcomes after experimental stroke. However, little is known about how the activation of α7nAChR modulates JAK2/STAT3 signaling after stroke. Hypothesis: We hypothesized α7nAChR activation by PNU120596 (PNU), an allosteric modulator will decrease JAK2/STAT3 signaling and improve neurological deficits (ND) after stroke. Methods: Young (8-12 weeks, n=5-6) male mice underwent 60-minute middle cerebral artery occlusion (MCAo) or sham surgery and euthanized at 24 hours. In second cohort (n=5-6), PNU or vehicle was administered for 3 consecutive days. One hour after the last injection, MCAo or sham surgery was done. Twenty-four hours after MCAo, body weight, ND scores were recorded and brains harvested for western blotting. Brain α7nAChR, pSTAT3, HMGB1 and RAGE expression was determined. Results: At 24 hours after MCAo, the brain expression of α7nAChR decreased (p=0.006) while pSTAT3 expression increased (p=0.001) as compared to sham. PNU treatment prevented a decrease in α7nAChR expression in the brain (p=0.017) and decreased pSTAT3 expression (p=0.005) compared to the vehicle group after MCAo. No difference in the HMGB1 expression was seen between groups. An increase in RAGE expression was observed in vehicle MCAo (p=0.006) compared to sham. A decline (p=0.08) in RAGE expression in PNU MCAo compared to vehicle MCAo was observed. NDS and percent body weight loss were lower in the PNU MCAo compared to vehicle MCAo. Conclusions: Activation of α7nAChR downregulated inflammatory JAK2/STAT3-HMGB1/RAGE signaling and may be a druggable target to improve recovery after stroke.

Introduction: Young women with stroke represent a unique patient population. There are a variety of sex-specific etiologies that warrant a different approach to evaluation and secondary prevention in young women. Secondary prevention begins during the acute hospitalization, thus, certain aspects of the history and evaluations may be indicated early after stroke. We describe our center’s experience with young women presenting with stroke over a 2.5-year period. Methods: We performed a retrospective review of women between 18-45 years, identified from our stroke registry (1/2015 to 5/2018). An assessment of key variables regarding common stroke risk factors (RFs), sex-specific RFs, (pregnancy history, estrogen use, migraine, autoimmune conditions) and workup (pregnancy test, hypercoagulable states, substance use) was conducted. Results: Of 198 women with acute ischemic stroke in our study period, medical RFs of hypertension, diabetes, hyperlipidemia, and atrial fibrillation were each ascertained for more than 98% of women. Lifestyle RFs of tobacco use and alcohol use were reported for 85.9% and 87.6%, respectively and urine drug screens were performed in 67%. Obstetric history (gravida/para) was obtained in only 19.7%, pregnancy tests were conducted in 69.2%, and hypertensive disorders of pregnancy were only reported in patients who were pregnant (2%) or post-partum (5.6%). Oral contraceptive (OCP) or hormone replacement (HRT) was reported in 6.6% and migraine with or without aura in 12.6%. Among 76 patients (38.4%) with cryptogenic stroke, obstetric history was reported in only 25%, OCP/HRT use in 5.3%, and antiphospholipid antibody syndrome was assessed in 64%. Conclusion: The acute stroke hospitalization is a critical period for identification of stroke etiology and assessment of cardiovascular risk. There are known sex disparities in standard evaluation and management of stroke. In pre-menopausal women, gaps in care may be even greater if the history and assessment are not completed with sex-specific variables in mind. We suggest a standard approach to the history and assessment of young women. We plan to implement this approach in our patient population while tracking the impact on care.

Cerebral amyloid angiopathy (CAA) is a small vessel disease, characterized by amyloid beta deposition in the cerebral vasculature. CAA increases the incidence of hemorrhagic stroke and dementia in the elderly. Evidence has revealed that aging alone induces disruption in B cell differentiation and immunosenescence by promoting the presence of age-associated B cells (ABCs) characterized by an upregulation of CD11b high . However, little is known on how these ABCs influence the neuroimmune landscape of vascular dementia, an immune-mediated age-associated disease. Hypothesis: CAA induces the differentiation of B cells in the brain resulting in neuroinflammation characterized by an exacerbated presence of microglia and lymphocytic infiltration. Pre-symptomatic (2mo) and symptomatic (18mo) Tg-SwDI (harboring Swedish, Dutch, and Iowa) mutations of human amyloid precursor protein male mice were used as a model of CAA. Through flow cytometry, we investigated the role of CD11b high B cells in promoting neuroinflammation in CAA. Age-matched C57BL/6 wild-type (WT) male mice were included to determine CAA-specific effects. Results: A remarkably heterogeneous CD19 + B cell population was identified in symptomatic CAA mice. Interestingly, CD19 + CD11b high B cells were significantly increased in the symptomatic CAA brain (n=4-6/gp, P =+ CD11b high B cells. Notably, CD19 + CD11b high B cells from CAA symptomatic mice had significantly lower expression of CD73 (n=4-6/gp, P =0.0004), CD80 (n=4-6/gp, P =0.0007), and CD138 (n=4-6/gp, P =0.0154) compared with those CD19 + CD11b high B cells from age-matched WT brain. Finally, CAA mice had significantly higher microglial counts (n=4-6/gp, P =0.0013) and lymphocytic infiltration (n=4-6/gp, P =0.0071) in the brain as compared to pre-symptomatic and WT aged mice. Here, we highlight the diversity of B cells in age-associated vascular dementia. Our data give us further perception into the influence of humoral immunity mediated by ABCs and their immune crosstalk in CAA.

Introduction: Social Isolation (SI) is a risk factor for a wide array of psychological disorders. SI significantly enhances the incidence of developing neurological diseases, including stroke within the elderly population. Recent studies have shown microRNA (miRNA) signatures in the brain and the circulation are altered in social defeat models and under stressful environmental conditions. Although SI has shown to influence post-stroke recovery, potential targetable interventions to mitigate these inflammatory events are limited. Hypothesis: Post-stroke SI influences temporally distinct miRNA networks in the brain that regulate acute microglial activation and contribute to chronic depressive phenotypes. Methods: Aged (18-20 month) C57BL/6 male mice were subjected to 60-minute middle cerebral artery occlusion (MCAO) followed by reperfusion. Mice were randomly assigned to either continued pair housing (PH), or SI three days after a 60-minute MCAO. Mice were euthanized at post-stroke SI Day 1, 4, and 27 and ipsilateral hemispheres were collected for miRNA sequencing and downstream analysis. Behavioral tests and brain cell analysis using flow cytometry were performed using independent experimental cohorts. Results: The top 10 differentially expressed miRNAs were identified in SI vs. PH mice across three time points (FC ≥ 1.5, FDR adjusted P P Conclusion: This is the first comprehensive miRNA expression profiling study using NGS technique in aging, stroke, and SI. Our data revealed that SI-specific miRNAs are involved in microglial activation and chronic depression in aged mice.

Introduction: Stroke is a sexually dimorphic disease. Stroke sensitivity in the elderly is altered by the decline of estrogen levels in females. Our previous studies suggested a X chromosomal effect on stroke sensitivity in aged mice; however, these mice were gonadal intact and the activational effect of hormones cannot be ruled out. In this study, we utilized the XY* mice model in which mice of four genotypes were produced (XX, XO, XY, and XXY) to dissociate the effect of X chromosome from that of Y. To rule out the activational effect of hormones, we gonadectomized all mice at 3 weeks old, and let them survive for 6-8 weeks to achieve “surgical post-menopause”. We hypothesized that the second X chromosome mediates the stroke sensitivity in the absence of activational sex hormones. Two X escapee genes ( Kdm6A and Kdm5C ) that escape from the X chromosome inactivation and involved in microglial activation, were also examined to explore the potential mechanisms. Methods: “Surgical postmenopausal” XY* mice were subjected to a 60-min middle cerebral artery occlusion (MCAO) (n=6-8/gp for stroke and = 4-5/gp for sham). Infarct volumes and behavior deficits were quantified 3 days after MCAO. Plasma inflammatory cytokine (TNFα, IL-1β, IL-4, iNOS, TGF-β1 and IL-10) levels were determined by ELISA. KDM6A and KDM5C expression in microglia was examined by IHC. Results: XX mice had significantly larger infarct size than XO mice in striatum (55.02±12.38% vs. 36.06±9.25%, P=0.0387) and the ipsilateral hemisphere (45.33±10.77% vs. 26.55±6.92%, p=0.0413); the same pattern was seen in XXY vs. XY group in striatum (58.47±14.26% vs. 34.22±15.24%, P=0.0027). Neurological deficit scores were higher in XX vs. XO mice. Mice with two copies of the X chromosome had significantly higher plasma levels of pro-inflammatory cytokines (TNFα, IL-1β) than the mice with one X. Significantly higher levels of KDM6A or KDM5C in Iba-1 positive microglial cells were also found in mice with two vs. one copy of X. Conclusion: The second X chromosome contributes to stroke sensitivity in the absence of activational sex hormones. Our future goal is to use Kdm6A and Kdm5C microglial conditional knockout mice to explore the molecular mechanisms underlying chromosomal factors in stroke sensitivity.

Intro & Background: Following the publication of the randomized clinical trials in 2015 and 2018 on endovascular stroke therapy (EVT), procedural utilization has increased. The effect of EVT utilization on clinical outcomes in acute ischemic stroke (AIS) at the population-level, however, remains incompletely characterized. Methods & Materials: We performed a retrospective cross-sectional analysis using the Texas Public Use Data File with records from January 2016 through the first quarter of 2020. This dataset includes full sample data on all patients hospital discharged from all state-licensed hospitals. AIS hospitalizations were identified as those with a primary diagnosis of AIS by ICD-10 diagnostic codes. Patients Results: Among 165,738 AIS cases, 61% were over 65 years old, 51% were female, 51% were White and 25% were Hispanic. Over the course of the study period, EVT utilization increased 0.25% per quarter [95% CI 0.23-0.27, P Conclusion: In this state-wide cohort, increasing usage of EVT was positively correlated with excellent clinical outcomes. These data provide evidence for population-level improvements in AIS care with improving EVT access and utilization.

Introduction: The relationship between pass number during endovascular therapy (EVT) and outcomes in anterior circulation large vessel occlusion (LVO) acute ischemic stroke (AIS) has been well studied. However, the association between number of thrombectomy attempts and outcomes for patients with posterior circulation LVO remains undetermined. Methods: From our prospective multi-institutional registry including 4 comprehensive stroke centers in the Houston area, we identified consecutive LVO AIS patients who underwent EVT (01/2018-06/2021). The number of thrombectomy passes and per-pass reperfusion grades (TICI) were recorded at time of the EVT procedure. The primary outcome was the effect of number of passes on functional independence (modified Rankin Scale 0-2) at 90 days. Secondary outcomes included likelihood of attaining substantial reperfusion (TICI ≥2b). Results: Among 894 LVO AIS patients who underwent EVT, median age was 68 [IQR 58-78], 49% were female, and median NIHSS was 16 [IQR 11-21]. Of this cohort, 86 had posterior circulation LVO including 65 with basilar artery occlusion and 9 vertebral artery. The diminishing benefit on reperfusion and functional outcome after additional EVT passes was similar for anterior and posterior circulation AIS (Figure 1). Among patients with posterior circulation LVO, successful reperfusion with fewer passes was associated with greater likelihood of good outcomes (41.9% vs 6.7% mRS 0-2 with 1-2 vs ≥3, p=0.012). The likelihood of good outcomes with 1-2 passes for patients with posterior circulation LVO was comparable to patients with anterior circulation LVO (41.9% vs 40.2%, p=0.164). Conclusions: Similar to patients with anterior circulation LVO, patients with posterior circulation LVO undergoing EVT are more likely to achieve functional independence with successful reperfusion in fewer EVT attempts. Overall clinical outcomes are significantly better with fewer passes regardless of occlusion location.

Introduction: Given the impact of endovascular therapy (EVT) on clinical outcomes and usage of pre-hospital bypass protocols to route acute ischemic stroke (AIS) patients with suspected large vessel occlusion (LVO) to EVT-performing hospitals (EPH), there is substantial interest in upgrading stroke center certification to become comprehensive centers. Here, we assess the effect of becoming EPH on total AIS and non-LVO AIS admissions in four hospitals in a large urban area. Methods: From our prospectively collected multi-institutional registry, we identified all consecutive patients with AIS at four EPHs between Nov 2017 - May 2019. Three of the EPHs (named EPH 1, EPH 2, EPH 3) became EPHs in early 2017, which allowed for preferential routing and pre-hospital bypassing of other non-EPH hospitals by regional emergency medical services protocols. Another EPH (named original EPH) had been an EPH for many years. The primary outcome was the volume of AIS admissions after upgrading to a EPH over time and was determined by linear regression. Results: Among 3,727 patients with AIS presenting to 4 hospitals across the Houston area, median age was 67 [IQR 57-78], 48% were female, median NIHSS was 5 [IQR 2-12], 21% had LVO, 11% underwent EST. AIS admissions increased at EPH 1 at a rate of 15 AIS per quarter (95% CI 5.5-25.3, p=0.012); EPH 2 increased at a rate of 8 AIS per quarter (95% CI 3.18 - 13.44, p=0.011); EPH 3 increased at a rate of 10 AIS per quarter (95% CI 2.4-18.6, p=0.023) AIS admissions at the original EPH did not change significantly during the study period (CI 0-11.9, p=0.47). Increases in AIS admissions were predominantly in non-LVO AIS (Figure A). Conclusions: In this observational cohort study, upgrading to EPH status resulted in continuously increasing AIS admissions over time, particularly for non-LVO patients; this growth was not observed in established EPH. These findings demonstrate and quantify the effect of upgrading stroke center certification on inpatient AIS volumes.

Introduction: Neonatal Hypoxic Ischemic Encephalopathy (nHIE) is a major cause of mortality and morbidity in infants, occurring in 1.5/1000 live births. Inflammation persists years after injury and detrimentally affects neurocognitive outcomes in children. Recent evidence shows that neuroinflammation and gut injury alters microbial populations in the gut (gut dysbiosis) and reduces the level of bacteria capable of producing beneficial, anti-inflammatory tryptophan (Trp) metabolites. Metabolites are detected by microglia (MG) with the aryl hydrocarbon receptor (AHR). Ligand specific AHR activation can influence MG behavior after CNS injury, potentially modifying recovery. We hypothesize that nHIE leads to chronic gut dysbiosis and reduced microbial-derived Trp metabolites, which exacerbates MG-induced CNS damage. We also hypothesize that Trp-derivatives bind MG AHR to reduce inflammation and improve neurological outcomes in males and females. Methods: We used a modified Rice Vannucci Model on PND9 C57BL/6J mice to investigate the role of MG AHR as a mediator of gut microbiome-brain communication after nHIE and demonstrated direct microbial metabolite modulation of MG using in vitro cell culture assays. Results: We found an increase in AHR and CD45 expression in MG 24hrs after nHIE (p=0.0169 and p=0.0047 respectively, n=5). RT-qPCR of SIM-A9 MG cells showed that pretreatment with indole-acetic-acid (IAA) followed by LPS injury produced significantly more Il10 than cells pretreated with AHR agonist FICZ, suggesting Trp-derivatives activate an anti-inflammatory response (p=0.013, n=8). IAA mildly reduced Il1β , Il6 , and Tnfα after LPS administration. 16S rRNA sequencing, behavior and metabolomics analysis of acute (24hrs) and chronic (7wks) cohorts showed detriments in behavioral tasks and dysbiotic changes after nHIE. Conclusion: nHIE results in increased AHR expression in MG, gut dysbiosis, and alterations in microbial-Trp metabolites in males and females. In vitro assays demonstrated that absence of Trp metabolites result in greater inflammatory activity of MG after injury via the AHR pathway, supporting our hypothesis that reduction of microbial-derived Trp metabolites after nHIE worsens MG-induced CNS damage.

Background: Clinically, ~65% of stroke patients are left with functional impairments after stroke and 15% die shortly after their stroke. Increasing evidence suggests that peripheral inflammatory responses after stroke play an important role in determining neurological outcome. Mast cells (MCs) are one of the most rapid responders to injury. MCs release histamine (HA), a pro-inflammatory transmitter that enhances inflammation. Gut MCs are a major source of HA. Hypothesis: We hypothesize that aged animals stroke will lead to robust gut mucosal MC-activation and HA release, with subsequent gut disruption and inflammation. Stabilizing peripheral MCs will decrease peripheral/central inflammation, MC trafficking, and improve stroke outcomes. Methods: We used a reversible middle cerebral artery occlusion (MCAO) model of ischemic stroke in aged (18mo) wild-type male mice to investigate the MC role in neuroinflammation post-stroke (PS). We stroke the aged animals and treated the animals with 25 mg/kg BW of cromolyn (MC stabilizer), oral gavage. Cromolyn was administered at 3-h, 10-h, 24-h and every other day PS. Positive control group that were stroked but treated only with saline. In total, four groups stroke and sham (surgery control), out of these animals one set received cromolyn and one set received saline. We sacrificed animals at 3-h, 24 –h and 3-days after cromolyn treatment post-stroke. Results: We found that cromolyn administration significantly reduced MC numbers in the brain at 24-hours (P Conclusion: Our results show that preventing MC-HA release post-stroke possess clinical value in preventing neuroinflammation PS. Future studies will focus on IgE and FcεR1 due to its cross-linking role in activating MC-HA release.

Microbiome-derived ligands of the aryl hydrocarbon receptor (AHR) including tryptophan-derived indole acetic acid (IAA) have anti-inflammatory effects in some tissues. However, their effect on neuroinflammation after stroke is unknown. Brain-derived ligands of AHR (e.g. kynurenine) increase post-ischemia and are detrimental. Consistently, pharmacological inhibition of AHR after stroke reduces deleterious effects of kynurenine-mediated activation of AHR and improves outcome. However, whether IAA-mediated activation of AHR is detrimental or beneficial after stroke is unknown. We hypothesized that post-stroke treatment with IAA will reduce neuroinflammation and improve outcomes via beneficial activation of microglial (MG) AHR. We used a reversible middle cerebral artery occlusion (MCAO) model in aged (18mo) WT male mice to investigate temporal changes in biome-derived (IAA) versus host-derived (kynurenine) AHR ligands. Using metabolomics analysis, we determined that plasma levels of IAA can be restored in naïve aged mice by oral probiotics administration of AHR ligand producers. We found that brain kynurenine increases but plasma IAA decreases as early as 3 hours after MCAO in aged mice (n=4/gp, p=0.0029 ) while brain IAA levels remain unchanged. Our 16S rRNA-sequencing shows that aging leads to reduction in AHR ligand-producers (e.g. Bifidobacterium [B] and Lactobacillus [L] ). Oral gavage with AHR ligand-producing BBL-probiotic cocktail restored the age-related decline in plasma IAA both acutely (24 hours post-treatment) and chronically (weekly for 6 weeks, n=8/gp, p=0.0086 and p=0.0073 , respectively). Further, the increase in plasma levels of IAA after probiotic bacteriotherapy with AHR ligand producers was associated with modulation of AHR activity in the brain (decreased AHR expression in MG, n=8/gp, p=0.0119 ) and reduced MG activation ( p=0.0069 ). Our results show that IAA modulates MG-mediated neuroinflammation after stroke. We plan to utilize post-stroke treatment with IAA in aged WT mice and in inducible knock-out mice with microglial Ahr deletion to further validate our hypothesis. Future studies are needed to focus on the regulatory function of other biome-derived AHR ligands in post-stroke neuroinflammation.

Introduction: Ischemic stroke results in activation of microglia, which may polarize towards a pro-inflammatory (M1) phenotype and/or an anti-inflammatory (M2) phenotype. Enhancer of zeste homolog (EZH) 2 is a histone-lysine N-methyltransferase enzyme, a key modulator of microglia polarization. We here investigated whether microglial-specific deletion of EZH2 leads to a beneficial protective effect in stroke and microglial over-expressing microglial EZH2 exacerbates stroke outcome in vivo . Methods: Aged male mice were subjected to 60-minutes middle cerebral artery stroke. Tamoxifen administration was started 30 days prior to stroke to induce genetic deletion of microglial EZH2 in CX3CR1-creER/EZH2-floxed mice. EZH2 floxed mice were used as controls. Microglial EZH2 over-expressing was performed using lentiviral vectors. Mice were sacrificed for immunohistochemistry and crystal violet staining (brain infarct assay) after behavior tests at 3 days after stroke. Results: The expression of microglial EZH2 was significantly abrogated with tamoxifen injection in KO mice compared to the control floxed mice (144±15.43 vs. 50.65±4.99 cells/mm 2 , P2 , P2 , P Conclusions: Genetic deletion of EZH2 in microglia improved stroke outcome in aged while overexpression of EZH2 exacerbated stroke outcome. The effect of protective effect of EZH2 inhibition may be due to limiting microglial M1 polarization and enhancing M2 polarization.

Introduction: Understanding the risk of aneurysmal subarachnoid hemorrhage in pregnant women with known history of Unruptured Intracranial Aneurysm (UICA) could potentially impact the delivery method. Due to presumed risk of intrapartum aneurysm rupture, understanding practice patterns of delivery methods and related complication risk is of importance. Methods: We used Nationwide Inpatient Sample database (1998-2014) to identify all the hospitalizations for childbirth in women with and without history of UICA. Patients with intracranial vascular malformation and traumatic intracranial hemorrhage were excluded. The cohort of pregnant women with known history of UICA was compared with the cohort of those without history of UICA with univariate analysis. Results: A total of 323 patients with known history of UICA were hospitalized for childbirth compared to 13,669,848 with no history of UICA. Higher rates of intracranial hemorrhage was observed in history of UICA group compared to no history of UICA [subarachnoid hemorrhage (0.31% Vs 0.001%, p Conclusion: Women undergoing childbirth with known history of UICA have higher risk of intracranial hemorrhage, peripartum complications, and longer hospitalizations compared to women without a known UICA. Aggressive management of risk factors for aneurysm rupture including hypertension and smoking could potentially decrease the rupture risk. Further research is needed to understand whether delivery method has an impact on decreasing risk of rupture.

Background and Purpose: Females exhibit a more robust immune response in many disease models, yet sex differences in the inflammatory response to ischemia remain largely unexplored. We conducted flow cytometry and RNA sequencing of blood from ischemic stroke patients, with follow-up studies in mice. Hypothesis: We assessed the hypothesis that there are sex differences in the acute immune response to ischemic stroke. Methods: Patient samples were drawn at 24 hours post-stroke for flow cytometry (n=6) and RNA sequencing Analysis (n=40). For murine studies, male and ovariectomized (OVX) female animals (n=14) were subjected to 90-minute middle cerebral artery occlusion or sham surgery and sacrificed at 24 hours. Murine blood was stained for leukocyte markers and CD62L (L-selectin). Results were analyzed by student T-test and two-way ANOVA. Results: RNA sequencing revealed that 24 hours after ischemic stroke, female patients had 79 significantly upregulated genes, compared to male patients with 6 significantly upregulated genes. Human flow cytometry revealed that male stroke patients had a significantly higher percentage of monocytes (p=.026), while females had a greater percentage of CD8+ T-cells (p=.023). Murine flow cytometry showed a post-stroke increase in peripheral myeloid cells at 24 hours in male mice only (p=.0046), whereas female mice had a higher CD8/CD4 T cell ratio (p=.0027). Neutrophils from male sham animals displayed greater L-selectin positivity, with stroke-induced shedding of L-selectin seen only in males (sex/stroke p=.0266). Male monocytes and lymphocytes also displayed higher L-selectin positivity (p=.0079, p=.0004). Conclusion: These results suggest that the immune response to ischemic stroke is different in male and female patients, a phenomenon that can be recapitulated in a mouse model of experimental stroke. Female immune cells exhibit a higher level of baseline activation (reduced L-selectin expression) and post-stroke activity, which may enable a quicker and more robust response to immune challenges. Understanding sex differences in the acute immune response is crucial to developing future immunomodulatory drugs for the safe and effective treatment of ischemic stroke in both sexes.

Background and Purpose: Eotaxin, a TH2 chemokine, has been shown to increase in mouse brain and plasma following experimental stroke. While eotaxin has been associated with age-related deficits in neurogenesis, little is known about its role in ischemic injury. Hypothesis: We tested the hypothesis that serum eotaxin levels are associated with long-term stroke outcome, with follow up mechanistic studies in a mouse model of ischemic stroke. Methods: Serum was taken from patients (n=158) 24 hours after ischemic stroke onset. Levels of serum eotaxin were quantified by ELISA, then analyzed for outcome association. For murine studies, animals (n=14) underwent 90-minute middle cerebral artery occlusion and were sacrificed at 24 hours, with sham surgery mice serving as controls. Blood was incubated with or without eotaxin (100 ng/ml) and stained for leukocyte markers and CD62L (L-selectin). Results: Although eotaxin protein levels were not significantly different between sexes, a multivariate analysis controlling for age, stroke severity and cardiovascular risk factors revealed a male-specific association between higher eotaxin levels at 24 hours post-stroke and a positive functional outcome at three months (p=.010). Analysis of peripheral leukocytes isolated from both sham and stroke mice revealed that addition of eotaxin to whole blood significantly increased the activation of myeloid cells in both in male (p=.0031) and female (p=.0048) animals, as measured by shedding of L-selectin. Further experiments demonstrated that shedding of L-selectin on CD8+ T-cells after treatment of whole blood with eotaxin was significant only in female animals (p=.0008). Conclusion: In conclusion, the results of this study suggest that eotaxin has a sex-specific association with improved stroke outcomes. Murine studies demonstrate that eotaxin causes activation of peripheral leukocytes (as measured by loss of L-selectin), with a sexually dimorphic effect on CD8+ T-cell activation that may alter the character of the post-stroke immune response and support a pro-recovery inflammatory phenotype in males. This research underscores the importance of studying both sexes in future ischemic inflammatory research.

Background: Stroke induced respiratory dysfunction (SIRD) is a common complication of stroke. SIRD delays recovery and increases mortality. Despite the high prevalence of SIRD, the magnitude of breathing dysfunction in stroke patients and its impact on functional recovery is unknown. Even less is known regarding age and sex differences in SIRD and respiratory instability due to the lack of an appropriate animal model of SIRD. Methods: Whole body plethysmography was performed in WT C57/B6 young (2-3 month)/aged (20 month) male and female mice to establish baseline frequency (BPM), tidal volume (TV), minute ventilation (MV-ml/min/g), periods of apnea, duration of apnea and approximate entropy of the system (ApEn). Mice were then subjected to 60 (aged) or 90 (young) minute MCAO, or sham surgery. 72 hours and 21 days after surgery whole body plethysmography was repeated to identify SIRD phenotype across cohorts. Results: Three days after stroke aged animals of both sexes exhibit hypoventilation and a reduced ventilatory response to CO2 (Air: male sham 2.13±.1 vs. stroke1.26±.13 MV, p=.01, female sham 2.89±.27 vs. stroke 1.73±.21 p=.001). A significant interaction of age and surgery was noted among males F(1,42)=10.69, p Conclusions: We found that both age and sex affect ventilatory response following stroke. Aged females showed higher pre-stroke MV and dropped to similar levels as males after stroke, suggesting a more severe effect on breathing in aged females. Aged males demonstrated a more pronounced hypoventilation following stroke than their young counterparts, with less robust recovery. Stabilizing respiratory dysfunction may improve post stroke recovery and cognitive outcomes.

Introduction: Periprocedural risk of ischemic stroke (IS) in patients undergoing endovascular treatment for unruptured intracranial aneurysms (EUIA) can be as high as 40%, with clinically significant strokes occurring in 3-10% of patients. There is limited data on how typical vascular risk factors can increase peri-procedural risk of IS and mortality in patients undergoing EUIA. Methods: We queried the Healthcare Cost and Utilization Project's (HCUP) Nationwide Inpatient Sample (NIS) data between 2004 and 2012. ICD-9 codes 437.3 (intracerebral unruptured aneurysm) and 39.72 (endovascular repair/embolization of head and neck vessel) were used to identify the study population. Multivariate logistic regression model was used to identify independent predictors of IS and mortality after controlling confounders including age, race, sex, atrial fibrillation (Afib), diseases of endocardium, alcohol, smoking, diabetes mellitus, hypertension, use of anticoagulants, use of antiplatelet agents, carotid artery stenosis, atherosclerosis and hyperlipidemia. We also investigated whether Afib, DM, hypertension and hyperlipidemia in possible combinations further increased the risk of IS and mortality. Results: A total of 50,478 EUC cases were identified, of which 10,600 (20.9%) reported an IS event. A total of 661 (0.01%) died of which 115 (17.4%) had an IS event. After controlling for confounders, age >70 (OR 1.521 95% CI 1.145- 2.020, P< 0.01) and diabetes (OR 1.426, 95%CI 1.092-1.861, p 70 (OR 2.472, CI 1.616-3.782, p Conclusions: In patients undergoing EUIA, Age >70 is an independent risk factor for IS and mortality. Diabetes independently increased risk of IS but not mortality; Afib independently increased risk of mortality but not IS.

Neurite plasticity is a critical aspect of functional recovery after stroke. Emerging data suggest that Rac1 plays a central role in neurite regeneration in the injured brain, specifically by stimulating neuronal intrinsic growth and counteracting the growth inhibitory signaling that leads to growth cone collapse. We assessed the hypothesis that Rac1 is an important stimulator of neurite regeneration after stroke. Transient focal ischemia was induced by middle cerebral artery occlusion for 90 minutes in 7 to 8 week old male wild type mice. A specific Rac1 inhibitor, NSC 23766 (NSC) and a selective inhibitor (IPA 3) of PAK1, a downstream kinase of Rac1, were injected starting 7 days after stroke. Pellet reaching test (success rate) was used to assess the integrity of sensorimotor function. Treatment with NSC worsened recovery assessed by pellet reaching test from day 14 to day 28 after stroke (Day 28: Vehicle 0.39 ±0.03 vs. NSC 0.18±0.03, n=7-9, p Conclusions: Inhibition of Rac1 signaling worsened functional recovery and reduced axonal density. This was associated with decreased activation of intrinsic pro-regenerative molecules and increased growth inhibitory signaling. Our data suggested that Rac1 signaling could enhance neurite regeneration and improve post-stroke functional recovery.

Background: There is paucity of data on safety outcomes in the subgroup of patients who are on anticoagulation (AC) that undergo mechanical thrombectomy (MT) for anterior circulation large vessel stroke (AS). Methods: The Healthcare Cost and Utilization Project's (HCUP) Nationwide Inpatient Sample (NIS) data was queried between 2006 and 2010 using validated ICD 9 diagnostic codes for acute ischemic stroke. ICD-9 99.1 for IV tPA and ICD-9 procedure code 39.71 (mechanical thrombectomy) were used. Patients with AS treated with MT were divided into three groups: 1. On AC not receiving iv tPA, 2. On AC receiving iv tPA, 3. Not on AC. Group 3 was subdivided into 3a. Received iv tPA and 3b. Did not receive iv tPA. Mortality, hemorrhagic transformation (HT) and disability at discharge were used to compare outcomes. In addition to demographics, comorbid vascular risk factors, concurrent use of antiplatelet agents, in-hospital complications (DVT, PE, sepsis, hospital acquired infections) were controlled. Patients on novel oral anticoagulants were excluded. Results: A total of 10,475 AS patients received MT: Group 1- 398 (3.8%), Group 2 - 439 (4.1%), and Group 3 - 9638 (92.1%). Group 3A - 3918 (37.4%) and 3B - 5719 (54.6%). After controlling for confounders, there was no statistically significant increase in the risk of mortality, hemorrhagic transformation and disability at the time of discharge (Table) in Cohort 1 vs. to other cohorts. Conclusion: The subgroup of patients who are on AC receiving MT for AS with or without tPA did not have an increased risk of HT, mortality or disability.

Background: IP-10 is a pro-inflammatory CXC chemokine that acts as a chemo-attractant for monocytes and T cells. Prior work has shown that IP-10 levels increase after experimental stroke. IP-10 levels are correlated with degree of neurological injury; lower levels are associated with better outcome. Importantly, although nearly three quarters of strokes occur in aged people, no studies have examined IP-10 levels in aged stroke patient or animal populations. As mice and humans age, there is a progressive increase in pro-inflammatory factors circulating in the blood. Consequently, we hypothesize that aged populations will have significantly higher post-stroke IP-10 levels. Methods: Young (2-3 months) and aged (20 months) male C57BL6 mice were subjected to 60 minutes (24hr and 28 day group) or 90 minutes (72hr group) of MCAO and randomly split into 3 groups (n=5/group). Mice were sacrificed 24hrs, 72hrs, or 28 days after sham or stroke surgery. We collected serum samples from the 24hr and 28-day group and brain samples from the 72hr group. We also collected young (37-69yrs; n=18) and aged (70yrs; n=23) human serum samples 24hrs after imaging-confirmed ischemic stroke. IP-10 levels were assessed via ELISA (mouse) and cytokine multiplex assay (human). Results: Aged mice had significantly higher serum IP-10 levels than young mice (102.43±9.21pg/ml vs. 61.99±8.98pg/ml; p Conclusions: This study demonstrates that aging exacerbates post-stroke brain and serum IP-10 levels in both mice and human subjects. Blocking IP-10 may be especially beneficial in improving post stroke recovery in aged populations.

Introduction: Inflammation plays a critical role in the pathophysiology of ischemia and atherosclerosis. TNFα plays a multifaceted role in stroke progression, having both protective and deleterious effects. Animal studies have shown that TNFα levels increase in cerebral tissue within the first 24 hours after experimental stroke. Hypothesis: Expression of TNFα is related to outcomes after acute ischemic stroke. Differences in expression will be seen with respect to patient gender and other stroke characteristics. Methods: Patients admitted to Hartford Hospital between January 2011 and March 2014 were considered for this study. Blood samples were collected within 24 hours of stroke presentation. Patients with a history of active cancer, primary or secondary neoplastic brain lesions, traumatic brain injury or intracranial hemorrhage were excluded from the study. Serum was analyzed using a multiplex human cytokine panel/Bio plex (Bio-Rad laboratories Inc.). Statistical analysis was performed using SPSS. Results: A total of 163 ischemic stroke patients were included in the study. Outcome variables including NIH stroke scale, modified Rankin scale (mRS) at discharge, 3 months, 12 months and modified Barthel’s index (BI) were collected. Composite outcome scores were determined using the mRS and BI. A negative outcome was defined as death or BI ≤ 14 or mRS >2.There was a statistically significant difference (p=0.003) in the expression of TNFα between male - 21.74 pg/ml (15.11-30.21) and female 27.9 pg/ml (19.07-35.497) stroke patients. A significant difference (p=0.009) was observed in TNFα expression in patients with a good outcome - 22.96 pg/ml (15.69-30.85) as compared to those with a poor outcome (discharge to worse/hospice or death) - 30.78 pg/ml (22.03-35.38) post stroke. Conclusions: Male- female sex differences are associated with changes in the post ischemic inflammatory cascade. Higher TNFα levels predict poorer outcomes post stroke. Cytokines like TNFα may serve as a potential therapeutic targets in patients with acute ischemic stroke.

Introduction: Aging remains the biggest risk factor for ischemic stroke. Initial observations into the role of aging on stroke outcome revealed that while aged mice had significantly smaller infarcts compared to young mice, they had greater long-term deficits in behavioral recovery. This study aimed to elucidate the role of systemic factors, such as intestinal stress, underlying the poor outcomes of aging mice after stroke. Methods: Young (8-12 wks) and aged (18-20 mos) wildtype mice were subjected to 90min of right middle cerebral artery occlusion (MCAO) followed by reperfusion. Intestinal permeability was measured with 4kDa FITC-dextran gavage. Mesenteric lymph node (MLNs) homogenates were plated on blood agar to measure bacterial growth. Bacterial endotoxin was measured using a LAL assay. Flow cytometry was used to assess immune cells. Plasma IL-6 was measured by ELISA. Results: Baseline gut permeability increased with age as evidenced by increased FITC-dextran extravasation across the intestinal lumina. Bacterial burden in MLNs and serum bacterial endotoxin levels were also elevated with age (p Conclusion: Our data suggest that the high mortality in aged mice after stroke is secondary to exacerbation of intestinal permeability, increased bacterial translocation, and subsequent induction of sepsis. This data points to gut-targeted interventions as a means to reduce the severity of peripheral inflammation and improve prognosis in aged stroke victims.

Introduction: Stroke is 4 th cause of death and one of the leading causes of disability in United States. Transforming growth factor β activated kinase-1 (TAK1) is a major contributor in the progression of injury post stroke. Pharmacological inhibition of TAK1 is neuroprotective after stroke, but the cell type responsible for these effects is unknown. Myeloid cells play a key role in inflammation after stroke and selective deletion of TAK1 in myeloid cells reduced neuro-inflammation in models of multiple sclerosis. Therefore, we hypothesized that the detrimental effects of TAK1 activation after ischemic injury were mediated by myeloid-specific activation of TAK1. Methods: Young male ovary-intact and ovariectomized female [litter mates TAK fl/fl and TAK fl/fl LysM cre (mTAK-/-)], (8-12 weeks) were randomly divided into sham and stroke group and subjected to 90 minutes of right middle artery occlusion. Neurobehavioral assessments were performed after 24 hours and brains were taken for analysis. Results: In sham males, there was a significant increase in the percentage of circulating macrophages and neutrophils in mTAK-/- (62.48 ± 2.43 and 33.33 ± 3.01 respectively) compared to TAK fl/fl (35.38 ± 3.09 and 17.04 ± 3.26) (pfl/fl (72.90 ± 4.63 and 57.78 ± 5.9, respectively) (pfl/fl females (3.0 ± 0.6and 0.9 ± 0.2) (pfl/fl females had similar infarct suggesting a role for estrogen in TAK signaling or post stroke inflammation. Conclusion: TAK1 deletion in myeloid cells leads to neuroprotection against ischemic injury. Further studies will be performed to determine the contribution of different cell types in the TAK1 signaling.

Introduction: Inflammation plays an important role in exacerbating injury following stroke, but current understanding of the factors regulating this inflammation is incomplete. Pregnane X Receptor (PXR) is a nuclear receptor which binds many xenobiotic targets including many commonly prescribed medications. Recent work has shown that PXR is crucial for maintaining the integrity of the intestinal epithelial barrier as well repressing inflammatory responses mediated by NFκB. Since both these pathways can modify the peripheral immune response, we hypothesized that PXR would play an important role in limiting inflammation following stroke. Using PXR knockout mice, we show that loss of PXR is detrimental for stroke recovery. Materials & Methods: Male PXR knockout mice and littermate controls underwent sham surgery or 60min middle cerebral artery occlusion (MCAO). Weight loss was monitored post-stroke and mice were sacrificed after 7 days for splenic weights, infarct analysis by cresyl violet staining, and bacterial translocation as assessed by culture of homogenized mesenteric lymph nodes on blood agar plates. Results: Following MCAO, PXR-/- mice lost significantly more weight than wild type littermate controls (n=6 vs. 4 respectively; p Discussion: PXR is known to negatively regulate NFκB signaling and maintain intestinal barrier integrity. Both of these mechanisms may contribute to delayed recovery following stroke with PXR acting as a master switch. In summary, this early work begins to explore the importance of a xenobiotic sensor, PXR, in stroke recovery.

HDAC4 has been identified as a transcriptional corepressor that catalyzes local histone deacetylation. It is highly expressed in neurons and usually trapped in cytosol. In vitro, translocation of HDAC4 from cytosol into nucleus induced neuronal death in response to glutamate toxicity. Here we assessed the hypothesis that accumulation of nuclear HDAC4 occurs after stroke, this translocation is detrimental in stroke outcome and regulated by calcium/calmodulin-dependent protein kinase IV (CaMK IV). Wild type (WT) and CaMK IV knockout (KO) mice were subjected to transient middle cerebral artery occlusion and were sacrificed 6 hours for molecule assessments or 72 hours post stroke for outcome assessments. Primary cortical neurons underwent oxygen-glucose deprivation (OGD) and viability was measured 24 hours after OGD. Lentiviral vectors were utilized in culture and mice. Stroke induced HDAC4 nuclear translocation in WT mice as assessed by both Western blots and immunohistochemistry. Knockdown of HDAC4 improved cell viability after OGD (HDAC4 SiRNA: 87.19±7.57% vs. control: 68.09±3.83%, p In conclusion, we showed that ischemia induces HDAC4 nuclear shuttling in neurons and this translocation is deleterious in stroke outcome. Our data suggested CaMK IV inhibits HDAC4 nuclear accumulation in stroke.

Backgrounds: Brain Derived Neurotrophic factor (BDNF) is a well-recognized neuro-protective and neuro-restorative protein of the neurotrophin family. Unfortunately, systemically administered native BDNF has minimal therapeutic effects due to poor blood brain barrier permeability and short half-life. Therefore, the aim of this study was to utilize a novel nano-particle formulation of BDNF for enhanced delivery to the brain in mice after middle cerebral artery occlusion (MCAO). Methods: Male C57BL/6J (8-10 weeks) mice were randomly assigned to saline or BDNF nano (Exp group 1, n=6/group) or saline vs. BDNF native vs. BDNF nano (Exp group 2, n=8/group) and subjected to 60min of MCAO. The mice in Exp. group 1 were treated twice with either saline or BDNF Nanoparticles (250μg/kg i.v) 3 and 24 hrs after the onset of MCAO. Exp group2 was also treated twice with saline vs. BDNF native vs. BDNF nano (250μg/kg i.v) but treatment was delayed until 12 hrs after MCAO with a second dose at 24 hrs. Mice were tested weekly on several behavioral tests and were sacrificed on day 15 and brains used for histological analysis. Results: Treatment with BDNF nanoparticles 3 hours after MCAo (Exp group 1) led to a significant (p Conclusions: This study reveals that BDNF nanoparticle formulation is neuroprotective when given within three hours of stroke, and enhances neuronal repair and behavioral recovery beyond that seen with native BDNF even in the absence of decreased infarct size when treatment is delayed.

Background: CaMKK β is a major kinase activated by elevated levels of intracellular calcium. Our previous data has suggested that CaMKK β is neuroprotective after stroke in young mice as inhibition of this kinase aggravated stroke outcome. As aging is an important determinant of stroke outcomes, here we evaluated the functional role of CaMKK β in stroke in aged mice. Methods: Aged wild type (WT) males received intracerebral injections of lentiviral vectors carrying either CaMKK β (LV-CaMKK β) or GFP (LV-GFP) 7 days before middle cerebral artery occlusion (MCAO, 90 minutes). Acute infarcts and neurological deficits scores were then analyzed at 72 hours post MCAO. In chronic survival studies, aged male CaMKK β knockout (KO) and WT mice were subjected to 60 minutes MCAO. Following stroke, long-term behavioral assessments were continuously performed for 3 weeks in KO and WT mice until the sacrifice for tissue loss assessments. Results: Baseline levels of CaMKK β in aged brain were significantly lower when compared to that in young mice measured by Western Blots (Aged WT 0.66±0.06 vs. Young WT 1.00±0.05, n=3 p/g, p Conclusions: Our studies demonstrated that CaMKK β is neuroprotective in stroke in the aged. As the elderly population has worse functional recovery after stroke, our data suggested that this protein may be a potential target for reducing long term disability of stroke patients.

Background: Age is an important non-modifiable risk factor for stroke. Stroke rates double every decade after age 55. Social isolation (SI) exacerbates behavioural deficits, slows functional recovery and worsens histological injury after stroke in young animals, primarily by increasing the inflammatory response. However, the inflammatory response differs in aging, and whether the detrimental effects of SI are seen in aged animals is unknown. We hypothesize that acute and chronic post stroke SI will worsen stroke pathology and recovery in aged mice and pair housing (PH) will reverse these effects. Methods: Eighteen-month-old male C57BL/6 mice were pair housed (PH) for two weeks prior to stroke and randomly assigned to various housing conditions immediately after stroke. Behavioral analysis was done weekly starting at day 7. Mice were sacrificed either at 72 hours or 4 weeks after 60-minute right MCAO or sham surgery (n=9-10/group). Results: Mice isolated after stroke (ST-ISO) mice had significantly greater hemispheric infarct volume and neurological deficit scores (p Conclusions: Social isolation immediately after stroke led to enhanced injury acutely. Despite similar infarcts at 4 weeks, SI mice had delayed behavioral recovery. Pair housing led to increased expression of BDNF and myelin protein expression. Therefore, the beneficial effects of pair housing may be related to BDNF and MBP expression and enhanced recovery after injury in aged animals.

Ischemic stroke, a leading cause of death and long-term disability, has far-reaching effects across the CNS beyond the region of infarct. As extensions of the brain, neural networks of the eye and spinal cord are affected by cerebral ischemia through the transmission of inflammatory and degenerative changes. Although neuropathological effects of MCAO have been reported in both regions, the contribution of the peripheral inflammatory response to the evolution of injury in these CNS regions is unknown. We evaluated local glial responses, vascular integrity, leukocyte infiltration, and cytokine expression after stroke in the eye and spinal cord. Adult male C57Bl/6 WT or GFP+ bone marrow chimeric mice were subjected to 90-min MCAO or sham surgery (n=3-6). Imaging of the retinal vasculature was performed by ophthalmoscopy. At 8h, 24 and/or 36h, 72h and 7d post-stroke mice were perfused with PBS and sacrificed. Organs were harvested for immunohistochemistry, Evans blue extravasation, flow cytometry, and RT-qPCR. Early (8h) after MCAO, over four-fold increases in TNFα and IL-1β gene expression were observed in the ipsilateral eye relative to sham (4.64±1.21, 1±0.17; 11.03±7.75, 1±0.21). Microglial activation indicated by increased CD11b, CD45, and cell granularity at 36h was accompanied by an influx of peripheral leukocytes predominantly comprised of neutrophils (81%). In contrast to BBB breakdown which was highest at 24h, blood-retinal barrier breakdown peaked in the ipsilateral eye at 36h, shown by a twelve-fold increase in Evans blue concentration (sham, 24h, 36h: 0.16, 0.41, 1.95 ug/mg tissue wet weight). Similarly, major blood-spinal cord barrier breakdown occurred at 36h (0.32 vs 0.052 ug/mg). This was followed by a largely monocytic response with corresponding increases in TNFα, IL-1β, and IFNγ gene expression. Together, our data show robust unilateral peripheral inflammatory responses after stroke in CNS regions outside of the brain. In addition, blood-parenchymal barrier permeability peaks later in the eye and spinal cord than in the brain. Given the distinct time course and inflammatory components associated with each region, these findings may have wide implications for treatments attenuating CNS-wide inflammatory damage after stroke.

Introduction: The impairment of energy metabolism during stroke is a major contributor to ensuing tissue damage and death. The NAD+-dependent histone deacetylase SIRT3 has been identified as a key regulator of mitochondrial energy metabolism. It has also been found to repress the transcription of nuclear genes via histone deacetylation. This study aimed to elucidate the function of nuclear SIRT3 in ischemic stroke. Methods: Focal cerebral ischemia was induced by transient right middle cerebral artery occlusion (MCAO) for 90min followed by 72hrs of reperfusion. Infarct area was quantified with Sigma ScanPro5 on 30μm coronal sections stained with Cresyl Violet. Expression of SIRT3 and acetylated lysine proteins was examined by immunohistochemistry (IHC) and western blotting with anti-SIRT3 and anti-acetylated lysine antibodies. NAD+ levels were measured with a colorimetric kit. Results: SIRT3 expression was increased in the ischemic hemisphere of wild-type (WT) mice at 72hrs. Higher magnification of the IHC image demonstrated localization of SIRT3 to both the nucleus and mitochondria of labeled cells. Subcellular fractionations confirmed this localization, and revealed a dramatic increase in nuclear translocation of SIRT3 with ischemia. With the same duration of MCAO and reperfusion, SIRT3 knockout (KO) mice displayed a reduction in striatal infarct from 60.6±7.2% to 24.9±9.7% (P Conclusions: Our findings suggest that SIRT3 contributes to brain injury during stroke via ischemia-induced upregulation and translocation to the nucleus, where it deacetylates both histone and non-histone proteins and represses nuclear gene transcription. Consistent with the neuroprotection afforded by the protein’s absence in KO mice, the inhibition of SIRT3 may serve as a potential therapeutic strategy for minimizing stroke damage.

Background and Purpose: Ischemic stroke is a sexually dimorphic disease. Events that occur during pregnancy can have lasting implications on both the mother and fetus for decades after parturition. In particular, fetomaternal microchimerism, the bidirectional exchange and persistence of cells between a pregnant female and her fetus, has been shown to play a role in various disease pathologies but has not been well studied in the brain. We hypothesized that microchimeric cells (MCs) home to sites of injury as part of the immune response to stroke and display a stem cell phenotype with potential to aid in repair. Methods: C57Bl/6 mice were bred with GFP transgenic mice using specific mating paradigms to generate fetal and maternal microchimeras. Young 10-12 week old maternal-microchimeric mice and 12-14 week old parous fetal-microchimeric mice were subject to a 90 min MCAO or sham surgery and sacrificed at 72hr or 30 day post-reperfusion (n=10). Blood, bone marrow (BM), and brain tissue were analyzed by co-labeling with anti-GFP antibody using immunohistochemistry, flow cytometry, and cytospin preparations. RNA was extracted from GFP+ cells in situ by laser capture microdissection to further characterize MCs. Results: At 72hrs after stroke, clusters of MCs were present in the ischemic area. Flow cytometry of maternal blood and BM identified a rare cell population (0.01-0.001%) of GFP+CD45+CD34+, indicative of umbilical cord stem cells. Engrafted MCs with stem-like phenotypes were also found in cytospin preparations of blood and BM. At 30 day after stroke, MCs in the brain displayed mature endothelial morphology in 60% of animals. This was validated by RT-qPCR, confirming the presence of MCs in the ischemic brain. Conclusions: We have shown that MCs display stem cell phenotypes, are actively recruited to the ischemic brain, and selectively aggregate at sites of injury. Our data show that MCs are mobilized in BM and blood early after stroke and later give rise to endothelial cells, indicating a potential angiogenic role in promoting revascularization. Further characterization of the functional contribution and mechanisms that underlie these responses may prove useful in assessing female responses to brain injury.

Introduction: Early after stroke local inflammatory changes are primarily mediated by microglia, the resident macrophages of the brain. As injury evolves and the vascular integrity becomes compromised, there is a robust infiltration of peripheral myeloid cells into the ischemic area. Given the common lineage ancestry and morphological similarity between bone marrow-derived (BM) macrophages and resident microglia, the two cell types become nearly indistinguishable from each other using standard immunohistological methods. To date, the most sensitive means of discerning between microglia and BM macrophages relies on the relative level of CD45 expression. In this study, ex-vivo analyses were performed to distinguish CD45intCD11b+ microglia and CD45hiCD11b+Ly6C+ BM macrophages. Functional studies were also performed. Materials & Methods: Young male C57Bl/6 mice were subject to sham surgery or middle cerebral artery occlusion for 90min followed by 72hr or 7d of reperfusion. Ipsilateral hemispheres were harvested from PBS perfused mice and fixed for immunohistological preparation or processed by dissociation into single cell suspensions for use in functional flow cytometry assays. Results: Significant microglia cell death occurred by 72hr, and inversely correlated with BM macrophage infiltration. Surviving microglia showed a low rate of proliferation in response to ischemic stress compared to BM macrophages (1% vs. 7%; p Discussion: There is a complex, biphasic macrophage response to stroke injury. The post-acute phase is characterized by widespread microglia activation and dysregulation, whereas the subsequent resolution phase consists of debris clearance in the necrotic core by infiltrating macrophages.

Background: MKP-1, a negative modulator of several MAP kinases including JNK and p38, is a critical regulator of inflammation and apoptosis in the CNS. Inhibition of MKP-1 worsens stroke outcome in mice, suggesting that MKP-1 is an endogenous protective molecule, although the underlying mechanisms remain unclear. Here we investigated the downstream mediators of MKP-1’s effects in stroke. Methods: Stroke was induced by 60 min transient middle cerebral artery occlusion (tMCAO) in male mice. Levels of p-JNK and p-p38 in MKP-1 KO mice and WT littermates 6 hours after stroke were assessed by Western blots. In a second cohort, the selective JNK inhibitor (SP600125) was administered intraperitoneally to both MKP-1 KO and WT mice 30 min before stroke then infarcts were analyzed at 72 hours post ischemia. In a third cohort, WT mice were intraperitoneally administrated the inhibitor or vehicle 30 min before stroke and sacrificed at 6 hours for protein analysis. Results: MKP-1 deletion produced significantly higher levels of p-JNK 6 hours after stroke (0.71±0.12 WT vs. 1.32± 0.14 KO, p0.05, n=3 p/g). There was no additive deleterious effects of pharmacological JNK inhibition in MKP-1 KO mice as measured by infarcts at 72 hours (cortex: 25.84±2.44%WT vs. 22.32±1.91%KO, striatum: 33.10±3.61% WT vs. 26.52±1.92% KO, total: 27.85±2.95% WT vs. 26.75±1.61% KO, p>0.05, n=6 p/g). In WT mice, JNK inhibition significantly decreased p-JNK (0.99 ± 0.14 vehicle vs. 0.44 ± 0.13 drug, p0.05, n=3 p/g). Conclusions: JNK is an important downstream mediator of MKP-1’s effects in stroke. In the ischemic brain, activating MKP-1 may restrain JNK activity, thereby producing neuroprotective effects.

Introduction: Evidence suggests that post-stroke inflammation and outcome is influenced by sex, yet the mechanistic processes behind these disparities remain largely unknown. Studies have shown that ischemic damage following experimental stroke is reduced in young female mice when compared to their male counterparts and that this protective effect can be diminished by ovariectomy (OVX). TGF-B activated kinase (TAK-1) is an important mediator in inflammatory signaling. TAK decreases following cerebral ischemia and inhibition of TAK reduces infarct size following experimental stroke. TAB2 is an activating binding partner that binds to TAK-1 following stimulation, allowing TAK-1 auto-phosphorylation and activation of the p38/MAPK and JNK inflammatory pathways. Post-stroke regulation of TAK levels and activation may contribute to the ischemic resistance seen in females. Hypothesis: We hypothesized that female mice would have a greater decrease in TAK1 and activated p-TAK1 than their male counterparts following experimental stroke. Methods: Male, intact female and ovariectomized female (OVX) mice were subjected to 90-minute middle cerebral artery occlusion (MCAO) and sacrificed at 24 hours post-stroke. Protein levels of TAK-1, TAB2, pTAK and JNK were examined by Western Blot. Results: TAK and pTAK levels were reduced following stroke in female, male and OVX mice. Baseline levels of both TAK1 and pTAK were lower in female mice and the loss of TAK, pTAK and TAB2 following stroke was more dramatic. JNK levels were lower in female mice than in male mice at baseline (p Conclusions: Female mice exhibit lower baseline JNK levels and do not demonstrate a stroke-induced increase in JNK, indicating a protective resistance against stroke-induced inflammation. The dramatic loss of TAB2 in females results in less binding and activation of TAK, reducing inflammation and conferring neuro-protection. OVX mice showed an intermediate phenotype, indicating that estrogen may play a role in TAK regulation following stroke.

Background and Purpose: Previous studies have demonstrated a rapid activation of peripheral immune responses following experimental stroke, marked by increases in pro-inflammatory cytokines, chemokines, and leukocyte entry into the brain, which may exacerbate tissue damage caused by reperfusion. In the CNS, a consequence of the local immune response to stroke is disruption of the immunosuppressant interaction between CD200 ligand (CD200), expressed by CNS-resident cells, and CD200 receptor (CD200R1) on microglia. Microglia are myeloid-derived cells, which serve as endogenous macrophages within the CNS. In models of CNS injury, disruption of the CD200-CD200R1 interaction leads to changes in intracellular signaling that can increase expression of several pro-inflammatory factors. In the present study, we compared gene expression of CD200 and CD200R1 in the bone marrow, a site of immune cell production, between sham and stroke mice. Additionally, we explored CD200 and CD200R1 protein expression in the primary and secondary lymphoid tissues between sham and stroke mice. Methods: Experimental stroke was induced by middle cerebral artery occlusion in male C57BL/6 mice for 90 minutes prior to reperfusion. Mice were sacrificed 5, 24, and 72 hours after stroke. Bone marrow was harvested from femur and RNA was extracted using Trizol®. cDNA was analyzed for CD200 and CD200R1 expression using qPCR. Western blots were performed on protein extracts from spleens and lymph nodes. Results: In the 5-hour cohort, a significant increase in CD200R1 and CD200 expression was observed in the bone marrow of stroke mice compared with sham. Expression of CD200 and CD200R1 was also elevated in secondary lymphoid tissue in stroke mice. In the 72-hour cohort, a significant decrease in CD200 expression was observed in stroke mice compared to sham mice. Within lymphoid tissue, similar trends were also observed for CD200R1. Conclusions: Immediately following stroke injury, CD200 and CD200R1 gene expression levels are altered in bone marrow, thereby enhancing pro-inflammatory signaling and increasing immune cells in peripheral circulation that can migrate into the brain. The subsequent decrease in expression of these proteins in the 72-hour cohort suggests that CD200- and CD200R-expressing cells are depleted in the bone marrow as a result of their activation, mobilization, and recruitment to the brain. Increased CD200 and CD200R expression in the spleen suggest a similar response by the peripheral immune system as the brain signals for the production of anti-inflammatory components. These results suggest that following stroke, bone marrow activation induces a mobilization signal which targets CD200/R-expressing cells to sites of ischemic brain injury, and is consistent with previous observations showing increased CD200R1 protein levels in the brain following stroke.

Background: Transforming growth factor-β activated kinase 1 (TAK1) activates several intracellular signaling cascades, affecting cell proliferation, inflammation, and apoptosis. It is also a putative kinase for adenosine monophosphate protein kinase (AMPK). 5Z-7-oxozeaenol is a selective inhibitor of TAK1. We and others have previously reported that this drug reduced infarct volume and levels of activated TAK1when administered prior to ischemia in a mouse model of ischemic stroke. We investigated the possible clinical utility of the drug by administering it after ischemia and measuring infarct volumes and behavioral outcomes at 7 days. We also explored the relationship between TAK1 and AMPK by examining outcomes of TAK1 inhibition in AMPK knockout (KO) mice. Methods: Stroke was induced by 90 minutes of reversible middle cerebral artery occlusion (MCAO) in male mice. In one cohort, wild-type (WT) and AMPK KO mice were treated with drug or vehicle via intraperitoneal (IP) injection 2 hours after stroke onset and stroke severity was measured by tissue staining at 24 hours. In a second cohort, WT and AMPK KO mice were treated with drug or vehicle 3.5 hours after stroke onset and stroke severity was measured using the novel-object recognition test (NORT) and by tissue staining at 7 days. Proteins were analyzed by western blot in a third cohort of mice that were sacrificed 4 hours after onset of ischemia. Results: IP injection of 5.0 mg/kg of drug 2 hours after onset of MCAO resulted in a significant reduction in whole-hemisphere infarct volume after 24 hours of reperfusion (49.18±4.18% vehicle vs.17.81±5.86% drug, p=0.001, n=7 vehicle, 8 drug) and after 7 days of reperfusion (57.16±2.38% vehicle vs. 38.35±8.97% drug, p=0.049, n=8 vehicle, 8 drug), and significantly increased the percentage of time that the mice spent exploring a novel object (59.85 ± 3.62% drug vs. 33.15 ± 5.04% vehicle, p=0.001, n=7 vehicle, 7 drug). In AMPK KO mice, there was also a reduction in whole-hemisphere infarct volume. The drug reduced levels of the TAK1 target JNK as well as the JNK target c-Jun. Conclusions: Post-stroke inhibition of TAK1 was neuroprotective and this effect was sustained for 7 days. It was also associated with improvement in the NORT, suggesting preservation of memory function. Together, these findings suggest that 5Z-7-oxozeaenol may have clinical utility for patients suffering ischemic stroke. 5Z-7-oxozeaenol reduced levels of the TAK1 target JNK and its target, c-Jun, suggesting that the benefits of inhibition are due to decreased levels of the transcription factor complex AP-1, of which c-Jun is a major component. The sustained protective effect in AMPK KO mice suggests that AMPK is not important to the mechanism of neuroprotection following TAK1 inhibition.