bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract WP358: A small peptide eCIRP inhibitor attenuates neuroinflammation and reduces brain injury in ischemic stroke.
Authors: Lapin Dmitriy , Sharma Archna , Wang Ping
Published: January 2025

Introduction:
Ischemic stroke (IS) is the 5
th
leading cause of death in the USA, but only one FDA approved drug exists. IS causes widespread neuron death and inflammatory injury. Microglial efferocytosis, the process to remove dead cells, limits inflammation and enhances brain recovery. Extracellular cold-inducible RNA-binding protein (eCIRP) is a pro-inflammatory alarmin that is deleterious in IS, increases infarct volume, neuroinflammation and microglial activation. eCIRP decreases efferocytosis and induces pro-inflammatory micro-RNA 155 (miR-155) in IS by binding to TLR4 on microglia. Our objective is to develop C23, a small peptide inhibitor of eCIRP/TLR4 binding, in a murine model of IS to reduce neuroinflammation and improve efferocytosis and IS outcomes.

Methods:
Primary microglia were isolated from mouse pups, seeded, and treated with eCIRP with or without C23, lysed for RNA, and RT-qPCR measurement of micro-RNA. 8-10-week-old male C57BL/6 mice, were subjected to transient middle cerebral artery occlusion (tMCAO) model of IS for 1 h, followed by reperfusion and retroorbitally injected with either normal saline (vehicle) or C23 (10 µg/g b.w.). 24 h post-reperfusion, behavior was scored, mice were sacrificed and tissues were collected for infarct volume measurement, immunofluorescence microscopy, or miRNA quantification.

Results:
As summarized in Table 1, eCIRP treated microglia upregulated miR-155 by 29-fold compared to controls, and pretreatment with C23 significantly attenuated eCIRP-induced miR-155 by 33%. Brain levels of miR-155 increased 1.8-fold in the vehicle tMCAO group vs. sham brains but were unchanged in the C23 tMCAO group. Similarly, plasma miR-155 levels increased 2.1-fold in vehicle tMCAO mice and returned to sham levels in C23 tMCAO mice. C23 treatment significantly increased efferocytosis in penumbra compared to vehicle in tMCAO brains (Fig. 1). Infarct volume was reduced in the C23 treated tMCAO group by 39% (100.3 mm
3
in vehicle tMCAO vs. 61.2 mm
3
in C23 tMCAO; Table 1&Fig. 2). Finally, C23 tMCAO mice had mild motor impairment with a mean Bederson score of 1.8 vs. severe impairment of vehicle tMCAO mice scoring 3.1 (Table 1).

Conclusion:
eCIRP induces pro-inflammatory miR-155 in primary microglia
in-vitro
and in tMCAO mice, and C23 attenuates this detrimental effect. C23 treatment improves microglial efferocytosis and reduces infarct volume and behavioral deficit in IS. Thus, C23 is a promising candidate therapeutic agent in IS.
Abstract WP360: The Detrimental Effects of Post-Stroke Social Isolation on Microglial Homeostasis are Mediated by MicroRNAs
Authors: Banerjee Anik , Mooz Patrick , Mohapatra Swati , Zhang Euan , Couture Lucy , Baskin Lynae , Lee Juneyoung , Fan Huihui , Ritzel Rodney , Venna Venugopal , McCullough Louise
Published: January 2025

Introduction:
Loneliness and social isolation (SI) are associated with both an increased risk of stroke and poor outcomes after stroke. However, the biological mechanisms that mediate how social factors affect stroke recovery are unknown. We hypothesized that the detrimental consequences of post-stroke SI result from differential miRNA expression that impacts neuroinflammation.

Methods:
To investigate the negative effects of post-stroke SI on brain miRNA profiles, aged (18–20 months) C57BL/6 male mice were utilized. Mice were randomly assigned to pair housing (PH) or single housing (SI) after middle cerebral artery occlusion (MCAO). We investigated the abundance of miRNA transcripts in isolated peripheral blood mononuclear cells (PBMCs) samples of lonely and socially integrated patients 24 hours after stroke (n = 140) using the UCLA loneliness and Lubben social interaction scales.

Results:
MiR-10a-5p, identified as a top differentially expressed target across mice and human-isolated stroke patients, was found to be a crucial node in the pool of miRNAs that interacted with the largest group of miRNAs for post-stroke at SI D4 in mice. At post-stroke, SI D4, significant microglial activation and suppression of cytokine production were observed as assessed by the median fluorescence intensity (MFI) of purinergic receptor P2Y12 (P2RY12), interleukin (IL)-4, IL-1β, and tumor necrosis factor (TNF)α, respectively, in microglial cells in the brain. The MFI of P2RY12 was significantly downregulated in post-stroke SI mice at D4 (n = 7-8/grp, *p<0.05) compared to PH mice. MFI of IL-4, IL-1β, and TNFα were significantly downregulated in post-stroke SI mice at D4 (n = 8–11/grp, *p<0.05, **p<0.01) compared to PH mice. Analysis of circulatory miRNA profiles revealed significantly altered sex-specific patterns of miRNAs (i.e., miR-24-3p, miR-143-3p, miR-223-5p, miR-192-5p, miR-331-3p, and miR-328-3p) (n = 40-50/grp, FDR adjusted *p<0.05) in isolated stroke patients.

Conclusions:
These findings validate our hypothesis that post-stroke SI exacerbates microglial activation, leading to differential expression of miRNAs that regulate microglial pathways. Further, alterations of sex-specific miRNA profiles in isolated post-stroke patients suggest its potential as a biomarker. Taken together, utilizing sex-specific miRNA-based therapies to restore miRNA profiles in the brain may be able to alleviate the detrimental impact of post-stroke SI on chronic depressive-like phenotypes.
Abstract WP368: Novel Brain Penetrant Manganese Porphyrin-Based SOD Mimic Improves Short-Term Outcomes of Subarachnoid Hemorrhage
Authors: Cisneros Oscar , Maeda Takuma , Uchikawa Hiroki , Nisson Peyton , Margaryan Tigran , Lawton Michael , Tovmasyan Artak , Hashimoto Tomoki
Published: January 2025

Introduction:
Oxidative stress plays an important role in both early brain injury and delayed cerebral ischemia after subarachnoid hemorrhage (SAH). Despite the beneficial effects demonstrated in preclinical studies with drugs targeting oxidative stress, their clinical translation has been hindered. There were critical issues in previous preclinical studies underpinning clinical translation, including inadequate demonstration of blood-brain barrier (BBB) penetration and insufficient assessment of drug concentration and biological activity in brain tissue. MnP-05 is a novel manganese porphyrin-based superoxide-dismutase mimic, which penetrates the BBB far better than previous compounds and exhibits superior anti-free radical properties compared to previous free radical scavenger. In this study, we evaluated the effect of MnP-05 on short-term outcomes of SAH in mice.

Methods:
We used 12-week-old male C57BL/6J mice. We induced SAH by an endovascular perforation of the right internal carotid artery. Mice (n = 31) were started on treatment with either MnP-05 or PBS 60 minutes after the induction of SAH. We set the dosing regimen as 1 mg/kg intravenous bolus followed by 5 mg/kg/day continuous intraperitoneal injection for 72 hours. We compared a composite neurological score, and rotarod performance (%baseline), between the two treatment groups for 7 days. We also compared the mRNA expression of oxidative stress and apoptosis markers in perihematomal brain tissue between the groups.

Results:
There were no specific side effects found in MnP-05 treatment group. There were no differences in blood pressure or body weight between the two groups. Seven-day neurological outcomes were better in MnP-05 group as evidenced by the significantly better composite neurological scores (22.9 vs. 19.4, P<0.05, Fig. A). Mice treated with MnP-05 showed better rotarod performance compared to vehicle group on day 7 (227% vs. 120%, P<0.01, Fig. B). MnP-05 significantly reduced the mRNA expression of oxidative stress markers (HO-1 and NRF) and apoptosis markers (Caspase-3 and BAX) compared to vehicle group (P<0.05, respectively, Fig C).

Conclusion:
MnP-05 improved short-term outcomes of SAH without specific side effects by reducing oxidative stress and neuronal apoptosis at the target site. In further studies, we will test how MnP-05 affects long-term outcomes of SAH. Our results serve as a basis for future clinical trials using MnP-05 to improve the neurological outcomes of SAH.
Abstract WP37: Predictors of Perioperative Stroke in Patients with Ischemic-type Moyamoya Disease Treated with Surgical Revascularization: A retrospective multicenter study
Authors: Musmar Basel , Roy Joanna , Abdalrazeq Hammam , Atallah Elias , El Naamani Kareem , chen jared , Jabre Roland , Saad Hassan , Grossberg Jonathan , Dmytriw Adam , Patel Aman , Khorasanizadeh Mirhojjat , Ogilvy Christopher , Thomas Ajith , Monteiro Andre , Siddiqui Adnan , Cortez Gustavo , Hanel Ricardo , Porto Guilherme , Spiotta Alejandro , Piscopo Anthony J. , Hasan David , Ghorbani Mohammad , Weinberg Joshua , Nimjee Shahid , Bekelis Kimon , Salem Mohamed , Burkhardt Jan-Karl , Zetchi Akli , Matouk Charles , Howard Brian , Lai Pui Man Rosalind , Du Rose , Abbas Rawad , Sioutas Georgios S , Amllay Abdelaziz , Munoz Alfredo , Herial Nabeel , Tjoumakaris Stavropoula , Gooch Michael , Rosenwasser Robert , Jabbour Pascal
Published: January 2025

Introduction:
Moyamoya disease (MMD) is a chronic cerebrovascular disorder characterized by progressive stenosis or occlusion of the internal carotid arteries and the development of collateral moyamoya vessels. Surgical revascularization is commonly used to prevent future ischemic events in ischemic-type MMD, but there remains a high rate of stroke perioperatively. This study aims to analyze the predictive factors for perioperative stroke in patients with ischemic-type MMD undergoing surgical revascularization using a large multicenter database.

Methods:
We conducted a multicenter retrospective study in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines. This study included patients with ischemic-type MMD who underwent surgical revascularization across 13 academic institutions in North America. Data were collected and analyzed on a per-hemisphere basis, covering patient demographics, disease characteristics, procedural details, and outcomes. Statistical analyses were performed using Stata (V.17.0), comparing baseline characteristics, and using univariable and multivariable logistic regression to identify predictors of perioperative stroke.

Results:
A total of 301 patients with ischemic-type MMD underwent surgical revascularization, with 34 patients (11.3%) experiencing perioperative stroke. Patients who experienced perioperative stroke had a mean age of 43.6 years (SD 14.0) compared to 40.0 years (SD 13.9) in those without perioperative stroke (P=0.16). Hypertension was significantly more prevalent in the perioperative stroke group (73.5% vs. 47.9%, P=0.005). Smoking was also more common in the perioperative stroke group (55.8% vs. 38.2%, P=0.04). Multivariate logistic regression identified hypertension as a significant independent predictor of perioperative stroke (OR 2.4, 95% CI 1.06 to 5.45, P=0.03).

Conclusion:
Hypertension is a significant predictor of perioperative stroke in patients with ischemic-type MMD undergoing surgical revascularization. Further prospective studies are needed to validate these findings.
Abstract WP374: Comparative Analysis of Human Microbiome in Acute Ischemic Stroke Patients
Authors: Kim Jun Yup , Kang Jihoon , Guk Hyung Seok , Kang Dong-wan , Jeong Han-gil , Han Moon-Ku , Lee Si Un , Lee Sang Hyo , Choi TaeWon , Oh Chang Wan , Lee Hyo-Jung , Kim Keun-Suh , Jeon JinSue , Jung Harry , Park Hyun Bong , Kim Woo Jin , Kim Dongin , Cho Heui Kyoung , Jeon Hyeong Kyu , Yun Ki Na
Published: January 2025

Introduction:
The human microbiome has been studied in various diseases, including inflammatory bowel disease, diabetes, obesity, and cardiovascular diseases. However, its role in acute ischemic stroke (AIS) remains underexplored. This study aims to investigate the microbiome profiles in stool and saliva samples from AIS patients compared to control groups to identify distinct microbial patterns associated with ischemic stroke.

Methods:
We enrolled 54 AIS patients, collecting stool and saliva samples within one week of admission. Saliva was either self-expectorated (n=44) or obtained via oral swab (n=10) for those unable to spit. The 40 control group was defined as those without cerebrovascular disease, in whom no abnormalities were found on brain MRI/A taken for health check-up purposes within the past year. DNA extraction and 16S rRNA gene sequencing were performed, and data were analyzed using QIIME 2 for diversity and taxonomy, with Linear discriminant analysis Effect Size (LEfSe) for differential abundance and functional predictions.

Results:
AIS patients demonstrated significantly higher alpha diversity in stool samples compared to controls (
p
=0.001), indicating increased microbial richness. Saliva samples, however, showed decreased microbial richness in AIS patients (
p
=0.024). Beta diversity analysis revealed distinct microbial community structures between AIS patients and controls, especially at the genus level in stool samples (
p
=0.001). LEfSe analysis identified several bacterial taxa enriched in AIS patients' stool, while saliva samples from AIS patients exhibited a higher number of depleted taxa compared to controls. Notably, the AIS microbiome showed reduced functional capabilities related to beneficial metabolic processes including
Proteobacteria, Gammaproteobacteria, Enterobacteriaceae
, and
Shigella, etc.
These findings highlight a potential link between microbiome dysbiosis and the pathophysiology of AIS.

Conclusions:
This study identifies significant microbiome dysbiosis in AIS patients, characterized by altered diversity and bacterial composition in stool and saliva samples. The findings suggest that gut and oral microbiomes may contribute to the pathophysiology of cerebral infarction, warranting further investigation into their roles as potential biomarkers or therapeutic targets.
Abstract WP376: Selective deletion of endothelial Piezo1 results in transcriptional changes in multiple cell types in the brain
Authors: WU TING , Doan Andrea , Andersohn Alexander , Kim Gab Seok , Chittet Elsa , Marrelli Sean
Published: January 2025

Introduction:
Piezo1 is a mechanosensitive ion channel that gates calcium and sodium influx in response to membrane deformation or elevated shear stress. We previously found that the chronic loss of endothelial Piezo1 leads to reduced cerebral blood flow, brain inflammation, and alterations in neurobehavioral tests. The present study was designed to better define the functional role of endothelial Piezo1 by determining the changes in the brain transcriptional landscape in response to selective Piezo1 loss-of-function in endothelial cells (EC).

Methods:
EC-specific inducible Piezo1 loss-of-function (LOF) mice (Cdh5(Pac)CreERT2;Piezo1 fl/fl) or control mice (Piezo1 fl/fl only) were injected with tamoxifen (75 mg/kg). After one month, brains were harvested for single-cell RNA sequencing (scRNA-seq) (10X Genomics) and analysis (SeqGeq V1.7, BD Biosciences).

Results:
We observed a broad differential distribution pattern across 10 principal brain clusters between control and EC Piezo1 LOF groups. Gene ontology (GO) analysis showed neuron, microglia, and astrocyte clusters associating with locomotory behavior, learning and memory, and gliogenesis. The EC cluster was linked to gliogenesis, while pericyte and mixed oligodendrocytes/ependymal cell clusters linked with GO terms associated with learning and memory. To explore whether EC Piezo1 LOF was linked to the emergence of specific endothelial cell population, we further sub-clustered the EC population. We found an endothelial cell subtype defined by
Igf1r
+ and
Ptprg
+ that was significantly increased (+36.9 %) in the EC Piezo1 LOF brain. Meta-analysis of DEGs in this population revealed their involvement in cell junction organization, fluid shear stress, atherosclerosis, and reactive oxygen species metabolism, while GO analysis highlighted "multicellular organismal response to stress" encompassing genes related to locomotory behavior, learning, memory, and cognition, and fear response behavior. Lastly, this EC population showed an association with the reactive oxygen species metabolic process and the Hif1 signaling pathway.

Conclusions:
In addition to promoting reduced cerebral perfusion, the specific loss of endothelial Piezo1 function causes widespread transcriptome alterations within the brain. Within the EC population, Piezo1 LOF results in a notable stress response with links to organismal dysfunction. These studies emphasize the functional importance of EC Piezo1 signaling in brain health.
Abstract WP379: Small Molecule Activator of the Sarcoendoplasmic Reticulum (ER) Ca
(2+)
-ATPase2 (SERCA2) Promotes Neuroprotection against Ischemic Stroke
Authors: Salman Mohd , Majid Abdul , Modaresi Mehrdad , Lebeche Djamel , Ishrat Tauheed
Published: January 2025

Background:
Ischemic stroke is a leading cause of high mortality and long-term disability. The current therapies for acute ischemic stroke remain limited to thrombolysis or thrombectomy, for which many patients are ineligible. Therefore, developing new treatments for stroke is a significant clinical need. Dysregulated calcium (Ca
2+
) homeostasis induces endoplasmic reticulum (ER) stress, mitochondrial dysfunction, microglial activation, inflammation, oxidative stress, and synaptic deficits implicated in stroke. Our recently developed molecular allosteric activator (CDN1163) of sarcoendoplasmic reticulum Ca
2+-
ATPase (SERCA2) plays a vital role in calcium homeostasis, alleviates ER stress and has emerged as a potential therapeutic approach for metabolic disorder and brain protection. Thus, this study aimed to investigate the protective role of CDN1163 against ischemic stroke in mice.

Methods:
Ischemic stroke was induced in adult male mice using the Rose Bengal photothrombosis method. CDN1163 (50mg/kg) was administered intraperitoneally at specific time points following the stroke. After 72 hours, the Catwalk test was performed to assess gait impairments. The mice were then euthanized, and their brain tissues were harvested for further analysis, including the measurement of infarct volume, brain edema, and molecular analysis. Neuronal apoptosis was determined using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeled (TUNEL) staining.

Results:
CDN1163-treated mice showed a significant improvement in gait impairments and reduction in infarct volume and brain edema compared to the vehicle group. CDN1163 treatment also attenuated vascular damage by reducing immunoglobulin extravasation and restoring tight junction protein. In addition, CDN1163 treatment attenuated oxidative damage by reducing 4-hydroxynonenal and nitrotyrosine protein expression levels. Furthermore, the expression levels of apoptotic proteins and numbers of TUNEL-positive cells were significantly decreased in the CDN1163-treated group compared to the vehicle group (
Table 1
).

Conclusion:
Our results indicate that CDN1163 reveals neuroprotection through attenuating vascular damage, oxidative stress, and neuronal loss against stroke pathogenesis. Further investigations into the therapeutic effects of CDN1163 are needed to determine whether it can be an effective therapeutic agent for stroke.
Abstract WP388: Plasma Eicosanoids Are Associated with Incident Stroke Over Decades in Participants in the Atherosclerosis Risk in Communities (ARIC) Study
Authors: Simpkins Alexis , Norby Faye , Alotaibi Mona , Yu Bing , Zhang Michael , Wang Wendy , Chen Lin , Fornage Myriam , Gottesman Rebecca , Cheng Susan
Published: January 2025

Introduction:
Eicosanoids (EIC) are lipid mediators that play a systemic role in inflammation and, more specifically, in the brain, contribute to the process of neurovascular coupling. Though EICs have been associated with immediate stroke risk and stroke risk factors, the long-term risk and associations with stroke risk factors have not been previously studied.

Methods:
Plasma EICs (214) were measured using non-targeted liquid chromatography mass spectrometry in conjunction with chemical networking of spectral fragmentation patterns among participants of the Atherosclerosis Risk in Communities Study at visit 2 (1990-92). Incident stroke (ischemic and hemorrhagic) was ascertained until the end of 2021 with adjudication by an expert panel. Cox regression was used to estimate the hazard ratio (HR) for incident stroke per doubling of EIC levels, and p-values were FDR corrected. Primary model 1 adjusted for age, sex, race/center and additional models adjusted for stroke risk factors (eGFR, BMI, hypertension, diabetes, current smoking status, total cholesterol, prevalent atrial fibrillation, prevalent heart failure, prevalent coronary heart disease, aspirin use, cholesterol-lowering medications, anticoagulation medication use, time-dependent incident atrial fibrillation and incident anticoagulant use).

Results:
Among the 9,444 participants included, the mean age was 57 (5.7 SD), and 43.2% were men. Over a mean of 22.7 (8.5 SD) years of follow-up, 950 participants had an incident stroke. In model 1, higher levels of 9 EICs were associated with a higher risk of incident stroke (11t LTD4 [HR 1.21, p=7.4E-05], 5S HpEPE [HR 1.21, p=0.005], LXA5 [HR 1.15 p=0.006], 13 HpODE_a [HR 1.17 p=0.009], 17S HpDHA_a [HR 1.16, p=0.018], 15 oxoEDE_a [HR 1.29 p=0.03], 11, 12 di-HETrE_a [HR 1.21 p=0.03], Arachidonic Acid_a [HR 1.17 p=0.03], and 5S HpETE_c [HR 1.18 p=0.04]; and 3 EIC analytes were associated with lower risk of incident stroke (PGF1a_b [HR 0.27 p=0.006], 13, 14 dihydroPGF1a [HR 0.37 p=0.02], 13,14 dihydroPGE1_b [HR 0.5 p=0.03]. Associations did not reach FDR-corrected significance after adjusting for risk factors.

Conclusion:
Our analysis of this large longitudinal community-based study demonstrates that the risk of incident stroke over decades is associated with alterations in the balance of increasing pro-inflammatory EICs likely interrelated with the presence of stroke risk factors over time and interestingly including an EIC associated with neurovascular coupling.
Abstract WP390: Induced Subarachnoid Hemorrhage Leads To Cortical Infarcts In a Mouse Model Of Cerebral Amyloid Angiopathy
Authors: Adetunla Damilola , Kim Sara Eugene , Vytla Sai Soumya , Lyle London , Maresco Lee , van Veluw Susanne , Kozberg Mariel
Published: January 2025

Background:
In cerebral amyloid angiopathy (CAA), amyloid-β accumulates within blood vessel walls, leading to hemorrhagic lesions such as convexity subarachnoid hemorrhage (cSAH), microbleeds, and lobar intracerebral hemorrhage. Cortical superficial siderosis (cSS), a chronic result of cSAH, is marked by iron-positive hemosiderin deposits within the superficial cortex and leptomeninges. cSS is associated with an increased risk of cortical microinfarcts, which may contribute to cognitive decline in CAA-related cSS. However, the mechanisms linking cSS to microinfarcts remain unknown. To explore this, we developed a subarachnoid hemorrhage model in the APP23 transgenic (Tg) mouse model of CAA. We hypothesized that mice with subarachnoid hemorrhage would exhibit more infarcts compared to controls, and that infarcts would occur more frequently in Tg mice than in wildtype (WT) littermates.

Methods:
After skull and dura removal, cranial windows with custom silicone ports were implanted in 11-month-old APP23 Tg and WT mice. One-month post-surgery, 10 µL donor arterial blood or sterile phosphate-buffered saline (PBS) was injected onto the cortex (blood: n=7 WT, 6 Tg; PBS: n=8 WT, 6 Tg). Mice were euthanized one-month post-injection, perfused, and brains were analyzed ex vivo. Serial brain sections were stained with H&E and for iron, and high-resolution images were acquired with a NanoZoomer slide scanner. Infarcts were first qualitatively analyzed by a blinded observer. QuPath, an open-source bioimage analysis software, was then used to quantify infarct volume and iron deposits in cortical tissue. The software was trained with pixel-level annotations to distinguish between healthy brain tissue and ischemic regions as well as to identify iron deposits.

Results:
Preliminary analysis revealed hemosiderin deposition in mice that received blood injections, similar to human cSS, and more iron deposits were observed in these mice compared to PBS controls. Qualitative analysis identified more cortical infarcts in regions with hemosiderin than in those without in individual mice. The results of ongoing analyses, including quantitative comparisons between infarcted tissue and iron deposition, will be presented.

Conclusion:
This study aims to establish a foundation for quantitatively assessing ischemic tissue and iron deposition in this novel mouse model of cSS, which may be useful for exploring underlying mechanisms and testing future interventions.
Abstract WP394: Homocysteine induces neurovascular injury and increases BBB permeability
Authors: Deng Wenjun , Yin Shanye , Wang Andrew , Chou Richard , Jacobson Jared , Wang Grace , McMullin David , Buonanno Ferdinando , Lo Eng , Ning MingMing
Published: January 2025

Background:
Our recently reported that patent foramen ovale (PFO) shunt enables the elevation of total homocysteine (tHcy) in patients’ circulation (Deng, Neurology 2021). However, the underlying molecular mechanism of how tHcy causes PFO-related injury is unclear. Considering that brain vascular endothelial cells are among the first to respond to the alterations in bloodstream, we hypothesized that elevated tHcy acts on brain endothelial cells, opens blood brain barrier (BBB) and leads to further neurovascular injury.

Method:
Brain microvascular endothelial cells were exposed to high concentration of tHcy for 72 hr and subjected to RNA sequencing analysis. The viability of the cells was evaluated using MTT method. Additionally, mass spectrometry was employed to measure tHcy levels in both plasma and cerebrospinal fluid (CSF) samples collected from 56 patients with cerebrovascular disease. BBB permeability was assessed by CSF/plasma albumin ratio, a highly reliable clinical indicator of BBB integrity.

Result:
Gene set enrichment analysis (GSEA) revealed significant activation of apoptosis in brain endothelial cells following exposure to high tHcy levels (NES: 1.80; p < 0.001) (Figure 1A). Many pro-apoptotic factors, such as Bax, Bak and FAS, were upregulated in response to tHcy (Figure 1B). Consistent with this, cell viability decreased with increasing tHcy concentrations (Figure 1C), suggesting that tHcy induces damages to brain endothelial cells. Supporting this, plasma tHcy levels in stroke patients were well correlated with those in CSF (r = 0.46, p = 0.001) (Figure 1D). Patients with high tHcy in plasma (> 12 μM) demonstrated increased BBB permeability (Figure 1E).

Conclusion:
Our findings suggest that elevated circulatory tHcy may injure brain endothelium and increase BBB permeability, potentially contributing to downstream neurovascular dysfunction. The impact of tHcy on BBB and other brain cell types are ongoing to explore its important role in PFO related neurovascular injury.
Abstract WP398: Synaptic origin of axonal injury in the mouse stroke model
Authors: Park Yujung , Osterli Emily , Yoon Jae Hyun , Al Nasser Hanoaf , Liu Chunli , Hu Bingren
Published: January 2025

Background:
White matter or axonal injury is a key predictor of long-term functional outcome following ischemic stroke in humans. However, this type of injury is significantly understudied in animal stroke research. The objective of this study aims to investigate the cause and origin of axonal injury in a mouse stroke model.

Methods:
Mice were subjected to sham surgery (sham n = 14) or 40 min middle cerebral artery occlusion (MCAO), followed by 4 h (n=5), as well as 1 (n=5), 3 (n=5), 7 (n=5), and 21 (n=5) days of reperfusion, respectively. The regional cerebral blood flow (rCBF) was monitored with a laser doppler flowmetry (LDF) needle probe and maintained below 10% of the pre-ischemia level via, when needed, adjusting the monofilament occluder position during the MCAO period. Rectal temperature was maintained at 36.5 +/- 0.5
o
C during the peri-MCAO periods. The swollen axon segments or dystrophic axons were used as indicators of axonal injury and were identified with antibodies against axon-specific proteins under confocal microscopy. The following categories of antibodies were used to identify the origin of these swollen or dystrophic axons after MCAO: (i) axonal markers; (ii) synapse markers; (iii) endolysosomal, mitochondrial, and endoplasmic reticulum (ER) markers, respectively; (iv) autophagy-specific markers; (v) synaptic adhesion molecules; and (vi) oligodendrocyte, myelin sheet, microglial, and astrocyte markers, respectively.

Results:
All axon-related antibodies used in this study could identify both normal axonal and dystrophic axonal segments. Synaptic cell adhesion molecules were significantly reduced in the stroke area probably due to stroke-induced postsynaptic neuronal death/damage. Most dystrophic axonal segments were co-labeled with antibodies against synapse-specific markers. In contrast, only a small fraction of dystrophic axonal segments were co-labeled with antibodies against endolysosomal, mitochondrial, or other organelle markers. Meanwhile, the levels or activities of oligodendrocyte and microglial markers appeared upregulated in the area surrounding the dystrophic axonal segments following focal brain ischemia.

Conclusion:
The post-MCAO dystrophic axonal segments are mostly swollen presynaptic structures or boutons. This type of post-stroke axonal injury is likely a result of the lack of support from synaptic adhesion molecules due to post-synaptic neuronal death in the mouse model of focal brain ischemia.
Abstract WP399: Effects of NLRP3 inhibitor on cerebral ischemia in chronic cerebral hypoperfusion model
Authors: Matsuyama Hirofumi , Hitotsuyanagi Norihito , Nishigaki Akisato , UTSUNOMIYA TAKAYA , Mizutani Akane , Ishikawa Hidehiro , Wakita Hideaki , Shindo Akihiro
Published: January 2025

Background:
The neuroinflammation triggered by acute cerebral ischemia can be mediated through inflammasome such as NLRP3 (NOD-like receptor family, pyrin domain containing 3) and ASC (apoptosis-associated speck-like protein). The NLRP3 inflammasome complex triggers caspase1-mediated maturation of cytokine. Chronic cerebral hypoperfusion induces upregulation of NLRP3 inflammasome and neuroinflammation. This study was undertaken to test our hypothesis that chronic cerebral ischemia in triggered inflammasome-mediated neuroinflammation, resulting in increased chronic ischemic injury. Furthermore, we undertook this study to demonstrate that NLRP3 inhibitor treatment reduces inflammasome-derived neuroinflammation and white matter damage caused by chronic ischemia.

Methods:
We used eight-week-old male C.B-17/Icr-Jcl mice in this experiment. The mice were subjected to sham or bilateral common carotid artery stenosis (BCAS) operation using microcoils with an internal diameter of 0.18 mm. At 3weeks after BCAS, these mice were sacrificed and examined by immunohistochemistry and western blotting (n = 5 for each group). In a separate set of experiments, mice were administered with 40 mg/kg of a NLRP3 inhibitor or vehicle daily for 3 weeks after BCAS. As before, the NLRP3 inhibitor and vehicle groups were compared by immunohistochemistry and western blotting (n = 5 for each group).

Results:
Cerebral hypoperfusion in BCAS mice induce upregulation of NLRP3, ASC, and upregulation of inflammasome-dependent IL-1β in the splenium of corpus callosum. In immunoblots and quantification of inflammasome proteins, the expression of NLRP3, ASC and IL-1β in ipsilateral brain lysates was increased after BCAS compared to the sham surgery group. Furthermore, the results of immunoreactivity showed that NLRP3 inhibitor treatment for BCAS mice reduced upregulation of NLRP3, caspase1 and IL-1β.

Conclusions:
These results suggest that chronic cerebral hypoperfusion induces upregulation of NLRP3 in the white matter lesion, likely leading to an increase of IL-1β. We also identified NLRP3 inflammasome inhibition as a novel mechanism to protective against chronic ischemic damage, can become a potential clinical benefit of therapeutic interventions.
Abstract WP400: SARS-CoV-2 Spike Protein Accelerates Alzheimer’s Disease-Related Dementia Through Increased Cerebrovascular Inflammation in hACE2 Mice
Authors: Hillard Parker , Vu Kaitlyn , Hermanns Veronica , Hafez Sherif , Coucha Maha , Abdelsaid Mohammed
Published: January 2025

SARS-COV-2 causes neurological and cognitive impairments and aggravates Alzheimer’s Disease-Related Dementia (ADRD). Yet, the molecular mechanism is not fully understood. We have previously shown that SARS-CoV-2 spike protein disturbs the brain's renin-angiotensin system (RAS) and increases cerebrovascular inflammation. We hypothesize that SARS-CoV-2 spike protein will accelerate hypoxia-induced ADRD via augmenting cerebrovascular inflammation and impairing blood-brain-barrier (BBB) functions. We propose that the pharmacological restoration of the RAS balance using Losartan, an AT
1
receptor blocker, will improve SARS-CoV-2 spike protein-induced ADRD.

Methods:
Hypoxia-induced ADRD was produced in humanized ACE2 mice, a COVID-19 mouse model, using a permanent unilateral common carotid artery ligation (UCCL). Cerebral hypoxia was confirmed by laser speckle imaging. hACE-2 mice received either vehicle, SARS-CoV-2 spike protein via jugular vein, or spike protein with Losartan (10 mg/kg) in drinking water after UCCL. ADRD was assessed via Novel Object Recognition at baseline, seven days, and fourteen days after surgery. Cerebrovascular inflammatory markers and tight junction proteins (TNF-α, Il-6, VEGF, MMP-9, and occludin) were measured in brain homogenate using RT-PCR and Western Blots.

Results:
Blood flow analysis confirmed cerebrovascular hypoxia in all groups. Spike protein further decreased cerebral blood flow, which was prevented with Losartan (P<0.05). Spike protein significantly increased inflammatory markers and worsened BBB functions after UCCL, which were mitigated by Losartan (P<0.05). Spike protein aggravated cognitive impairment after UCCL, which was attenuated by Losartan (P<0.05).

In conclusion,
the SARS-CoV-2 spike protein accelerates Alzheimer’s Disease-Related Dementia through increased cerebrovascular inflammation and deterioration of BBB in hACE2 Mice. The AT
1
R blocker Losartan attenuated the detrimental effect of the spike protein by reducing cerebrovascular inflammation and improving cerebral blood flow.
Abstract WP54: Longitudinal Proteomics and Fatigue Assessment Demonstrates a Persistent Association Between Post-Stroke Fatigue and Stress Granule Related Proteins Years After Ischemic Stroke
Authors: Aslan Alperen , Tartici Alp , Chung Chastin , Xue Lei , Drag Lauren , Mlynash Michael , Zera Kristy , Aghaeepour Nima , Altman Russ , Allan Stuart , Smith Craig , Buckwalter Marion
Published: January 2025

Introduction:
Post-stroke fatigue is prevalent and significantly impacts quality of life chronically after stroke. Its underlying biological mechanisms remain largely unknown. Here we set out to understand the persistence of post-stroke fatigue and to use plasma proteomics to identify candidate mechanisms.

Methods:
We acquired neurocognitive assessments and blood draws from 250 stroke survivors from two sites at baseline (median 8 mo after stroke, range 5-120 mo), and 131 participants one year later. The Functional Assessment of Chronic Illness-Fatigue (FACIT) scale was used to assess fatigue, with no fatigue defined as FACIT>41, any fatigue as FACIT≤41, and severe fatigue as FACIT<31. We used binary logistic regression to examine associations with clinical variables. Aptamer based proteomics (Somalogic) was used to quantify 6344 proteins in 97 participants at baseline and 51 one year later. We employed machine learning with rigorous feature selection to predict FACIT from proteomics. We performed Gene Set Enrichment Analysis (GSEA) comparing no fatigue to any fatigue or to severe fatigue at both visits.

Results:
At the baseline visit, fatigue was present in 44% of our cohort with no difference between sites. 41% were female and the median age was 67 [IQR 58,74]. When adjusted for age, sex and NIHSS, diabetes (OR=8.4, 95% CI=3.1-22) and a prior diagnosis of cancer (OR=4, 95% CI=1.2-12) were associated with fatigue. If a patient had fatigue at baseline, they were more likely to have fatigue at year 1 (OR=32, 95% CI=10-97). Our machine learning model achieved high predictive performance of FACIT at baseline, with an AUROC of 0.81 for distinguishing fatigue and a Spearman correlation of 0.62 for direct FACIT score prediction. GSEA comparing no fatigue with either any fatigue or severe fatigue revealed fatigue-associated increases in pathways involved in stress granule formation and clearance (9 and 8 of the top 20 pathways, respectively). Excitingly, one year later 7 and 11 of the top 20 fatigue-associated pathways were also directly related to stress granules.

Conclusion:
We observed high prevalence and persistence of post-stroke fatigue and proteomics demonstrated associations with stress granules at two consecutive visits. Stress granules broadly dysregulate protein translation and are associated with both neurodegeneration and myopathy. Future studies will seek to replicate these findings, identify affected cell types, and define therapeutic targets.
Abstract WP55: Increasing White Matter Hyperintensity Volume After Ten Years Correlate with Declining Manual Dexterity
Authors: Sasannia Sarvin , Wang Shimeng , Matsyuk Mykola , Zhang Jinwei , Walker Keenan , Shin Hyeong-Geol , Leigh Richard , Prince Jerry , Becker Lewis , Yanek Lisa , Vaidya Dhananjay , van Zijl Peter , Knutsson Linda , Nyquist Paul
Published: January 2025

Introduction:
We previously reported an association between ischemic white matter hyperintensities (WMH) lesion volume on brain MRI and declining manual dexterity as measured by the grooved peg board test (GPT). Here we investigated this relationship longitudinally using repeated measures of total WMH, periventricular white matter (PVWMH) and subcortical deep white matter lesions (DWMH) volumes over ten years and compared them to changes in repeated GPT over 10 years.

Hypothesis:
We hypothesized that increases over time in total WMH, PVWMH, and DWMH volume over time would be associated with declining repeated GPT performance.

Methods:
We conducted a longitudinal study of 100 volunteers from GeneSTAR, an ongoing study of families enriched for vascular risk factors and cardiovascular disease, age = 63.3 ± 9.5 years, 60% females, 63% hypertension 22% African Americans. Participants underwent two 3T brain MRI lesion volume measurements and repeated manual dexterity testing using GPT at baseline and after 10–13 years. Total WMH, PVWMH, and DWMH volumes were quantified using automated segmentation software. GPT was calculated as the total time to complete the test with both dominant and non-dominant hand. Changes in total WMH, PVWMH, and DWMH, together with changes in GPT performance over time were analyzed using linear regression adjusting for age, systolic blood pressure, race, sex, and education.

Results:
For the dominant hand (Figure 1), changes in PVWMH volume (p = 0.028, R-squared = 0.167) and total WMH volume (p = 0.03, R-squared = 0.168) were significantly associated with changes in GPT performance. In contrast, changes in DWMH volume (p = 0.7, R-squared = 0.0878) were not associated with GPT performance. No significant association were found for the nondominant hand. (Figure 2)

Conclusions:
In middle-aged adults enriched for risk of for cardiovascular disease, progression of total WMH volume and PVWMH volume are correlated with declining manual dexterity. No significant correlation was observed for the non-dominant hand suggesting differences in the impact of WMH progression on motor function between dominant and non-dominant sides.

Future research should investigate whether interventions targeting vascular risk factors can slow WMH progression and associated motor decline in this at-risk population.
Abstract WP56: Dental Flossing associated with reduced White Matter Hyperintensity and Intracranial Atherosclerosis
Authors: Logue Makenzie , Martin Ansley , Suri Fareed , Wasserman Bruce , Rosamond Wayne , Moss Kevin , Marchesan Julie , Beck James , Sen Souvik
Published: January 2025

Introduction:
Prior studies have shown the association between oral infection, white matter hyperintensity (WMH), a marker of cerebral small vessel disease (CSVD), and asymptomatic intracranial atherosclerosis (ICAS). However, the impact of preventative oral behavior such as dental flossing is unknown. The relationship between flossing CSVD,&ICAS was tested using the Atherosclerosis Risk in Communities (ARIC) cohort study.

Methods:
3T MRI and MRA were performed in visit 5. CSVD was assessed using the log of WMH volume. WMH volume was derived from a semiautomated segmentation of FLAIR images, 3D time-of-flight MR angiogram and 3D high-isotropic resolution black blood MRI. ICAS was graded according to the criteria established by the Warfarin-Aspirin Symptomatic Intracranial Disease trial. In this study, we evaluated the relationship between flossing and ICAS, defined as ≥50% stenosis. Flossing was assessed by a medical history questionnaire performed in visit 4. Student t-tests were performed to test continuous variables, and X
2
test was used to compare categorical variables. Analysis of covariance (ANCOVA) was used for multivariate testing of continuous variables and multivariable logistic regression analyses for categorical variables.

Results:
Among subjects who underwent vascular imaging, 1033 (90%) had <50% ICAS and 112 (10%) had ≥50% ICAS. Dental flossing was significantly associated with lower rates of ICAS≥50% (Crude OR 0.59 95% CI 0.39-0.90). The association remained significant after adjustment for age, race, gender, hypertension, diabetes, smoking status, education level and regular dental care use (Adjusted OR 0.61 95% CI 0.38-0.97). The log WMH volume was higher (9.5±0.85 vs. 9.3±0.89), the difference being statistically significant (p=0.003). The difference remained significant after adjustment for age and SBP (p=0.002).

Conclusion:
We report a significant association between flossing, reduced WMH volume and ≥50% ICAS. This may be one of the first reported associations between dental flossing, and reduced WMH volume (a CSVD indicator) and moderately severe ICAS. These results emphasize the importance of dental flossing in reducing the rates of CSVD and moderately severe ICAS, both known risk factors for ischemic stroke.
Abstract WP72: A Stroke System’s Implementation and Analysis After Transitioning to Tenecteplase, a Novel Thrombolytic
Authors: Montgomery Kelly , Campbell Meghan , Fussner Julie , Traeger Jessica , Sarraj Amrou
Published: January 2025

Background:
Our system, comprised of 21 Northeast Ohio hospitals includes 3 Stroke Ready, 10 Primary and 1 Comprehensive Stroke Center (CSC). Alteplase was the standardized thrombolytic until 2022. Clinical trials support the efficacy and safety of Tenecteplase for the treatment of acute ischemic stroke (AIS). Tenecteplase administration is efficient with less room for error and is cost-effective. The transition from Alteplase to Tenecteplase for AIS patients eligible for thrombolysis occurred on June 6, 2022. Implementation transpired over 9 months.
Purpose:
To evaluate a health system’s experience transitioning to a novel thrombolytic.

Methods:
Stroke program and pharmacy stakeholders reviewed literature and agreed upon Tenecteplase 0.25mg/kg. The legal department approved as our new standard of care for AIS. Alteplase remained on formulary for pediatric stroke and other diagnoses. Guidelines and references were updated. A dosing table was created for rounding accuracy. Nurses were provided with dosing table and care instruction badge backers. Due to Tenecteplase package labeling for MI, supplies were repackaged into “TNK Kits”. Kits include a dosing table, visual step-by-step reconstitution instructions, 10mL syringe, 5mL syringe, 2 flush syringes and medication vials. EHR Alteplase references were changed to “thrombolysis”. Tenecteplase Stroke and MI order sets with auto-calculations were developed for safety. All impacted disciplines received education, including nursing skills demonstration. A retrospective review of system Alteplase administrations during 6/2020- 5/2022 was compared to Tenecteplase administrations throughout 6/2022- 5/2024.

Results:
Thrombolytic utilization increased from 464 patients (10.3%) treated with Alteplase versus 521 patients (12.3%) with Tenecteplase. The percentage of door to thrombolysis
<
30 minutes improved from 23.3% to 34.8% and
<
45 minutes from 60.4% to 73.2%. Door to puncture times for CSC direct arrivals that received Alteplase had a 93 minute median and CSC direct arrivals given Tenecteplase had a median of 82 minutes. Door to reperfusion median decreased by 23 minutes. The system Alteplase hemorrhagic complication rate was 2.8% compared to 2.5% for Tenecteplase. Current Alteplase wholesale cost is $10,560 and Tenecteplase is $8,854. Hence, a cost-savings of >$500,000 annually.

Conclusion:
Our system’s experience was successful in our goal to achieve high quality patient outcomes and improved care efficiency.
Abstract WP75: Evaluating the Efficacy of EMS Integration and CSC Triage in Improving Stroke Patient Outcomes
Authors: Ramage Michael
Published: January 2025

Background and Issues:
Timely diagnosis and treatment are essential for optimal outcomes in acute stroke care. The lack of EMS integration into the stroke system of care is leading to delays in treatment resulting in poor outcomes. The absence of centralized resources at the Comprehensive Stroke Center (CSC) results in delayed patient care and worsened outcomes.

Purpose:
This project aimed to enhance EMS stroke recognition and improve triage to appropriate stroke centers, thereby reducing treatment times. The successful integration of EMS with an optimized CSC triage protocol seeks to reduce hospital stays, mortality rates, direct costs, and long term disability.

Method:
Effective integration of EMS into the stroke system of care was achieved by developing triage protocols with local EMS leaders. Adoption of a standardized stroke triage assessment tool was implemented. Centralizing resources at the CSC was a multidisciplinary approach to streamline the identification and treatment of stroke patients in a definitive location near CT scan. Stroke triage education and mock drills were provided to local EMS agencies and the acute stroke team. Outcomes were measured using pre and post intervention data for TNK administrations and endovascular therapy.

Results:
Outcomes were measured in two groups of acute stroke patients who received TNK, mechanical thrombectomy, or both. 115 patients from 2022 prior to project initiation and 131 patients from 2023 after its initiation. TNK door-to-needle time decreased from 37 to 32 minutes. Thrombectomy door-to-revascularization time dropped from 118 to 85 minutes. Average hospital stay reduced from 8 to 7 days. Mortality rates fell from 13.04% to 3.94%. Direct costs decreased from $27,989 to $24,109. Thrombectomy mortality decreased from 17.14% to 6.06%. 90-day modified Rankin score 2 or less improved from 45% to 49%. Average EMS first contact to revascularization went from 152 minutes to 120 minutes. Average EMS first contact to TNK administration went from 70 minutes to 67 minutes.

Conclusion:
The implementation of standardized EMS triage protocols and centralized resources at the CSC led to significant improvements in acute stroke care. The interventions resulted in faster treatment times, reduced hospital days, lower mortality rates, decrease in direct costs, and improved long-term functional outcomes. These findings highlight the significance of EMS integration and resource centralization to improve patient outcomes.