bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract TP328: The Association between Post-ICH Continuation of Statin Use and Major Adverse Cardiovascular Events
Authors: Bako Abdulaziz , Schaefer Caroline , Potter Thomas , Pan Alan , Borei Karim , Wozny Joseph , Nicolas Charlie , Caballero Elizabeth , Nair Rejani , Ganduglia-Cazaban Cecilia , Vahidy Farhaan
Published: January 2025

Objective:
The role of statins in the management of intracerebral hemorrhage (ICH) remains controversial. We evaluated the association between post-ICH statin continuation and the development of major adverse cardiovascular events (MACE) in a cohort of Medicare fee-for-service ICH survivors.

Methods:
We identified Medicare-insured patients discharged alive with radiologically confirmed ICH who were taking statin pre index ICH event from the registry of a large stroke-certified healthcare system serving a diverse metropolitan area in Texas, United States (2016-2020). We longitudinally followed the patients by linking the registry data to the Center for Medicare&Medicaid Service (CMS) claims data. We used multivariable logistic regression models to report the adjusted odds ratio (aOR) and 95% confidence interval (CI) of predictors of post-ICH statin continuation, as well as the association of post-ICH statin continuation with the development of MACE (a composite outcome, including any stroke, acute myocardial infarction [AMI], systemic embolism, or vascular death) within 1-year of follow-up. Model fit was assessed using the Hosmer-Lemeshow test.

Results:
Among 142 eligible pre-ICH statin users (median age [IQR]: 77.5 [72-86] years; 57.0% female; 66.0% non-Hispanic White [NHW]), 59 (41.6%) continued using statin post-ICH (median age [IQR]: 76 [73-85] years; 57.6% female; 69.5% non-Hispanic White [NHW]), and 9 (6.3%) experienced MACE within 365-days. Patients with diabetes (aOR, 95% CI: 2.99, 1.25-7.15) and those with a history of anticoagulant use (2.54, 1.00-6.43) were significantly more likely to continue using statin post-ICH. However, obesity (0.30, 0.11-0.85) and higher comorbidity burden, as measured by the Charlson Comorbidity Index (CCI) (0.78, 0.68 – 0.90), were associated with lower odds of post-ICH statin use. Conversely, a higher age-adjusted CCI was significantly associated with lower odds of post-ICH statin use (0.92, 0.86 – 0.98). Post-ICH Statin continuation (vs. discontinuation) was associated with a non-significant decrease in the odds of developing MACE (5.1% vs. 7.2%; aOR, 95% CI: 0.67, 0.12-3.80). All the multivariable logistic models had a Hosmer-Lemeshow test p-value > 0.05, indicating a good fit.

Conclusions:
Continuation of statin post-ICH was associated with a non-significant decrease in the odds of developing MACE. Results from ongoing large clinical trials will elucidate the risks and benefits of the use of statins among ICH survivors.
Abstract TP329: Hospital encounters in the 7 days and 30 days before first-ever stroke: An observational study using data linkage
Authors: Kalpakavadi Serah , Rehman Sabah , Chappell Katherine , Dalli Lachlan , Olaiya Muideen , Kilkenny Monique , Gall Seana
Published: January 2025

Background/Aim:
Up to 20% of people have a hospital encounter in the 30 days before a first ever stroke, suggesting opportunities for better primary prevention. The reasons for these healthcare encounters before stroke have been largely unexplored. We aimed to describe diagnoses in hospital encounters within 7 days and 30 days before a first-ever stroke.

Methods:
We used a statewide (Tasmania, ~500,000 population) linked dataset comprising emergency department [ED] presentations, hospital admissions and deaths in Australia from 2007-2020. An 8-year look back period was used to identify first ever strokes. ICD 10-AM codes in the principal diagnosis field were used to determine diagnoses in hospital encounters before stroke. We describe the distribution of diagnosis groups for each hospital encounters, separately for ED presentations and hospital admissions, in the 7 days and 30 days before stroke.

Results:
Out of 4907 first-ever stroke patients, 202 (4.12%) people had 234 hospital admissions within 7 days before stroke and 507 (10.33%) people had 724 hospital admissions within 30 days before stroke. Among 339 (7%) people, there were 351 ED presentations within the 7 days before stroke and among 676 (13.78%) people, there were 765 ED presentations in the 30 days before stroke. In event-level analyses, the most common diagnoses for hospital admissions were dialysis, diseases of circulatory system (e.g. carotid artery stenosis and unruptured cerebral aneurysm), abnormal symptoms or pathological findings (e.g. convulsions and headache), nervous system disorders (e.g. migraine) and respiratory diseases (Table 1). The most common diagnoses for ED presentations were circulatory system disorders, abnormal pathological findings and nervous system disorders in the 7 days and 30 days before stroke.

Conclusion:
Hospital encounters in the period immediately before stroke are common and associated with a broad range of health conditions. These may represent potentially high-risk groups requiring more intensive primary stroke prevention interventions.
Abstract TP332: Assessing the Role of Thyroid Function in Ischemic Stroke: A Retrospective Analysis Across TOAST Classification
Authors: Singh Zamir , Bozkurt Subutay , Haimi Joshua , Siddiqui Mariah , Briski Nicholas , Strauss Sara
Published: January 2025

Introduction:
Subclinical hyperthyroidism has been linked to an increased risk of atrial fibrillation, which elevates the likelihood of cardioembolism, cryptogenic stroke (CS), and embolic stroke of undetermined source (ESUS). However, the broader relationship between thyroid function abnormalities and acute ischemic stroke remains uncertain. This study aims to investigate whether patients with acute ischemic stroke due to cardioembolism, CS, or ESUS are more likely to have abnormal thyroid-stimulating hormone (TSH) levels compared to those with ischemic strokes from other etiologies, as classified by the TOAST criteria.

Methods:
Utilizing data from the Get With The Guidelines Database, we conducted a retrospective chart review of patients admitted to our single comprehensive stroke center, diagnosed with an acute ischemic stroke between August 2022 and January 2023. The Kruskal-Wallis rank sum test was employed to assess differences in TSH, Free T4, and, if available, one year follow-up TSH levels among patients with ischemic stroke etiologies of small vessel disease, large artery atherosclerosis, cardioembolism, CS, and ESUS.

Results:
A total of 211 patients were included in the analysis, the distribution of stroke etiologies included 33 (16%) with small vessel disease, 51 (24%) with large artery atherosclerosis, 77 (36%) with cardioembolism, 13 (6.2%) with an undetermined/cryptogenic etiology, and 25 (12%) with ESUS. The majority of patients had normal TSH levels, accounting for 82% of the total cohort, the overall mean TSH level was 2.52 (reference range 0.27-4.2uIU/mL). No significant differences were observed in the mean TSH (p = 0.066), Free T4 (p = 0.13), or follow-up TSH (p = 0.79) levels across each stroke subtype.

Conclusions:
Our findings suggest that thyroid function does not significantly differ across the various etiologies of acute ischemic stroke, as classified by TOAST criteria. While further large-scale studies are warranted, our results indicate that routine evaluation of thyroid function may not be essential in the determination of stroke etiology.
Abstract TP338: Functional recovery during the chronic phase after cerebral infarction via CSF-derived microRNA
Authors: Xu Hai-Bin , Kenichiro Hira , INABA TOSHIKI , Kijima Chikage , Yoshifumi Miyauchi , Ueno Yuji , Miyamoto Nobukazu , Hattori Nobutaka
Published: January 2025

(Background):
Stroke has become a leading cause of mortality and disability worldwide. However, except rehabilitation, there is no effective therapy during chronic phase after ischemic stroke.

Interestingly, it has been reported that young cerebrospinal fluid (CSF) restores oligodendrogenesis and memory in aged mice. However, if specific components in CSF could contribute to functional recovery during chronic phase of ischemic stroke remains unclear. We tried to distinguish a specific microRNA in CSF and identify its potential therapeutic functions.

(Method):
Rats were subjected to permanent left middle cerebral artery occlusion (pMCAO). Brains and CSF were collected at 3, 7, 14, 28 days after pMCAO. MicroRNA in CSF was evaluated by microRNA Array. A specific microRNA was selected based on heatmap and volcano plot. IPA analysis was used to distinguish possibly related signaling pathways.

Mimic/ inhibitor of this microRNA was applied respectively to primary cultured astrocytes and neurons soon after 3 hours’ oxygen glucose deprivation (OGD). Associated proteins were tested through western blot at 96 hours after OGD.

In vivo, time course of pStat3 was assessed in peri-infarct cortical area. At 7 days after pMCAO, mimic of this microRNA was applied to CSF through Cisterna Magna. behavioral tests were assessed at 7, 14, 21, 28 days after pMCAO. At 28 days after pMCAO, brains were harvested for ischemia volume calculation and immunofluorescence.

(Result):
miR-204-5p was significantly upregulated in CSF at 7 days after pMCAO and selected as target microRNA. According to IPA analysis, miR-204-5p might be associated with Stat3 signaling pathway.

Based on results of astrocyte culture, GFAP was significantly decreased by applying miR-204-5p mimic. Regarding neuron culture, pStat3, MAP2 showed a tendency to increase by applying miR-204-5p mimic.

In vivo, pStat3 significantly increased at 3 days and began to decrease from 7 days after pMCAO. miR-204-5p mimic group showed a tendency of ameliorated functional dysfunction, smaller ischemia volume at 28 days after pMCAO. In peri-infarct area, compared with non-treatment group, GFAP was significantly decreased, and pStat3 showed tendency to increase in miR-204-5p mimic group.

(Conclusion):
Applying miR-204-5p mimic into CSF contributed to functional recovery via activating Stat3 signalling pathway and reducing astrocytosis, which might be a candidate of exosome or liposome preparation for future clinical trials.
Abstract TP341: Longitudinal Analysis of Peripheral Blood Dynamics During the First Week after Ischemic Stroke: Biomarkers and Correlation to Functional Outcome
Authors: Couture Lucy , Do Trang , Khan Romeesa , Cahuiche Ana , Guzman Gary , West Bryce , Devlin Patrick , Delevati Colpo Gabriela , Ritzel Rodney
Published: January 2025

Introduction:
Peripheral blood-based biomarker development for neurological diseases such as ischemic stroke has traditionally focused on measuring alterations in leukocyte number and composition. Deeper immunophenotyping of cells using fluorescent probes that detect alterations among organelles and structures can provide a better understanding of the biological function of leukocytes after ischemic stroke.

Methods:
We evaluated the utility of BODIPY 493/503 and LysoTracker as fluorogenic probes to assess cellular changes in lipid content and lysosomal burden. Flow cytometric assessments were performed on peripheral mononuclear blood cells from healthy volunteers (HV, N=31), and from ischemic stroke patients (N=43) at 3d and 7d to capture the temporal profile of these biomarkers.

Results:
We found decreased frequency of CD3+ T cells 3d after stroke, which was also seen at 7d after stroke. We found CD4:CD8 ratios were significantly and persistently increased in stroke patients compared to HV controls. Within the CD4 subset, we found a persistent increase in the frequency of effector CD4 T cells after stroke, this finding is consistent with previous literature. Interestingly, we noted a delayed, but profound increase in lipid content of all cell types at 7d after stroke compared controls. This difference was more pronounced among females. Lysosomal content was differentially altered by cell type, with increased levels in CD4 naïve T cells, but decreased levels in CD4 central memory, CD8 naïve, CD8 effector, CD8 central and effector memory T cells after stroke. While we did not see an association with increase in severity at admission, high lipid content at the acute phase was significantly associated with increased disability at 3 months after hospitalization.

Conclusion:
Taken together, our findings provide greater context to the previously documented compositional changes in the blood leukocyte compartment after stroke. Ischemic stroke causes a pervasive increase in cellular lipidosis. Lysosomal content has also been significantly positively correlated to functional disability among stroke patients. Using cell-based functional assays with flow cytometric immunophenotyping may provide meaningful insights into biological health and function of circulating leukocytes after brain injury. Understanding the effects of these compositional changes seen in the acute phase in respect to functional outcomes should be a priority for researchers moving forward.
Abstract TP342: Associations Between Pericarotid Fat Density and Carotid Plaque Inflammatory Cytokine Levels
Authors: Kalina Samuel , Nogami Kai , Mahmoudi Hamidabad Negin , Mahmoudi Elham , Benson John , Saba Luca , Lanzino Giuseppe , Nardi Valentina , Lerman Amir
Published: January 2025

Background:
Previous research has suggested pericarotid fat density to be significantly linked to atherosclerotic plaque components and cerebrovascular events. Inflammatory markers within the plaques are critical in the pathogenesis and progression of carotid artery disease. However, the association between pericarotid fat and inflammatory processes within atherosclerotic plaques remains underexplored. This study hypothesizes that pericarotid fat density is directly associated with the levels of inflammatory markers within atherosclerotic plaques.

Methods:
Carotid plaques were prospectively collected from patients with carotid artery disease undergoing carotid endarterectomy (CEA). The levels of inflammatory plaque cytokines (MCP-1, IL-8, IFN-γ, IL-10, IL-1β, IL-6, PDGF-AA, PDGF-AB/BB, TNF-α, VEGF) were measured in tissue homogenates using a customized 10-plex Luminex assay. Baseline demographics and clinical data of our population were extracted from electronic health records. We analyzed preoperative neck computed tomography angiography (CTA) images placing regions-of-interest (ROI) in the pericarotid fat on the slice showing maximal stenosis. For each patient, we calculated the average of Hounsfield Units (HU) within the ROI. Spearman correlation test was used to assess the correlation between variables.

Results:
Among the 43 patients included, the median age was 73 (67, 78) years, and 33 (76.7%) patients were male. Plaque levels of PDGF-AA and VEGF showed significant positive correlations with HU (r=0.330, p=0.030; r=0.380, p=0.011, respectively), as illustrated in
Figure 1
. Although plaque levels of IFN-γ demonstrated a significant negative correlation with HU (r=-0.390, p=0.010), the most cases exhibited values close to zero.

Conclusions:
The assessment of pericarotid fat density on CTA may allow for the estimation of plaque inflammation, suggesting that more fat density is associated with increased inflammatory activity within atherosclerotic plaques.
Abstract TP343: Co-Expression Modules In The Periphreal Blood Transcriptome Following Subarachnoid Hemorrhage Associate With 90-Day Outcome
Authors: Knepp Bodie , Lenz Garreck , Hull Heather , Ko Nerissa , Jickling Glen , Choi H , Ander Bradley , Sharp Frank , Stamova Boryana
Published: January 2025

Background:
Subarachnoid Hemorrhage (SAH) accounts for 2-7% of strokes and has high mortality and morbidity. We sought to identify peripheral blood transcriptome changes in the acute SAH phase that associated with 90-day outcome, as measured by the modified Rankin Scale (mRS), to gain insights about potential mechanisms contributing to long term outcome.

Methods:
We sequenced the peripheral blood transcriptome of SAH patients within 3 days post
ictus
and stratified the patients into patients with Good (mRS≤2, n=37) and Poor (mRS≥3, n=23) outcomes at 90-day follow up. We generated the co-expression networks using the Weighted Gene Co-Expression Network Analysis (WGCNA) package to determine modules (groups of co-expressed genes) associated with 90-day SAH outcome. The outcome-significant modules (p<0.05) were further analyzed for their biological relevance using pathway analysis.

Results:
We identified two outcome-significant modules, the Pink module and the Purple module. The Pink module was enriched (corrected p<0.05) in Monocyte- and Granulocyte-specific genes, while the Purple module and its hubs were enriched in Monocyte-specific genes. The hub genes are the most interconnected genes in each module, which are therefore potential master regulators. The Pink module was enriched (corrected p<0.05) in Neutrophil Degranulation, an inflammatory pathway which was predicted to be activated in patients with worse outcome, in Histone Modification Signaling, which is involved in epigenetic control of gene expression, and in SUMOylation of transcriptional cofactors, which confers post-transcriptional modifications of these cofactors. The Purple module was enriched in 45 canonical biological pathways, including numerous inflammatory pathways predicted to be activated in SAH patients with worse outcomes, such as Neutrophil Degranulation, Phagosome Formation, IL-8 Signaling, and Macrophage Classical Activation Signaling.

Conclusions:
Early peripheral blood changes in the transcriptome architecture following SAH are associated with long-term outcome. The identified genes and networks may guide the search for potential biomarkers of outcome and novel treatment targets.
Abstract TP345: Electronic Cigarette Exposure Worsens Stroke Induced Inflammation in Female Rats
Authors: Sinder Sophie , Cabeda Diaz Indy , Sharma Harry , Perez Gina , Raval Ami
Published: January 2025

Introduction:
The rise in popularity of nicotine-containing electronic cigarettes (ECs) globally necessitates a deeper understanding of their effects on stroke outcomes. This study investigates the influence of EC exposure on post-ischemic inflammation.

Methods:
Adult Sprague-Dawley rats of both sexes were randomly assigned to either air or EC vapor exposure (5% nicotine Juul pods) for 16 nights, followed by transient middle cerebral artery occlusion (tMCAO; 90 minutes) or sham surgery. Post-surgery, animals were divided into two cohorts. In the first cohort, cortical brain tissue was collected 24 hours after tMCAO or sham surgery for Western blot analysis of inflammasome proteins, including Caspase-1, Interleukin-1β (IL-1β), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and gasdermin-D (GSDMD). The second cohort was sacrificed 21 days post-tMCAO for immunohistochemical analysis of microglial marker ionized calcium-binding adapter molecule-1 (Iba-1). De-ramified microglia were quantified across three serial coronal sections (10 μm) using the optical fractionator probe in StereoInvestigator software, with a counting frame of 100x100 μm and approximately 40 sampling sites per section. Microglial morphology and Sholl analysis were performed by capturing three cortical images per slide, each containing at least three microglia, and across three slides per subject, resulting in 18 images for peri-infarct and contralateral areas. Analysis was conducted using ImageJ software.

Results:
Western blot analysis revealed a significant increase in Caspase-1 and ASC protein levels in the ipsilateral cortex of EC-exposed female rats compared to males after tMCAO (p<0.05). Quantification of Iba-1 indicated an increase in de-ramified microglia in the cortex of EC-exposed female rats as compared to air-exposed after tMCAO, potentially exacerbating post-stroke infarction. Microglial morphology analysis showed increased cell soma size and decreased branch length in EC-exposed female rats. Sholl analysis demonstrated reduced microglial complexity in EC-exposed group as compared to air-exposed after tMCAO, with a smaller area under the curve and fewer peak intersections in the cortex.

Conclusion:
EC exposure exacerbates stroke-induced inflammation and cognitive deficits in female rats. Future research should explore whether EC withdrawal mitigates ischemic severity and determine the necessary duration of withdrawal for potential recovery.
Abstract TP356: Nucleocytoplasmic shuttling of Sirt1 and Sirt2 during cerebral ischemia
Authors: Lee Seeun
Published: January 2025

Background and Rationale:
Acute hyperglycemia caused by cerebral ischemia occurs when there is a lack of oxygen, resulting in anaerobic glycolysis. The Sirt1 and Sirt2 initiate the influence on glucose metabolism and cellular homeostasis through nucleocytoplasmic shuttling. We observed nuclear-cytoplasmic translocation of Sirt1 and Sirt2 during anaerobic glycolysis in the brain.

Methods:
We investigated differences between young and aged mice in a non-fasting group of cerebral ischemia model. Young mice were subjected to time-dependently ischemia induction (0, 1, 2, or 3 h) without reperfusion, and aged mice underwent ischemia for 1 h. All mice measured relative cerebral blood flow using a Laser Doppler. To evaluate the cerebral cortex was separated from nuclear and cytoplasmic fractions by Western blotting.

Results:
Aged mice had a more rapid and dramatic decrease in relative cerebral blood flow compared to young mice. Cytoplasmic-Sirt1 was elevated and cytoplasmic-Sirt2 did not change in young mice. In both young and aged mice in the ipsilateral cerebral cortex during 1 h ischemia, Sirt1 activity shifted from the nucleus to the cytoplasm, and Sirt2 activity shifted from the cytoplasm to the nucleus.

Conclusion:
Cellular senescence can contribute to the accelerating glucose metabolism and aging processes in cerebral ischemia. We demonstrated that ischemia accelerates aging-like processes in the brain. These processes are mediated through the upregulation of the cytoplasmic-Sirt1 and nuclear-Sirt2 pathways.

Key words:
Stroke, Ischemia, middle cerebral artery occlusion, nucleocytoplasmic shuttling, Sirt1, Sirt2
Abstract TP357: Mitochondrial intercellular transfer via platelets after physical training exerts neuro-glial protection against cerebral ischemia.
Authors: Miyamoto Nobukazu , INABA TOSHIKI , Kenichiro Hira , Kijima Chikage , Hattori Nobutaka
Published: January 2025

Background:
Despite the effectiveness of immediate treatment, such as thrombolytic therapy, after a stroke, many patients are unable to benefit due to time restrictions. In an aging society, sarcopenia, a condition characterized by reduced muscle volume, is emerging as a significant issue, often contributing to poor recovery after stroke. Our study examined the role of mitochondria, which are abundant in muscle, and their migration during exercise in the context of stroke recovery.

Methods:
We used mice models to simulate chronic hypoperfusion and distal middle cerebral artery occlusion. We also conducted in vitro studies with rat primary cells, co-cultured with a nonlethal concentration of CoCl
2
and oxygen-glucose deprivation.

Results:
We found that 28 days post-hypoperfusion, there was a progression in white matter injury, including myelin loss, astroglial formation, and memory disorder. However, treadmill training protected against these damages. In cases of acute ischemia, training improved complications and reduced glial activation. We observed increased mitochondria in muscle and blood due to training, which migrated between tissues using platelets. In vitro analysis revealed that adding muscle mitochondria improved the survival of neurons, astrocytes, and oligodendrocytes. Most importantly, we found that mitochondrial transfusion from treadmill-trained mice platelet improved ischemic white matter injury and post-stroke complications.

Conclusions:
The study showed mitochondria as part of the secretome, essential for regulating cell interactions. It explored mitochondrial secretion, transfer between cells, and dynamics influencing ischemic tissue. The protective effects of remote ischemic preconditioning might be linked to muscle-derived mitochondria, a recent phenomenon. Unlike conventional platelet transfusion, mitochondria administration even from cryopreserved platelets shows brain protection effect. Therefore, this suggests that the advantage of this therapy is that mitochondria can be extracted from platelets that could not be used for transfusion and used for treatment. Our study suggests it could be a promising new treatment for reducing post-stroke complications and vascular dementia.
Abstract TP359: Peripheral blood
GATA2
expression increases
RNF213
mutation penetrance and clinical severity in moyamoya disease
Authors: Mineharu Yohei , Kamata Takahiko , Tomoto Mei , Sato Noriaki , Tamada Yoshinori , Funaki Takeshi , Oichi Yuki , Harada Koji , Koizumi Akio , Kimura Tetuaki , Inoue Itsuro , Okuno Yasushi , Miyamoto Susumu , Arakawa Yoshiki
Published: January 2025

Background:
The p.R4810K founder mutation in the
RNF213
gene confers susceptibility to moyamoya disease (MMD) and non-MMD intracranial artery disease. However, penetrance is incomplete, and the underlying molecular mechanism remains unknown.

Methods and Results:
Transcriptome analysis of peripheral blood was conducted with 9 MMD patients and 5 unaffected mutation carriers from 4 familial MMD pedigrees. Bayesian network analysis identified upregulated gene modules associated with lipid metabolism and leukocyte development (including
GATA2
and
SLC45A3
), and EGFR signaling (
UBTD1
). It also identified downregulated gene modules related to mitochondrial ribosomal proteins (
RPS3A
and
RPL26
), and cytotoxic T cell immunity (
GZMA
and
TRGC1
). The
GATA2
network was replicated through WGCNA analysis and further examined in a case-control study, comprising 43 MMD patients, 16 non-MMD patients, 19 unaffected carriers, and 35 healthy controls.
GATA2
exhibited a significant linear correlation with
SLC45A3
and was significantly higher in MMD patients compared to age- and sex-matched unaffected carriers or wild-type controls. Among patients with the p.R4810K mutation, higher
GATA2
expression was associated with an earlier age of onset, bilateral involvement, and symptomatic disease onset.

Conclusions:
Peripheral blood
GATA2
expression was associated with increased penetrance of the
RNF213
mutation and more severe clinical manifestations in MMD.
Abstract TP361: Type 2 Diabetes Enhances Leukocytes And Brain Endothelial Cells Adhesion Interactions Following Ischemic Stroke
Authors: Kato Yuya , Sato Yoshimichi , Gu Wanjun , Liu Jialing
Published: January 2025

Introduction:
Stroke is a leading cause of death and disability worldwide, with diabetes being a significant risk factor that exacerbates stroke outcomes. While previous studies have identified various cell populations characteristic of stroke and other neurological diseases through transcriptomic analysis of brain and blood cells, the interaction between leukocytes and brain endothelial cells during stroke remains inadequately understood. This study aimed to investigate these intercellular interactions in the context of diabetes and stroke.

Methods:
We utilized a permanent ischemic stroke model in leptin receptor-deficient db/db mice (a model for type 2 diabetes) and their non-diabetic counterparts, db/+ mice. Three days post-stroke, brain cells and circulating leukocytes were isolated and subjected to single-cell RNA sequencing (scRNAseq). The CellChat tool was employed to analyze the intercellular communication between these cells. Additionally, in vivo two-photon microscopy was used to observe real-time interactions between leukocytes and brain endothelial cells in cortical veins.

Results:
scRNAseq analysis revealed that diabetic mice had a higher proportion of myeloid cells in the blood compared to controls. Post-stroke, both diabetic and non-diabetic mice showed an increased presence of microglia and macrophages in the brain. CellChat analysis indicated that both diabetes and stroke significantly elevated the number and strength of cell-cell interactions, with the highest levels observed in the diabetic stroke group. Notably, interactions involving the integrin family and ICAM/VCAM, known for their roles in cell adhesion between blood leukocytes and brain endothelial cells, were particularly prominent in the diabetic mice. In vivo imaging confirmed significant leukocyte accumulation in the cortical veins immediately after stroke in both genotypes, with delayed clearance observed in the diabetic group.

Conclusion:
The findings suggest that diabetes exacerbates the inflammatory response in stroke by enhancing leukocyte-endothelial interactions, leading to prolonged leukocyte retention in the cerebral vasculature, which likely contributes to increased inflammation and worsened stroke outcomes in diabetic conditions.
Abstract TP364: Nasal Delivery of Nogo-66 Receptor (NgR1) Antagonist Decreases Axonal Growth Cone Collapse in Response to Diesel Exhaust Exposure
Authors: Tang-Tan Angela , Shkirkova Kristina , Demetriou Alexandra , Chen Selena , Bent Christine , Daggupati Sindhu , Zhao Lifu , aguilar Jennifer , Gopalakrishna Rayudu , Mack William
Published: January 2025

Background:
Patients chronically exposed to air pollution suffer myelin damage and neuroinflammation that can inhibit axonal regeneration. Our group has demonstrated that diesel exhaust particulate (DEP) inhalation increases expression of axonal outgrowth inhibitors in mice. The objective of this study was to examine how pharmacologically targeting the Nogo-66 receptor (NgR1) through a novel intranasal route can mitigate this effect.

Methods:
Female and male 8-week-old C57BL/6 mice were exposed to 6 weeks of inhaled Filtered Air (FA) and DEP (NIST SRM 2975) at 100μg/m3. Within a week of the exposure ending, we began 30 days of nasal administration of either NEP1-40, an NgR1 antagonist inhibiting peptide, or a scrambled peptide control. There were four groups: NEP 1-40+Filter (NF), NEP 1-40+DEP (ND), Scrambled NEP 1-40+Filter (SF), Scrambled NEP 1-40+DEP (SD). Mice were humanely euthanized. We confirmed intranasal absorption of NEP 1-40 and scrambled peptide with fluorescence analysis of the cortex. Corpus callosum levels of Oligodendrocyte myelin glycoprotein (OMgp), Chondroitin sulfate proteoglycans (CSPGs), Nogo-A membrane protein, Nogo Receptor 1 (NgR1), Paired immunoglobulin-like receptor B (PirB), Aquaporin 4-(AQP4), and degraded myelin basic protein (dMBP) were assayed via immunofluorescence.

Results:
In fluorescence analysis of peptide absorption in the cortex, there was no difference between the FA and DEP groups within the active peptide and scrambled peptide arms. In the scrambled peptide arm, DEP exposed mice (SD) had higher levels of NgR1 (p<0.01) compared to the FA (SF) group, with no difference observed in the active peptide arm between DEP (ND) and FA (NF). The SD group had higher levels of OMgp compared to the SF group (p=0.05), with no difference in the active peptide arm. There was a trend towards an increase in PirB in the SD group compared to the SF group (p=0.06), with no difference in the active peptide arm (Figure 1). No significant effects were observed in Nogo-A, CSPG, AQP4, and dMBP between groups.

Conclusion:
Nasal administration of NEP 1-40 successfully penetrated into the brain. NEP1-40 peptide administration mitigated expression of axonal outgrowth inhibitor OMgp and axonal outgrowth inhibitor receptors NgR1 and PirB compared to scrambled peptide administration. This suggests that intranasally targeting the NgR1 pathway may be a viable pharmacologic mechanism of facilitating neuronal recovery in patients exposed to air pollution.
Abstract TP365: Understanding neutrophil recruitment and lifespan following ischemia/reperfusion using a novel tracking method
Authors: Arias Erika , Haynes Maureen , Nadkarni Neil , Muller William , Sullivan David , Batra Ayush
Published: January 2025

Introduction:
Polymorphonuclear leukocytes (PMNs) are among the first leukocytes recruited to the infarct following reperfusion in ischemic stroke. Although transendothelial migration (TEM) blockade in preclinical studies was found to reduce infarct size, TEM blockade in clinical trials has been unsuccessful. Studying PMN recruitment and lifespan in ischemic stroke may offer insight into these failures in clinical trials and advance therapeutic development.

Methods:
PMNs were briefly pulse-labeled with 5-Ethynyl-2’-deoxyuridine (EdU) to determine PMN lifespan and migration pattern following ischemia/reperfusion in stroke. Large vessel occlusion and reperfusion were simulated using the transient middle cerebral artery occlusion model (tMCAO, 90 minutes of ischemia followed by reperfusion). Infarct size was determined via triphenyl tetrazolium chloride staining. The percentage of EdU+ PMN in circulation was assessed each day following tMCAO and compared to the percentage of EdU+ PMNs in the ischemic hemisphere at sacrifice by flow cytometry. The position of PMN in coronal sections was examined by wide field fluorescence microscopy.

Results:
Large cortical ischemic strokes involving over 20% of cerebral volume were induced in mice. A majority of PMN recruitment and extravasation was localized to the cortical surface of the infarct at early time points (12 and 24 h post stroke), contrasting other vascular beds where there is PMN migration deep into the infarcted tissue by 24 h. EdU+ PMNs persisted in the ischemic hemisphere until 96 h post stroke despite their clearance from circulation at least 48 h prior, indicating PMN can survive for days in the infarct. Additionally, the percentage of EdU+ PMN in the ischemic hemisphere was similar to the percentage of EdU+ PMN in circulation at 24 h post stroke, suggesting PMN recruitment centers around 24 h.

Conclusions:
Our findings demonstrate PMN recruitment and infiltration post stroke evolves over several days. This data suggests that the majority of PMNs are recruited to the infarct within the first 48 hours and likely can persist within the infarct for days. Modulating the timing of PMN labeling via EdU can lead to a better understanding of PMN spatiotemporal infiltration and persistence and will help inform future therapeutic interventions targeting leukocytes.
Abstract TP369: Alpha7 nicotinic acetylcholine receptor plays a beneficial role in long-term cognitive recovery after stroke.
Authors: Nguyen Dustin , Hall Cassie , Chauhan Anjali
Published: January 2025

Introduction:
The cholinergic anti-inflammatory pathway regulates immune responses through the alpha7 nicotinic acetylcholine receptor (α7nAChR), found in neurons, macrophages, and monocytes. α7nAChR activation via agonists or Vagus nerve stimulation (VNS) reduces pro-inflammatory cytokines in disease models. In young mice, pharmacological activation or stimulation of the Vagus nerve has been shown to mitigate ischemic stroke injury by reducing brain and peripheral inflammation and oxidative stress. However, the role of α7nAChR in long-term stroke outcomes remains unclear.

Methods:
Young (8-12 weeks) male wild-type (WT) and α7nAChR knockout (KO) mice underwent middle cerebral artery occlusion (MCAO) for 60 minutes. After 24 hours, brain acetylcholine levels and α7nAChR expression were assessed by mass spectrometry and western blot respectively. Microglia, macrophage counts, and TNF-α expression were evaluated using flow cytometry. Long-term behavioral tests included the Barnes maze (days 7 and 30), novel object recognition (day 10), and object location tests on day 20. A second cohort was euthanized on day 7 for brain-infiltrated immune cell analysis.

Results:
At 24 hours post-MCAO, brain α7nAChR expression decreased significantly without changes in acetylcholine levels. WT MCAO mice showed reduced microglia, increased microglial TNF-α expression, and fewer α7nAChR-positive microglia compared to shams (p<0.05). In the spleen, no difference in F4/80 positive macrophages was observed, but macrophage TNF-α expression increased, and α7nAChR-positive macrophages decreased in WT MCAO mice (p<0.05). By day 7 post-MCAO, α7nAChR KO mice had more infiltrated myeloid cells and showed poorer performance in novel object and location tests than WT MCAO mice. However, no significant differences were found in the Barnes maze escape latency or incorrect entries between groups on days 7 and 30.

Conclusion:
α7nAChR plays a beneficial role in maintaining long-term functional outcomes after stroke in young animals.
Abstract TP371: Effects of Empagliflozin on Ischemic Stroke in a Rat Model: Time-Frequency Electroencephalogram Features and Cerebral Infarction Size
Authors: Li Jiajun , Alavi Rashid , Dai Wangde , Carreno Juan , Pahlevan Niema , Arakaki Xianghong , Gharib Morteza , Kloner Robert
Published: January 2025

Background:
Emerging evidence suggests that empagliflozin (EMPA) may offer additional benefits beyond its primary use in diabetes. Ischemic stroke is known to cause distinctive alterations in EEG signals. However, the influences of EMPA on the stroke-induced EEG changes has not been investigated. Here, we study such influences via EEG time-frequency features using the Hilbert-Huang Transform (HHT) and examine how these features correlate with the cerebral infarction.

Methods:
n= 47 male SD rats were randomized into 3 groups: 1] Control (n = 16, regular diet); 2] Acute EMPA treatment (n = 16, EMPA 10 mg/kg, IV given at 10 mins prior to middle cerebral artery occlusion (MCAO) and 1 min prior to reperfusion); 3] Chronic EMPA treatment (n = 15, EMPA by food, 20 mg/kg for 7 days before MCAO). To induce ischemic stroke, standard MCAO was performed for 1 hour followed by 3 hours of reperfusion. Post-surgery, triphenyltetrazolium chloride (TTC) staining was used to measure volume of cerebral infarction. EEG signals were continuously recorded throughout the procedure. We applied the Hilbert-Huang Transform (HHT) to analyze the EEG signals. HHT includes 2 steps: 1] empirical mode decomposition (EMD) to decompose the EEG signal into a set of intrinsic mode functions (IMFs); 2] the Hilbert Transform is applied to each IMF to compute analytic signals. Such analytic signals are represented in the complex plane, where they often form circular or elliptical contours (Fig. 1). The area of these contours in the complex plane contains unique information about variations in the signal properties. We computed the HHT area metric for the first four IMFs by using EEG recordings at 3 hours post-reperfusion (duration: 2 minute).

Results and Conclusions:
Our results showed the control group demonstrates higher correlations between the HHT area metric and the cerebral infarction size, compared to acute or chronic EMPA treatments (Fig. 2), with an especially significant correlation for IMF1in controls (R= -0.76). No significant difference was found in the HHT area metric between control, acute, and chronic EMPA (Fig. 3). It revealed that while EMPA treatments do not alter the EEG time-frequency features compared to controls, control group exhibits a stronger correlation between EEG features and cerebral infarction size. This indicates that EMPA's neuroprotective effects might not be directly reflected in EEG changes, highlighting the need for further investigation of its mechanisms.
Abstract TP372: Establishing a Physiologically Variable Model of Ischemic Stroke to Recapitulate Patient Heterogeneity
Authors: Schappell Laurel , Zoumpanidopoulos Eleftherios , Varghese Jeremiah , Polizu Claire , DiPersio James , Tang Meiyi , Madeira Miguel , Tsirka Stella , Nadkarni Neil
Published: January 2025

Introduction:
Ischemic stroke is highly heterogeneous, with patient-to-patient differences in infarct location, severity, and degree of reperfusion, among other factors. Incorporating this variability in preclinical stroke models is useful to more comprehensively recapitulate the patient population. Prior studies employ Longa (complete reperfusion) or Koizumi (chronic hyporeperfusion) murine models of stroke interchangeably over a range of ischemic durations. These models represent distinct stroke phenotypes, but the unique features of each model warrant further definition. Therefore, we sought to differentiate the pathology of these models to more accurately model patient heterogeneity.

Methods:
Transgenic mice expressing a fluorescent neutrophil marker (Ly6G-TdTomato) were subject to the Longa or Koizumi temporary middle cerebral artery occlusion (tMCAO) model of ischemic stroke. Ischemic duration was varied to model strokes of mild (30 min), moderate (60 min) and high (90 min) severity (n = 6-10 per experimental model). Laser speckle contrast imaging (LSCI) was performed at baseline, prior to and after reperfusion, and at endpoint to quantify cerebral blood flow (CBF) using a custom-made pixel assignment algorithm. Brains were collected at 24h or 72h and imaged via confocal microscopy to evaluate neutrophil infiltration in the ischemic hemisphere.

Results:
Mortality significantly increased with ischemic duration in the Koizumi model at 24h, 48h, and 72h, but did not differ across the Longa models. LSCI demonstrated a 2-to-5-fold increase in the area of profound ischemia (lowest quintile of pixel values) in the Koizumi model compared to the Longa model and with increasing ischemic duration. Additionally, the Koizumi model exhibited less restoration of CBF following reperfusion and more variability in CBF within the ischemic hemisphere regardless of ischemic duration. The number of infiltrating neutrophils increased 10-fold between the 30- and 90-min Longa and Koizumi models, but the Koizumi model demonstrated increased variability in the number of neutrophils at 24h and 72h.

Conclusions:
Our findings demonstrate differences in mortality, CBF, and immune response between stroke models of varying ischemic duration and reperfusion status. These unique pathological features can be linked to the physiological perturbations defining each model to establish a phenotypic spectrum that can be exploited to more thoroughly represent human stroke patient heterogeneity.
Abstract TP373: Impaired resting state functional connectivity in CADASIL mutant mice
Authors: Aykan Sanem A , Lai James Han , Sugimoto Kazutaka , Chung David , Aykan Orhan , JOUTEL ANNE , Sakadzic Sava , Ayata Cenk
Published: January 2025

Objectives:
Cerebral autosomal-dominant arteriopathy, subcortical infarcts, and leukoencephalopathy (CADASIL) is a leading monogenic cause of cerebral small-vessel disease. This study aimed to evaluate the functional integrity of long white matter tracts in CADASIL transgenic mice and assess the potential of tocotrienol (T3) as a therapeutic agent.

Methods:
Optical resting-state functional connectivity imaging and behavioral examinations were employed to characterize white matter tracts in CADASIL transgenic mice, both with and without focal white matter lesions in the corpus callosum. Functional connectivity and behavioral outcomes were compared between Notch3
R169C
and Notch3
WT
mice. The efficacy of T3, a neuroprotective vitamin E derivative, was also evaluated based on findings from previous clinical trials in small vessel disease.

Results:
After performing stepwise elimination using the Akaike Information Criterion (AIC), the diet variable was removed from the model and we pooled the data in the two diet arms for subsequent analyses of interhemispheric homotopic connectivity. At baseline, resting-state interhemispheric functional connectivity was significantly reduced in Notch3
R169C
mice compared to Notch3
WT
mice (p=0.004). No significant differences between genotypes were observed on day 1 and day 7 post-lesion. Behavioral analysis using the grid walk test revealed an increased number of foot faults in Notch3
R169C
mice at baseline, with similar increases observed in both genotypes one week after lesion induction. Lesion volumes on day 7 did not differ between genotypes.

Conclusions:
The Notch3
R169C
CADASIL model demonstrated impaired resting-state functional connectivity and increased foot faults, indicating significant functional and behavioral deficits. Future research will focus on evaluating therapeutic and preventive interventions in CADASIL models using these parameters.
Abstract TP374: Spleen Immune Cell Changes in Stroke and Hyperglycemia: Insights from Flow Cytometry
Authors: Chen Hansen , Chiang Terrance , Cheng Michelle , Bliss Tonya , Steinberg Gary
Published: January 2025

Introduction:
The cross-talk between the brain and peripheral immune organs plays a crucial role in the response to stroke injury. Spleen responses are implicated in stroke pathology and inflammatory responses. Hyperglycemia is known to worsen stroke outcomes, with increased immune cell infiltration into the brain. However, it remains unclear whether hyperglycemia exacerbates spleen immune cell responses and whether this contributes to the heightened immune response in hyperglycemic stroke.

Method:
Male C57/BL6 mice (10-12 weeks) were subjected to transient middle cerebral artery occlusion (MCAO) for 30 minutes, followed by reperfusion to mimic ischemic stroke. Acute hyperglycemia was induced by glucose injection 10 minutes before MCAO. The study included three groups: sham, MCAO only, and MCAO with hyperglycemia (n=6-7 per group). At 24 hours post-stroke, spleen and brain immune cell populations were analyzed using our established 12-color flow cytometry technique, which simultaneously analyzes myeloid and lymphoid subpopulations.

Results:
Compared with sham controls, stroke significantly decreased the numbers of dendritic cells (2.33 ± 0.48 vs. 1.50 ± 0.42 million; p<0.05), NK cells (2.30 ± 0.37 vs. 1.21 ± 0.37 million; p<0.01), Ly6C+ macrophages (0.29 ± 0.12 vs. 0.11 ± 0.06 million; p<0.01), and total lymphocytes (56.9 ± 16.6 vs. 37.0 ± 5.1 million; p<0.05) at 24 hours post-stroke in the spleen, whereas there were no significant changes in neutrophils and Ly6C+ monocytes cell counts. Hyperglycemia during stroke did not alter spleen immune cell numbers compared to stroke alone. However, hyperglycemia significantly increased brain-infiltrating immune cells at 24 hours after stroke, including neutrophils, B cells, and CD8+ T cells compared to normoglycemic mice.

Conclusion:
This study provides insights into spleen immune cell responses to acute ischemic stroke and hyperglycemia. Significant changes in spleen immune cell populations after stroke confirm their potential roles in stroke pathology and recovery, warranting further investigation. Acute hyperglycemia, however, does not significantly affect spleen immune cell numbers after stroke, implying that the spleen is not the primary target of hyperglycemia-exacerbated immune cell infiltration into ischemic stroke brains. These results pave the way for exploring other mechanisms driving immune cell infiltration under hyperglycemic conditions.