bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P704: Associations of Intersectional Discrimination, Microaggressions, and Perceived Stroke Risk in Lesbian, Gay, Bisexual, Transgender, and Queer People of Color
Authors: Abbas Maleeha , Serpas Dylan , Zacahula Denaly , Sandoval Samantha , Garcia James J
Published: March 2021

Introduction:
As a psychosocial stressor, intersectional discrimination may contribute to adverse cardiovascular/stroke health for racial/ethnic LGBTQ (Caceres et al., 2019). This study tested this by examining the associations of intersectional discrimination and microaggressions as unique predictors of stroke risk in a sample of racial/ethnic LGBTQ.

Method:
Racial/ethnic LGBTQ (
N
= 307) were recruited online and completed the LGBT PoC microaggression scale (LGBT-PCMS), the Intersectional Discrimination Index (InDI), and the Perceived Stroke Risk subscale of the ABCD Risk Questionnaire for this study. To account for high depression levels typically found in online samples (Ophir et al., 2019), depression (measured by the Patient Health Questionnaire-9) was included as a covariate. A hierarchical linear regression model was used, with depression (i.e., PHQ-9) in block 1, intersectional microaggressions subscales of the LGBT-PCMS in block 2, and intersectional discrimination subscales of the InDI in block 3 to predict stroke risk.

Results:
Participants were 19-68 years of age (
M
= 34.63,
SD
= 9.91), majority Cisgender (92%), with 6% Transgender, and 2% Genderqueer/Gender Non-Conforming. Most were Bisexual (76%), with 14.6% identifying as Gay, 8.7% as Lesbian, and .7% as Heterosexual. The sample was 72% Hispanics, 17.2% non-Hispanics Black, 6.5% non-Hispanic Asians, 2.6% Native American/American Indians, and 1.7% identifying as two or more Hispanic groups. For block 1, depression was positively and significantly associated with stroke risk (
β
= .612,
p <
.001). In block 2, above and beyond depression, the LGBT PoC heterosexism subscale of the LGBT-PCMS was positively and significantly associated with stroke risk (
β
= .216,
p
=.015). In block 3, anticipated intersectional discrimination (
β
=.241,
p
< .001) and major intersectional discrimination (
β
=.265,
p
< .001) of the InDI were positively and significantly associated with stroke risk.

Discussion:
Findings provide evidence for overt and covert intersectional discrimination as unique predictors of perceived stroke risk for this racial/ethnic LGBTQ (majority Hispanic and Black Bisexual) sample.
Abstract P709: Risk Factors Associated With Atrial Fibrillation Detection by Mobile Cardiac Outpatient Telemetry Following Acute Ischemic Stroke
Authors: Inamullah Ovais , McConnell Alec , Al-khalidi Hussein , Bloomfield Gerald S , Shah Shreyansh
Published: March 2021

Background:
Mobile Cardiac outpatient telemetry (MCOT) is often used for patients (pts) with cryptogenic ischemic stroke following hospital discharge to detect atrial fibrillation (AFib) but criteria for patient selection remains a subject of debate.

Methods:
We identified 297 pts hospitalized with acute ischemic stroke who had an inpatient transthoracic echocardiogram (TTE) and underwent MCOT upon discharge between 2016 and 2018 at a large academic comprehensive stroke center. Pts characteristics between AFib vs. no AFib were compared by Fisher’s exact test for categorical and Wilcoxon rank-sum test for continuous variables. A multivariable stepwise logistic regression model was developed to determine the predictors of AFib detection. Statistical hypotheses were tested as two-sided at 0.05 level of significance.

Results:
Of the 297 pts, AFib was detected in 24 (8.1%) on 30-day MCOT. Pts with AFib detected were older, white, and have had a larger left atrial area (Table). The final logistic model demonstrated that white race (vs. non-white) (OR 4.86, 1.53-15.41), left atrial area (OR 1.15, 1.05-1.25) and left ventricular internal diameter in diastole (OR 0.33, 0.16-0.67) were associated with AFib detection by MCOT.

Conclusion:
Although rates of AFib detection on 30-day MCOT post-discharge was low, there are important patient characteristics and TTE features that can improve patient selection. Further studies are needed to determine if this data can be used prospectively to clinically decide which pts with cryptogenic stroke should be given 30-day MCT to detect atrial fibrillation.
Abstract P713: Early Apixaban Therapy After Ischemic Stroke in Patients With Atrial Fibrillation
Authors: Alrohimi Anas , Buck Brian H , Jickling Glen , Shuaib Ashfaq , Thirunavukkarasu Sibi , Butcher Ken
Published: March 2021

Background:
The optimal timing of anticoagulation after stroke in patients with atrial fibrillation (AF) is unknown.

Patients and methods:
A prospective, open label study (NCT04435418) of patients with AF treated with apixaban within 14 days of ischemic stroke/TIA onset was conducted. Baseline and follow-up CT scans were assessed for hemorrhagic transformation (HT) and graded using European Cooperative Acute Stroke Study (ECASS) criteria. The primary endpoint was symptomatic HT. Incident HT rates were assessed as Objective Performance Criteria.

Results:
One-hundred AF stroke patients, with a mean age of 79±11 years were enrolled. Median infarct volume was 4 (0.5-10.75) ml. Median time from index event onset to apixaban initiation was 2 (1-6) days, and median baseline NIHSS was 4 (1-9). Asymptomatic HT on baseline imaging was present in 15 patients. Infarct volume (OR= 1.1, [1.02-1.12], P <0.0001) and NIHSS (OR= 1.11, [1.03-1.20], P =0.007) were both associated with baseline HT. No patients developed symptomatic HT or systemic hemorrhage. Incident asymptomatic HT was seen on follow-up CT scan in 3 patients. Patients with incident HT were functionally independent (mRS=0-2) at 90 days. Recurrent ischemic events occurred within 90 days in 13 patients, 4 of which were associated with severe disability (mRS 3-5) and 4 with death.

Discussion:
Early apixaban treatment did not precipitate symptomatic HT after stroke. All HT was asymptomatic identified on imaging. Recurrent ischemic events were common and clinically symptomatic.

Conclusions:
Symptomatic HT rates are likely to be low in randomized trials of DOAC initiation post-stroke. Recurrent ischemic stroke may be the major clinical outcome.
Abstract P716: Factors Associated With Functional Dependence at Hospital Discharge in Patients With Low NIHSS Strokes Who Do Not Receive Intravenous Alteplase
Authors: Antzoulatos Eleni , Sucharew Heidi , Stanton Robert J , Demel Stacie L , Haverbusch Mary , Alwell Kathleen , De Los Rios La Rosa Felipe , Coleman Elisheva R , Mackey Jason , Ferioli Simona , Mistry Eva , Jasne Adam , Slavin Sabreena J , Walsh Kyle B , Star Michael , Flaherty Matthew L , Martini Sharyl R , Broderick Joseph P , Adeoye Opeolu M , Khatri Pooja , Kissela Brett M , Woo Daniel , Kleindorfer Dawn O
Published: March 2021

Introduction:
Patients without prior functional deficits who suffer mild stroke (NIHSS <6) have a 20-30% likelihood of disability (mRS ≥2). Predictors of disability have been described mostly in clinical trials and single center registries. We identified variables associated with functional dependence (mRS ≥3) in mild stroke using a retrospective population-based sample.

Methods:
Hospitalized strokes from the Greater Cincinnati Northern Kentucky Stroke Study were used. Included patients had an initial NIHSS <6 and baseline mRS 0, both extrapolated from chart review. To minimize the inclusion of patients with disabling symptoms, tPA treatment was excluded. Demographic and clinical characteristics were analyzed by discharge disability status. A multivariable logistic model with least absolute shrinkage and selection operator (lasso) regression analysis identified independent predictors of disability.

Results:
Of 1268 ischemic strokes, 353 (28%) were functionally dependent at discharge. Increased baseline NIHSS was associated with worse outcome on the mRS. Leg, LOC questions, and sensation NIHSS subscores were the best predictors of outcome. Multivariable analysis identified age, race, hypertension, chronic kidney disease, heart failure, and post-stroke dysphagia as independently associated with discharge mRS ≥3.

Discussion:
Our results agree with and complement the results of prior studies. They are not limited by inclusion/exclusion criteria or referral bias. Rather, our major limitation is the retrospective estimation of NIHSS and mRS based on physician descriptive documentation rather than direct score assessment. Our results may allow for modeling to better predict outcome which in turn can inform clinical decision making and trial design.
Abstract P720: Primary Stroke Prevention by Primary Care Providers: An Unmet Need
Authors: Reichwein Raymond , Richardson Alicia , Velasco Cesar
Published: March 2021

Introduction:
The majority of patients who present with acute ischemic stroke (AIS) have known stroke risk factors which are not optimally managed. It has been suggested that a CHADS-VASC score can assist with primary prevention by calculating future stroke risk. This however, has not been widely adapted in the primary care setting.

Methods:
From 2018-2019, 686 AIS patients were included in retrospective analysis. Data elements included: historical stroke risk factors, historical CHADS-VASC score, antiplatelet/anticoagulant use at time of presentation, discharge location, and mRS.

Results:
Of the 686 AIS patients, 77% were age
>
60, and 52% were male. Etiology subtypes were small vessel/lacunar 20%, large vessel 22%, cardioembolic 20%, undetermined 31% (cryptogenic 15%), and other determined 5%. On presentation, the majority of patients had 2 or more stroke risk factors and a calculated historical CHADS-VASC score
>
2 (Table 1). Over half of the patients with large vessel or small vessel/lacunar etiology were not on any antiplatelets and 53% of patients with known history of atrial fibrillation weren’t on anticoagulants. Forty-nine percent of patients had a mRS
>
3 at discharge.

Conclusion:
Patients with several stroke risk factors are sub optimally managed by primary care providers. Primary prevention education for PCPs in management of higher stroke risk individuals and additional analysis of the CHADS-VASC tool for this setting is needed. If widely adapted, this tool may prevent strokes by providing adequate risk reduction in the primary care setting.
Abstract P727: Single-Cell RNA Sequencing Identifies Ifi27l2a as a Novel Gene Involved in Regulating Microglial Inflammation in the Context of Aging and Stroke
Authors: Kim Gab Seok , Stephenson Jessica , Wu Ting , Cantu Gutierrez Manuel E , Wythe Joshua D. , Marrelli Sean P
Published: March 2021

Microglial cells (MG) serve as resident immune cells in the brain and play a critical role in the acute response and chronic recovery to stroke. However, how the transcriptional signature of these cells is altered in aging and stroke remains poorly defined. Here, we compared the MG transcriptomic response to stroke in young and aged mice by single-cell RNA sequencing (scRNAseq). Our unbiased approach identified Ifi27l2a (a member of the interferon signaling family) as one of the most highly upregulated genes in MG with aging or ischemic stroke. Further analyses suggested that this gene may be a novel regulator of MG inflammation in aging and stroke.

Methods:
Permanent distal middle cerebral artery occlusion (pdMCAO) or sham surgery was performed in young and aged mice of both sexes (3 & 20 months). scRNAseq was performed to explore the transcriptional signature in brains at 14-days post-stroke and sham controls. We used qRT-PCR to validate our scRNAseq findings and compare regional transcriptional changes. Human microglial cells (HMC3; subjected to OGD) and human autopsy brain sections (stroke versus non-stroke) were used to examine IFI27l2 protein expression.

Results:
Using comprehensive scRNAseq analysis, we identified Ifi27l2a as the most highly up-regulated gene in aged MG following stroke. Further analysis showed a positive correlation between Ifi27l2a and multiple “MG activation” genes. In contrast, a negative correlation was shown with genes involved in promoting the phagocytic MG phenotype (e.g. Spp1, Cst7, Lpl, and Itgax). Our qRT-PCR results validated the central scRNAseq findings and further showed that Ifi27l2a was significantly up-regulated in thalamus of aged brains (p = 0.0412) and trending in cortex (p = 0.23) (n=4 young/aged). Following stroke, Ifi27l2a and other markers of MG activation were significantly increased in both brain regions (n= 4-6). In human tissues, we observed an increase in IFI27l2 protein in HMC3 cells with OGD and in brain of stroke patients.

Conclusions:
We identified Ifi272la as a highly up-regulated gene in MG in aging and following stroke. Our findings suggest that Ifi27l2a may be a novel regulator of neuroinflammation in MG, and thus provide the basis for a new therapeutic target to reduce inflammation in aging and stroke.
Abstract P732: Genetic Ablation of Endothelial C3a-Receptor Confers Cerebrovascular Protection Independent of the Dynamics of Reperfusion in Stroke
Authors: Kindelin Adam , Bowen Sara , Ahmad Saif , Waters Michael F , Hoda Nasrul , Ducruet Andrew F
Published: March 2021

Background:
Stroke enhances endothelial C3a Receptor (C3aR

End

) expression and humoral levels of C3a which is further exacerbated by intravenous thrombolysis (IVT). Therefore, it is critical to investigate the role of C3aR

End

in post-stroke neurovascular injury.

Hypothesis:
Genetic depletion of C3aR is vasculoprotective in stroke.

Methods:
Using a
loxP
-
Cre
approach (C3aR

Flox/Flox

xCdh5

Cre

), we generated mice conditionally deficient or sufficient in C3aR

End

(C3aR

End-/-

or C3aR

End+/+

), and subjected males (6±0.5-mo old) to thrombotic stroke (TS). Cerebral blood flow (CBF), behavioral outcomes, infarct volume, blood brain barrier (BBB) leakage, brain hemoglobin (Hb) content, brain tissue oxygen (PbtO
2
) , neutrophil polarization (
Proinflammatory N1:
Li6G
+
CD206
-
vs.
Antiinflammatory N2:
Li6G
+
CD206
+
) and their brain infiltration were analyzed. P<0.05 was considered statistically significant.

Results:
TS resulted in similar degree of cerebral ischemia in both groups (N=10/gp); however, CBF, behavior, and infarct volume were significantly improved in C3aR

End-/-

vs.
C3aR

End+/+

mice at 72h post-TS. BBB-leakage and brain-Hb at 72h were less in C3aR

End-/-

vs.
C3aR

End+/+

mice but this did not meet significance (N=5/gp). Interestingly, C3a infusion 3h post-TS significantly enhanced BBB-leakage and brain-Hb in C3aR

End+/+

but not in C3aR

End-/-

mice at 72h (N=5/gp), demonstrating that C3a in conjunction with C3aR

End

exacerbates neurovascular injury in TS. Moreover, late-IVT at 4h post-TS (2 mg/kgbwt; N=8/gp) significantly enhanced PbtO
2
in C3aR

End-/-

vs.
C3aR

End+/+

mice when assessed at 6h. Finally, C3aR deficiency significantly enhanced the N2/N1 ratio relative to the C3aR sufficient group at 24h post-TS (N=3/gp); thus inducing an anti-inflammatory effect and reduced neutrophil brain infiltration.

Conclusion:
C3aR

End

plays a critical role in stroke injury. Future studies targeting brain specific C3aR

End

are warranted.
Abstract P734: Reactive Astrocytes Facilitate Vascular Repair and Remodeling After Stroke
Authors: Williamson Michael R , Fuertes Cathleen Joy A , Drew Michael R , Jones Theresa A
Published: March 2021

Astrocytes and blood vessels interact closely in the neurovascular unit. After stroke, reactive astrocytes surrounding the infarct undergo structural and functional changes. Simultaneously, considerable plasticity of vascular structure occurs, including formation and elimination of microvascular segments, and is associated with restoration of blood flow and behavioral recovery. Reactive astrocytes have established protective functions soon after injury, but less is known about how they influence later reparative processes, which include vascular remodeling. Here we investigated whether reactive astrocytes support vascular remodeling in a mouse model of stroke. Analysis of gene expression data from reactive astrocytes during the first week after stroke showed substantial upregulation of genes and pathways implicated in regulating vascular and basement membrane remodeling. With repeated
in vivo
2-photon imaging, we found that astrocytes contacted new vascular segments formed in peri-infarct cortex after photothrombotic infarcts, suggesting close interactions between reactive astrocytes and new blood vessels. Next, we used GFAP-thymidine kinase (TK) transgenic mice to chemogenetically ablate proliferating peri-infarct astrocytes after stroke (N=8 GFAP-TK; N=8 wildtype mice). Astrocyte ablation following photothrombotic stroke in motor cortex caused dysfunctional vascular repair and remodeling characterized by reduced vascular density, diminished endothelial cell proliferation, prolonged blood flow deficits, and sparse coverage of vessels by glycocalyx, basement membrane, and pericytes. Moreover, peri-infarct vascular permeability was exacerbated in GFAP-TK mice, and this increased permeability was correlated with heightened cell death in the subacute phase after stroke. Tests of forelimb use asymmetry and locomotor coordination both showed worsened recovery in GFAP-TK mice. In contrast to the effects of peri-infarct astrocyte ablation, vascular structure was unaffected by adeno-associated virus-mediated ablation of astrocytes in the otherwise intact brain. Our findings implicate reactive astrocytes as crucial cellular mediators of functionally important vascular repair and remodeling after stroke.
Abstract P737:
Human Homologs Of Differentially Expressed Plasma MicroRNAs In Preclinical Murine Models Target Fundamental Mechanisms Of Cerebral Cavernous Malformation
Authors: Romanos Sharbel , Srinath Abhinav , Moore Thomas , Li Ying , Koskimaki Janne , Zhang Dongdong , Li Yan , Sone Je Yeong , Lightle Rhonda , Hobson Nick , Shen Le , McCurdy Sara , Shkoukani Abdallah , Stadnik Agnieszka , Piedad Kristina , Shenkar Robert , Lopez-ramirez Miguel A , Kahn Mark L , Marchuk Douglas A , Ginsberg Mark H , Girard Romuald , Awad Issam A
Published: March 2021

Introduction:
There is a current need for sensitive and specific biomarkers of Cerebral Cavernous Malformation (CCM) that can be readily translated from preclinical to human models to accurately diagnose and monitor disease states and response to novel therapeutics. MiRNAs are small non-coding RNAs that influence gene expression and whose levels can be affected by disease states. We hypothesize that there are human homologs of differentially expressed (DE) miRNA in the plasma of CCM murine models that can be identified in CCM patients. We further hypothesize that these miRNAs have gene targets within previously published CCM transcriptomes, mechanistically linking them to CCM disease.

Methods:
Plasma miRNAs from homozygous and heterozygous
Ccm1
and
Ccm3
mice, as well as their respective
wild type
controls were sequenced and analyzed. Putative gene targets of DE miRNAs [p<0.05, false discovery rate (FDR) corrected] were queried in previously published mouse CCM transcriptomes. The human homologs of the DE miRNAs in the plasma of
Ccm1
and
Ccm3
mouse models were identified and assessed in the plasma of healthy controls (n=13),
CCM1
(n=11), and
CCM3
(n=11) patients using RT-qPCR.

Results:
5 miRNAs in homozygous and 10 in heterozygous for
Ccm1
, while 45 in homozygous and 2 in heterozygous for
Ccm3
were DE in the plasma of mouse models (p<0.05, FDR corrected), had gene targets within CCM mouse transcriptomes, and have a human homolog. Preliminary results show
mmu-miR-375-3p
as DE in both

Ccm1
+/-

and

Ccm3
-/-

mice. RT-qPCR assays show that plasma relative quantification values of the human homolog
hsa-miR-375-3p
were higher in
CCM3
than in
CCM1
patients (
p
<0.001) and in healthy controls (
p
<0.05).

Conclusion:
DE plasma miRNAs identified in mouse models with human homologs and putative gene targets mechanistically implicated in CCM disease may be used as candidate biomarkers for specific clinical contexts.
Abstract P742: Nanoparticles Targeting the Inflammatory Network in Acute Neurovascular Inflammation: Lungs to Brain Axis
Authors: Marcos-Contreras Oscar Armando , Glassman Patrick M , Nong Jia , Walsh Landis , Zuluaga-Ramirez Viviana , Rodriguez-Garcia Alba , Kiseleva Raisa Yu , Greineder Colin , Hood Elizabeth , Villa Carlos H , Myerson Jacob W , Brenner Jacob , Persidsky Yuri , Muzykantov Vladimir R
Published: March 2021

After acute ischemic stroke, the cerebral vasculature is known to become more permeable to circulating leukocytes (WBC). Several laboratories attempted to use this for co-opting WBC do deliver into the central nervous system drugs and nanoparticle (NP) loaded into WBC ex vivo or in the peritoneal cavity. In theory, intravascular loading WBC may provide an interesting alternative.

We hypothesize: i) direct
in vivo
targeting of WBC accessible to NP in the bloodstream (mainly pulmonary intravascular monocytes and marginated neutrophils in lungs), followed by carrier WBC migration to the brain will allow NP delivery to the brain and, ii) that targeted NP against WBC would be a relevant in treatment of acute brain inflammation. In fact, intravenous injection of ICAM-targeted NP 2h after the injection of tumor necrosis factor (TNF) in the brain initially showed a low uptake of the carrier in the brain that increased with time, as shown by radiotracing (5 times increase) and intravital microscopy of the brain. However, ICAM targeted NP were rapidly accumulated in the lungs and declined with time. Complete blood counts and flow cytometry revealed: i) an increase in circulating WBC counts following injury, ii) >60% of the ICAM-targeted NP were uptaken by WBC in the lungs, iii) ICAM targeted NP were almost exclusively in WBC in the brain (>98%; panel A shows the cellular distribution). Finally, we observed that the anti-inflammatory drug dexamethasone reduces brain edema only when encapsulated in ICAM-targeted nanoparticles (panel B).

In conclusion, ICAM targeted NP: i) can be taken up by WBC, ii) migrate to the inflamed brain and iii) can reduce brain edema when encapsulating dexamethasone.
Abstract P75: Cerebrovascular Events and Outcomes in Hospitalized Patients With Covid-19: The Society of Vascular and Interventional Neurology Multinational Registry
Authors: Siegler James E , Portela Pere , Arenillas Juan F , Chavarria-Miranda Alba , Guillen Ana , de Lera Mercedes , Khandelwal Priyank , Bach Ivo , Patel Pratit , Singla Amit , Requena Manuel , Ribo Marc , Jillella Dinesh V , Nogueira Raul G , Haussen Diogo C , Vazquez Alejandro R , Urra Xabier , San Roman Luis , Thon Jesse M , Then Ryna , Perez de la Ossa Natalia , Millan Monica , Mansour Ossama , Tiu Cristina , Nguyen Thanh , Kaliaev Artem , Czap Alexandra L , Zha Alicia , Liebeskind David S , Ortega-Gutierrez Santiago , Farooqui Mudassir , Hassan Ameer E , Bushnaq Saif , Zaidat Osama O , Jovin Tudor G
Published: March 2021

Background:
Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has been associated with a significant risk of thrombotic events in critically ill patients.

Aims:
To summarize the findings of a multinational observational cohort of patients with SARS-CoV-2 and cerebrovascular disease.

Methods:
Retrospective observational cohort of consecutive adults evaluated in the emergency department and/or admitted with coronavirus disease 2019 (COVID-19) across 31 hospitals in 4 countries (2/1/2020 - 06/16/2020). The primary outcome was the incidence rate of cerebrovascular events, inclusive of acute ischemic stroke, intracranial hemorrhages (ICH), and cortical vein and/or sinus thrombosis (CVST).

Results:
Of the 14,483 patients with laboratory-confirmed SARS-CoV-2, 172 were diagnosed with an acute cerebrovascular event (1.13% of cohort; 1130/100,000 patients, 95%CI 970-1320/100,000), 68/171 (40.5%) of whom were female and 96/172 (55.8%) were between the ages 60-79 years. Of these, 156 had acute ischemic stroke (1.08%; 1080/100,000 95%CI 920-1260/100,000), 28 ICH (0.19%; 190/100,000 95%CI 130 - 280/100,000) and 3 with CVST (0.02%; 20/100,000, 95%CI 4-60/100,000). The in-hospital mortality rate for SARS-CoV-2-associated stroke was 38.1% and for ICH 58.3%. After adjusting for clustering by site and age, baseline stroke severity, and all predictors of in-hospital mortality found in univariate regression (p<0.1: male sex, tobacco use, arrival by emergency medical services, lower platelet and lymphocyte counts, and intracranial occlusion), cryptogenic stroke mechanism (aOR 5.01, 95%CI 1.63-15.44, p<0.01), older age (aOR 1.78, 95%CI 1.07-2.94, p=0.03), and lower lymphocyte count on admission (aOR 0.58, 95%CI 0.34-0.98 p=0.04) were the only independent predictors of mortality among patients with stroke and COVID-19.

Conclusions:
COVID-19 is associated with a small but significant risk of clinically relevant cerebrovascular events, particularly ischemic stroke. The mortality rate is high for COVID-19 associated cerebrovascular complications, therefore aggressive monitoring and early intervention should be pursued to mitigate poor outcomes.
Abstract P751: Global Analyses of Protein and Microrna Cargo in Neural Stem Cell Derived-Exosomes After Brain Ischemia
Authors: LIU Xianshuang S , Fan Baoyan , Li Chao , Levin Albert , Zhang Rui Lan , Chopp Michael , Zhang Zheng Gang
Published: March 2021

Background:
Adult neurogenesis facilitates brain remodeling after stroke. Exosomes derived from neural stem cells (NSCs) promote ischemic neurovascular remodeling including angiogenesis and axonal outgrowth, potentially by transferring their cargo proteins and miRNAs to recipient cells. However, cargo profiles of proteins and miRNAs in NSC exosomes have not been investigated.

Methods:
Exosomes were isolated from supernatants of cultured NSCs harvested from the subventricular zone of rats subjected to 7 day middle cerebral artery occlusion (MCAO) and non-MCAO rats, respectively. Mass spectrometry and miRNA array were utilized to determine the protein and miRNA profiles of NSC-derived exosomes (NSC-Exos). Bioinformatic pathway analyses were performed using Ingenuity Pathway Analysis (IPA).

Results:
Exosome markers and size distribution (50-200nm) were validated with Western blot, transmission electron microscopy and Nanosight measurements, respectively. Proteomics analysis yielded a total of 1,770 proteins in ischemic NSC-Exos. Bioinformatics analysis identified 24, 23 and 23 proteins that were related to neuronal cell proliferation, migration and differentiation, respectively. Intriguingly, enrichment signaling pathway analysis revealed cargo proteins in ischemic NSC-Exos were highly associated with dysfunction, membrane, and permeability of mitochondrion, indicating a critical role of extracellular mitochondrion in stroke-induced neurogenesis. In addition, 318 miRNAs were detected in ischemic NSC-Exos. Gene ontology analysis demonstrated that differentially expressed miRNAs between ischemic and non-ischemic NSC-Exos were highly related to inflammation, cell proliferation, cell cycle, and differentiation. The top 3 upregulated miRNAs including miR-106b, miR-542,miR-125b were validated in ischemic NSC-Exos using RT-PCR. The functions of these miRNAs are related to the induction of angiogenesis.

Summary/Conclusion:
Our results for the first time demonstrate that ischemic NSC-Exos contain a robust profile of protein and miRNA effectors, which may provide new insights into the function of NSC-Exos in stroke-induced neurogenesis and potentially lead to new therapeutic targets against stroke.
Abstract P758: Ischemic Stroke Induced Cardiac Dysfunction in Aged Mice
Authors: Manwani Bharti , Venna Venugopal Reddy , Finger Carson , Zhou Zhen
Published: March 2021

Introduction:
Acute ischemic stroke patients with cardiac complications have increased in-hospital mortality. Most of these cardiac complications have been attributed to pre-existing cardiac comorbidities. Aging is a known risk factor for both stroke and myocardial injury. It is being increasingly recognized that ischemic injury to the brain in itself may lead to cardiac injury. However, the post stroke brain- heart interaction is not well defined in the aged. This study was designed to fill this gap in our knowledge.

Methods:
C57BL6/J young (3 months) and aged (18 months) male mice were subjected to 60 minutes of middle cerebral artery occlusion (MCAO). Mice had electrocardiogram (EKG, using rodent surgical monitor) and Echocardiogram (using vevo 3100 imaging system) pre-stroke, post stroke day 3, 7, and 14. All mice were sacrificed at day 14. EKG and ECHO results were analyzed using generalized estimated equation method with post hoc group comparison test at each time point, Bonferroni correction.

Results:
There was a significant decrease in cardiac output after stroke in aged but not young mice at three days post stroke, as compared to baseline (young, baseline:18.5±1.6ml/min vs day 3:16.6±5.16 ml/min, n=8; aged, baseline 25.4±6.8 6 ml/min vs. day 3, 16.8±6.8 n=6, p=0.0038). The cardiac output normalized to baseline in young but remained significantly low in aged mice even at day 14. Interestingly, the Left ventricular (LV) mass decreased significantly after stroke in aged mice at day 3, 118.4±23.2 mg as compared to baseline, 160.7±22.9 mg. This remained significantly lower than baseline at post stroke day seven, 124.6±32.9 mg and fourteen, 129.5±37.2 mg, p<0.05. A similar trend was seen in left ventricular anterior and posterior wall thickness. There was no significant change in LV mass in young mice, p>0.05.

Conclusion:
Our data indicates that cardiac output decreases after stroke in aged mice. Interestingly, there is a significant decrease in left ventricular mass and ventricular wall thickness in aged mice after MCAO, indicating cardiac atrophy. We speculate this may be secondary to cardiac ischemia caused by stroke. Ongoing work in our laboratory is aimed at examining the histological changes in cardiomyocytes after MCAO.
Abstract P759: Microglial EZH2 Inhibition Leads to Neuroprotection After Stroke in Aged Mice
Authors: Bu Fan , Min Jia-wei , Xu Yan , Li Qi , Koellhoffer Edward C , McCullough Louise D , Li Jun
Published: March 2021

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. Accumulating evidence has suggested EZH2 is key modulator of microglia polarization by epigenetically regulating gene expression. We here investigated whether microglial-specific deletion of EZH2 leads to a beneficial protective effect in stroke
in vivo
. We further tested the therapeutical potential of an EZH2 inhibitor after stroke.

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. EZH2 inhibitor GSK343 was I.P. injected (once a day for two consecutive days), starting three hours after stroke onset. 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 in KO mice compared to the control floxed mice (144±15.43 vs. 50.65±4.99 cells/mm
2
, N=5/each group, P<0.01). EZH2 deletion reduced brain infarct volume (29.27±2.23% vs. 6.07±0.88%, N=7/each group, P<0.001) and improved functional outcome assayed by adhesive removal test (59±13.1 sec, N=7 in floxed control vs. 26.28±4.1 sec, N=12 in KO, P<0.01). Mechanistically, microglial EZH2 deletion led to a decrease in expression of M1 marker iNOS (170±14.78 vs. 76.65±11.38 cells/mm
2
, N=4/each group, P<0.05), an increase in M2 marker Arg1 (96.64±11.48 vs. 203.3±22.02 cells/mm
2
, N=4/each group, P<0.05) co-stained in microglia (Iba1). Finally, GSK343 treatment robustly reduced infarct volume (37.1±2.97% vs. 21.9±2.92%) and increased latency to fall in hang-wire test (23.5±3.5 vs. 45.9±7.1 sec) (N=8/group, p<0.05) 3 days after stroke.

Conclusions:
Both genetic deletion of EZH2 in microglia and pharmacological inhibition of EZH2 improved stroke outcome in aged. The effect may be due to limiting microglial M1 polarization and enhancing M2 polarization.
Abstract P761: Reducing Reactive Astrocytes Improves Cognitive Function in a Mouse Model of Vascular Cognitive Impairment and Dementia
Authors: Liu Qian , Bhuiyan Mohammad Iqbal , song Shanshan , Begum Gulnaz , Liu Ruijia , chen fenghua , Cao Guodong , He Li , Sun Dandan
Published: March 2021

Inflammation and reactive astrocytes are pathologic features in vascular cognitive impairment and dementia (VCID). However, there are no effective therapies to reduce chronic hypoperfusion-induced reactive astrocytes and neuroinflammation and cognitive deficits in VCID. Activation of Na
+
/H
+
exchanger 1 (NHE1) in astrocytes mediates Na
+
influx in exchange of H
+
efflux, causing hypertrophy and swelling of reactive astrocytes after acute brain injury. Whether pharmacological blockade of NHE1 protein can reduce reactive astrocytes and improve cognitive deficits remains untested. Bilateral carotid artery stenosis (BCAS) with microcoils as a murine VCID model was induced in adult male C57BL/6 mice. BCAS procedures led to sustained reduction (30-40%) in cerebral blood flow (CBF) and deficits in special working memory by 30 days post-surgery (p < 0.05). Compared with sham control mice, BCAS mice displayed demyelination in corpus callosum (CC) and external capsule (EC) at 30 days post-surgery (p < 0.05). There were significantly increased GFAP
+
astrocytes and Iba1
+
microglia in CC, EC and hippocampus in BCAS brain at 30 days post-surgery (p < 0.05). Interestingly, those GFAP
+
astrocytes and Iba1
+
microglia exhibited elevated NHE1 protein expression in these brain regions (p < 0.05). These results suggest that NHE1 protein may play a role in chronic hypoperfusion-induced reactive astrocyte transformation and microglia activation. We administered saline vehicle (Veh) or NHE1 inhibitor HOE642 during 30 days post-BCAS (via either i.p. or osmotic mini-pump). The Veh- and HOE642-treated BCAS mice caused similar reduction in CBF. Compared to the Veh controls, both HOE642 treatment regimens significantly improved cognitive performance at 30 days post-surgery (p < 0.05). Causative relationship between blockade of NHE1 protein activation and reducing glia-mediated inflammation and demyelination are under investigation by immunostaining assessments and ex-vivo MRI DTI analysis. Taken together, our study suggests that elevation of NHE1 protein activity plays a role in glial activation in the murine VCID model. Pharmacological blockade of NHE1 protein shows efficacy in reducing cerebral hypoperfusion-induced chronic brain injury and cognitive impairment.
Abstract P762: Hippocampal Plasticity Deficits After Cerebellar Stroke
Authors: Vigil Jose , Moreno Myriam , Minjarez Crystal , Orfila James E , Quillinan Nidia
Published: March 2021

Cerebellar stroke-induced cognitive and affective symptoms (CCAS) have been observed in patients with infarcts localized to the posterior regions of the cerebellum. Our lab has developed a mouse model of cerebellar stroke in which mice with posterior infarcts display hippocampal-dependent memory deficits. Synaptic plasticity in the hippocampal area CA1 is essential for episodic memory consolidation. There is evidence of functional connectivity of cerebellum with contralateral hippocampus. Therefore, we hypothesize that changes in after cerebellar stroke are the result of synaptic plasticity impairments within the hippocampus. We used a photothrombotic model to induce stroke in the posterior cerebellar hemispheres. Seven days after stroke, we performed extracellular recordings of field excitatory postsynaptic potentials (fEPSP) within the CA1 region of acute brain slices. In shams, theta-burst stimulation (TBS) of Schaffer collateral inputs produced long-term potentiation (LTP). TBS failed to evoke an increase in fEPSP slope in contralateral hippocampus of mice at 7 days post-injury. These data provide strong evidence demonstrating that injury to the cerebellum can produce changes in synaptic function within the hippocampus. The lack of LTP impairment in ipsilateral hippocampus after cerebellar stroke suggests that the observed plasticity deficits are activity dependent. We also observed reduced NMDA receptor, Fos and Arc expression. Brain-derived neurotrophic factor (BDNF) signaling through TrkB receptors is required for LTP and memory formation. We tested whether enhancement of TrkB signaling with the agonist 7, 8 dihydroflavone (DHF) could improve hippocampal plasticity. We observed a full restoration of hippocampal LTP in slices from cerebellar stroke mice that were incubated with DHF (250 nM) prior to TBS. We provide strong evidence for hippocampal dysfunction following cerebellar stroke. Our data have implications for benefit of strategies to enhance neurotrophic signaling not only at the site of injury, but in non-injured regions as well.
Abstract P763: A Precision Medicine Approach to Intracerebral Stem Cell Transplantation Utilizing Lesion Topology in a Porcine Model of Ischemic Stroke
Authors: Scheulin Kelly M , Jurgielewicz Brian J , Saini Monika , Spellicy Samantha E , Waters Elizabeth S , Baker Emily W , Kinder Holly A , Simchick Gregory A , Sneed Sydney E , Zhao Qun , Stice Steven L , West Franklin D
Published: March 2021

Despite the devastating global impact of ischemic stroke, there are few Food and Drug Administration (FDA) approved treatments, none of which are capable of regenerating or replacing infarcted brain tissue. Recently, our group demonstrated that induced pluripotent stem cell derived neural stem cells (iNSCs) are a regenerative and neuroprotective cell replacement therapy in an ischemic stroke porcine model that closely resembles human stroke pathophysiology. Historically, intracerebral stem cell transplantations have been largely guided by the injury site without taking individual neuroanatomical structures and their functions into consideration. Utilizing magnetic resonance imaging (MRI), the current study aims to identify key structures lesioned by a permanent middle cerebral artery occlusion (MCAO) that impact stroke recovery in a preclinical porcine model and determine an ideal transplantation location for future stroke iNSC transplant studies. A porcine MRI brain atlas was registered to identify stroke lesion location, and linear regressions between infarcted brain structures and functional data were completed to evaluate the predictive capacity of individual brain structure lesion on neurological outcome. MCAO resulted in prominent lesion volumes and decreased white matter integrity. Highly lesioned brain structures included the insular cortex, somatosensory cortices, visual cortices, temporal gyri, and putamen. MCAO severely impaired translational gait parameters, decreased voluntary movement in open field testing, and resulted in increased modified Rankin Scale (mRS) scoring. Linear regression analysis determined that lesions in the secondary visual cortex, claustrum, amygdala, and superior temporal gyrus were highly prognostic of overall gait and behavioral outcomes. This regression analysis approach identified neuroanatomical structures that were predictive of stroke outcome, and these structures may be key iNSC transplantation locations to facilitate optimal functional recovery.
Abstract P767: Understanding the Role of Microglial Il-1α After Stroke
Authors: Lemarchand Eloise , Ma Sarah , Rahman Abir A , Narayanappa Amruta , Coutts Graham , Bix Gregory J , Pinteaux Emmanuel
Published: March 2021

The role of inflammation driven by the pro-inflammatory cytokine interleukin-1 (IL-1) during poststroke injury has been the focus of intense research. Indeed, pre-clinical studies have demonstrated the deleterious actions of IL-1 after stroke, whilst blocking its actions is beneficial in pre-clinical and clinical settings. Whilst most studies have focused on the role of IL-1β, the role of IL-1α during poststroke inflammation has been largely overlooked and very little has been done to examine the selective contribution of each IL-1 isoform in ischaemic stroke. In this study, we have investigated the contribution of IL-1α to ischaemic and haemorrhagic stroke.
Methods
:
in vivo
model of middle cerebral artery thrombosis through topical application of FeCl3 (40%) (n=4/group (4, 24, 72h) histology, n=6 /group (4, 24, 72 ) qPCR)
in vivo
model of haemorrhagic stroke induced by using the collagenase model (intra-striatal injection of collagenase VII-S, 0.045U) (n=4/group (4, 24h) histology). We first investigated the spatio-temporal expression of IL-1 in the brain after ischaemic stroke using an
in vivo
model of middle cerebral artery thrombosis through topical application of FeCl3 (n=4-6/group). We observed IL-1α positive microglia as early as 4 hours after ischaemic stroke. At 24 hours we observed IL-1α and also IL-1β positive microglia, moreover, IL-1β was also expressed by neutrophils and monocytes. These results have been confirmed by qPCR. Interestingly, we observed similar results after haemorrhagic stroke, induced by using the collagenase model, whereby IL-1α expression in microglia precedes IL-1β expression (n=4/group). The early expression of IL-1α in microglia in both models suggest a critical role for the microglial response during sterile inflammation. To further investigate the role of microglial IL-1α, we have generated a conditional IL-1α mouse mutant crossed with CX3CR1 Cre-ERT2 mice to induce a specific deletion of IL-1α in microglia. Future experiments using this genetic model will clarify the role of IL-1α during the acute phase of strokes.
Abstract P772: Complement C3a Receptor Deletion is Neuroprotective in a Murine Model of Vascular Cognitive Impairment
Authors: Ahmad Saif , Nadeem Muhammed , Kindelin Adam , Mahady Laura , Bhatia Kanchan , Fuentes Alberto , Turner Gregory H , Fulzele Sadanand , Waters Michael F , Hoda Nasrul , Ducruet Andrew F
Published: March 2021

Background:
Chronic cerebral hypoperfusion (CCH) leads to vascular contributions to cognitive impairment and dementia (VCID). We and others have reported that either the genetic deficiency of complement C3a receptor (C3aR) or its pharmacological inhibition in rodents protect against cerebral ischemia.

Hypothesis:
CCH augments VCID
via
overactivation of C3aR in brain.

Methods:
Male C3aR knockout (C3aR
-/-
) and wild-type (C3aR
+/+
) mice (Age:12 weeks; N=8-10/gp) were subjected to either VCID with bilateral common carotid artery stenosis (BCAS) or sham surgery. At 4-mo post-BCAS, changes in cerebral blood flow (CBF), hippocampal atrophy (HA), white matter degeneration (WMD) and ventricular size were determined with laser speckle contrast analysis and magnetic resonance imaging. Behavioral outcomes were evaluated using Morris water maze (MWM) and novel object recognition (NOR). Histopathology with Luxol-Fast Blue (LFB) staining, and protein biochemistry with Western blotting were also performed.

Results:
BCAS resulted in reduced CBF, inducing HA, WMD and ventricle enlargement in both groups compared to their sham controls. These deleterious findings were attenuated in C3aR
-/-
compared to C3aR
+/+
mice. Moreover, the C3aR
-/-
-BCAS group performed significantly better than C3aR
+/+
-BCAS group in MWM and NOR tests; however, mice subjected to BCAS never outperformed their sham-groups. WMD demonstrated by LFB-staining and reduced expression of myelin basic protein and tight junction proteins (ZO-1 and Occludin) in mice subjected to BCAS was mitigated in C3aR
-/-
as compared to C3aR
+/+
mice. C3aR
+/+
-BCAS also showed significantly higher expression of C3a in plasma and C3aR in brain lysates compared to their sham-controls.

Conclusion:
CCH augments C3aR activation leading to poor outcomes in VCID. Therefore, future studies in cell-specific mutant mice are warranted to understand the pathological role of C3aR in VCID.
Abstract P775: Ascorbate Prevents Secondary Brain Damage Following Stroke Through Epigenetic Gene Regulation
Authors: Morris-Blanco Kahlilia , Kim TaeHee , Chokkalla Anil , Bertogliat Mario , Vemuganti Raghu
Published: March 2021

The antioxidant ascorbate is an enzyme cofactor with established roles in maintaining brain function and providing neuroprotection in response to oxidative stress. Recently ascorbate has been identified as an epigenetic regulator through its ability to induce the activity of the ten-eleven translocase (TET) enzymes that produce 5-hydroxymethylcytosine (5-hmC), a CNS-enriched epigenetic modification that is associated with transcriptional activation and neuroprotection. In the current study, we evaluated the role of ascorbate on 5-hmC and its therapeutic potential against ischemic brain injury in comorbid conditions associated with stroke in mice. Adult mice subjected to transient middle cerebral artery occlusion (MCAO) were injected intraperitoneally (i.p.) with ascorbate at 30 min of reperfusion. Young adult male and female mice showed robust induction of 5hmC in the peri-infarct region of the cortex, reduced infarct size and improved motor function. Knockdown of TET3 by intracerebral injection using siRNA blocked the ascorbate-induced increases in 5hmC and led to increased brain degeneration and motor function deficits. Genome-wide sequencing analysis of differentially hydroxymethylated regions (DhMRs) revealed that ascorbate increased 5hmC at the promoters of genes associated with protection against ischemia pathophysiology. Furthermore, ascorbate treatment reduced infarct and improved functional recovery in aged, obese diabetic and hypertensive male and female mice. Delayed ascorbate treatment at 6h of reperfusion was also protective against secondary brain damage and motor deficits following experimental stroke. Collectively, these results indicate that ascorbate regulates the 5hmC epigenetic modification in a neuroprotective manner and may represent a promising therapeutic target for stroke treatment.