Abstract WMP117: SARS-CoV-2 infection worsens neuroinflammation and brain senescence in an endothelial nitric oxide synthase deficient model of vascular dementia.

Stroke, Volume 56, Issue Suppl_1, Page AWMP117-AWMP117, February 1, 2025. Introduction:SARS-CoV-2 causes various neurological sequelae in COVID-19 survivors including fatigue and cognitive dysfunction. Endothelial dysfunction, a key mechanism in COVID-19 illness, is also a major risk factor for vascular dementia (VaD). Clinical evidence suggests that reduced nitric oxide (NO) bioavailability is a likely pathogenic factor of endothelial dysfunction in COVID-19 patients, and eNOS levels decline with advancing age, a risk factor for both COVID-19 morbidity and VaD . We hypothesize that endothelial nitric oxide synthase (eNOS) deficiency contributes to brain endothelial dysfunction in SARS-CoV-2 infection and that SARS-CoV-2 infection accelerates the onset of VaD in eNOS-deficient mice.Methods:6-month-old eNOS+/- (pre-cognitively impaired experimental VaD) and WT male mice were infected with 1X104-pfu mouse-adapted (MA10) SARS-CoV-2 intranasally, and animals were evaluated acutely out to 3-days-postinfection (3dpi) for changes in body weight and clinical signs of illness. Viral copy numbers and nuclear capsid were also quantified in lung and brain. Quantitative PCR and immunofluorescence were used to analyze markers of brain inflammation and senescence.Results:eNOS+/- infected mice exhibited more disease-associated weight loss (~15%) than WT-infected mice (~5 %). While infected WT and eNOS+/- had comparable pulmonary viral load, neither had detectable virus in the brain. Quantitative PCR analysis of whole brain-isolated mRNA showed increases in multiple proinflammatory mediators such as CCL2 and IL-6 and senescence markers such as p53 and p21 (eNOS+/- > > WT). Similarly, immunofluorescent analysis showed increased Iba1 (microglia marker) fluorescent intensity in the cortex of infected mice (eNOS+/- > > WT).Conclusions:eNOS+/- deficiency, a clinically relevant model of VaD, worsens acute SARS-CoV-2-associated morbidity, neuroinflammation and markers of brain senescence despite comparable pulmonary viral load (to WT-infected animals) and absence of virus in the brain. While the potential effects of SARS-COV-2 on cognitive decline in this model will be assessed in future studies, this is the first experimental evidence demonstrating a link between eNOS and neuropathology associated with COVID-19.

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Abstract 90: Astrocytic Cluster of Differentiation 38 Contributes to Hippocampal Synaptic Plasticity Deficits following Focal Cerebral Ischemia

Stroke, Volume 56, Issue Suppl_1, Page A90-A90, February 1, 2025. Introduction:Post-Stroke Cognitive Impairment (PSCI) is increasingly recognized as a major factor in long-term disability. Therefore, new therapies that enhance post-stroke brain function (plasticity) are appealing in translational research. Activation of the cation channel TRPM2 after middle cerebral artery occlusion (MCAO) induces PSCI-like symptoms, such as memory impairment and deficits in Long-Term Potentiation (LTP). Determining the mediator of chronic TRPM2 channel activation may implicate a novel contributor to LTP deficit. The enzyme CD38 may be this mediator, as it produces the TRPM2 activator ADPr. Here, we investigate astrocytic CD38’s role in MCAO and TRPM2-induced deficits.Methods:LTP was assessed through Extracellular Field Recordings from CA3-CA1 synapses of Acute Hippocampal Slices. Immunofluorescence stained for GFAP and CD38 in hippocampal sections. Wt or CD38-/-mice were subjected to 60-minute MCAO, or Sham, and assessed for LTP 7 days later. Wt or TRPM2-/-mice received Intracranial Administration (200nL, 1012gc/mL) of AAV-CD38-eGFP (astrocyte CD38 overexpression) or AAV-eGFP (control) to the right ventral CA1 and assessed for LTP after 21 days. All mice were 8-12-week-old males. Differences between groups were determined with either Welch’s T-Test or One-Way ANOVA followed by post-hoc test.Results:We observed increased expression of CD38, colocalized with GFAP (astrocyte marker), in the hippocampus 7 days post-MCAO. Sham Wt mice exhibited robust LTP, not observed in Wt MCAO (Wt-Sham: 176% +/- 33.9 vs Wt-MCAO: 135% +/- 23, p < 0.05). Conversely, CD38-/-mice displayed robust LTP after either Sham or MCAO, depicting protection of synaptic plasticity (CD38-/--Sham: 175.2% +/- 24.1, CD38-/--MCAO: 184.8% +/- 27.3). AAV-CD38-eGFP astrocytic overexpression vector produced impairment in LTP in the Wt mice compared to the empty vector control (Wt-eGFP: 195.6% +/- 46 vs Wt-CD38:141.5% +/- 38.7, p < 0.05). No differences were observed in TRPM2-/-mice injected with either virus. (TRPM2-/--eGFP: 180.3% +/- 39.4, TRPM2-/--CD38 174% +/- 54.1)Conclusions:Our study implicates astrocytic CD38 as a novel inducer of hippocampal synaptic plasticity impairment. CD38 is increased following injury and required for MCAO-induced LTP deficit. Additionally, overexpression is sufficient to impair LTP in uninjured mice, in a TRPM2-dependent manner. Astrocytic CD38 is a novel target for PSCI and the likely upstream mediator of chronic TRPM2 activation.

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Abstract TP127: Isolation and Stroke: A Retrospective Analysis of Outcomes Amidst Solitude

Stroke, Volume 56, Issue Suppl_1, Page ATP127-ATP127, February 1, 2025. Introduction:Stroke is the leading cause of long-term disability and fifth leading cause of death in the United States. Social isolation (SI) and loneliness are known risk factors for stroke and may be linked to worse functional outcomes. Previous animal studies have demonstrated enhanced outcomes associated with socialization. The impact of SI following stroke may be clinically relevant for therapeutic intervention.Hypothesis:Stroke patients experiencing social isolation will exhibit worse outcomes compared to those with social support networks.Aim:This retrospective analysis aims to compare stroke outcomes during the COVID-19 visitation restrictions with outcomes during the preceding years when visitation was permitted.Methods:Data were collected from the Patient Cohort Explorer, a de-identified database within our institution’s Research Data Warehouse. Patients were divided into two groups based on date: the isolation group and the control group. The control group included patients admitted from December 1, 2018, to January 1, 2019, during normal visitation policies, while the isolation group comprised patients admitted from December 1, 2020, to January 1, 2021, during visitation restrictions. Two-proportion Z-tests were conducted to analyze differences in demographic data, and two-sample T-tests were used to assess outcomes, including length of stay and discharge disposition.Results:A total of 725 unique patients met the inclusion criteria, with demographic characteristics such as sex and race well-matched between the isolated and control groups, except for a notable age difference (p-value .011). Significant differences in mortality rates were observed, with the control group showing a higher likelihood of returning home (p-value < .001) and the isolation group having a greater proportion of deaths (p-value .003). Additionally, there was a statistically significant difference in hospital stay length, with the control group able to discharge earlier (p-value .001).Conclusion:Social isolation can result in longer hospital stays, poorer outcomes, and increased mortality for patients with acute infarcts. Since the data was gathered during the COVID-19 pandemic, it's challenging to eliminate the virus as a confounding factor in these outcomes. However, the findings suggest that patients lacking social support may face worse functional recovery and different recovery trajectories compared to those with support.

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Abstract 159: Cerebral endothelial cells generate ceramides that worsen outcomes of acute ischemic stroke in mice.

Stroke, Volume 56, Issue Suppl_1, Page A159-A159, February 1, 2025. Acute ischemic stroke (AIS) elevates plasma ceramides that correlate with the prevalence and severity of neurological and motor deficits displayed by these patients. Our preclinical murine model of AIS [60-min transient middle cerebral artery occlusion (tMCAO) followed by 23-hr reperfusion (R)] increases ceramides (targeted mass spectrometry) in plasma and ipsilesional (ischemic) brain hemispheres compared to sham-operated controls. Notably, AIS-induced ceramides accumulated in cerebral endothelial cell (EC)-enriched but not non-EC (NEC)-enriched fractions (magnetic activated cell sorting). These findings inspired the hypothesis that preventing AIS-induced cerebral EC ceramide accrual mitigates infarct volume, neurological and motor deficits in mice. Dihydroceramide desaturase 1 (DES1, gene:Degs1) catalyzes toxic ceramide generation from inert dihydroceramide. We deployed a cerebral EC-specific adeno-associated virus BR1 serotype (AAV-BR1) to either carry Cre-recombinase (AAV-BR1-Cre) or Null virus (AAV-BR1-Null) to adult, maleDegs1fl/flmice, enabling selective depletion ofDegs1in cerebral ECs (aka Cre-Degs1) or not (Null-Degs1) [n=5 each]. Fourteen days after tMCAO+R,Degs1gene expression was reduced (p

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Abstract WP30: Detection of Somatic Mutations in Brain Arteriovenous Malformation Patients Using Non-Invasive Cell-Free DNA Screening

Stroke, Volume 56, Issue Suppl_1, Page AWP30-AWP30, February 1, 2025. Introduction:Brain arteriovenous malformations (bAVMs) are vascular anomalies resulting from defective morphogenesis of blood vessels in the brain. Kirsten rat sarcoma virus (KRAS)somatic activating gene mutations have been identified in the majority of bAVMs using digital droplet PCR-based assays (ddPCR). Currently, bAVM somatic mutation genetic characterization requires sequencing surgically excised lesion DNA, but recent advancements have overcome some conventional biopsy limitations through sequencing cell-free DNA (cfDNA) which is directly released into the blood circulation from cell breakdown and turnover at the site of the lesion. Therefore, cfDNA released from the mutated bAVM tissue cells may be detectable in a peripheral blood sample, providing a non-invasive approach for somatic mutation screening.Hypothesis:We hypothesize that somaticKRAS G12Dmutations in bAVM patients can be detected using non-invasive cfDNA screening.Methods:We selected six bAVM patients whose surgically-resected bAVM lesions screened positive for somaticKRAS G12Dmutation using ddPCR and had contributed a peripheral blood sample for research within 2 months prior to surgery. For each patient, cfDNA was isolated from 1.0 mL of banked plasma using the Circulating cfDNA/RNA Isolation Kit (Qiagen). We used the ddPCRKRAS G12Dassay (Bio-Rad) to screen cfDNA samples for presence of the mutation. Samples were screened in duplicate using 8 uL and 4 uL of cfDNA eluate and assays included both positive controls (syntheticKRAS G12Doligo sequence (Integrated DNA Technologies)) and negative controls (no template and water). The variant allele frequency was estimated for each sample as the (target concentration)/(target + reference concentration).Results:Of the six bAVM cases, five screened positive forKRAS G12Dmutation in the cfDNA sample. TheKRAS G12DcfDNA variant allele frequency ranged from 0.20 – 0.54% for the five positive samples.Conclusions:We detected somaticKRAS G12Dmutations in bAVM patients using non-invasive cfDNA screening. While further studies are needed to validate these findings, these exciting results suggest that we may be able to perform non-invasiveKRASsomatic mutation screening using cfDNA in a greater number of bAVM cases (e.g., not just those undergoing surgery), which may be useful to clinically stratify bAVM patients and provide targeted therapies based on specific genetic defect.

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Herpes

To the Editor I read with great interest the recent JAMA Insights article on genital herpes. However, the authors did not mention the Tzanck smear test in the Diagnosis section. Indeed, this technique described in 1947 by Arnault Tzanck is a neglected, but rapid, reliable, and cost-effective bedside technique to establish the diagnosis of acantholytic dermatoses, such as herpes simplex virus (HSV) and varicella zoster virus infections. Gentle scraping of the base of vesiculobullous lesions (incised if intact or stripped of their crust if ulcerated) with a scalpel allows for cytologic spreading on a glass slide, air-drying, and quick Giemsa staining. Instant microscopic examination by a trained cytologist enables identification of acantholytic and multinucleated keratinocytes with typical nuclear inclusion bodies indicating diagnosis of HSV and varicella zoster virus infections in 80% to 90% of patients, respectively. However, a Tzanck smear cannot differentiate between HSV-1 and HSV-2 infections. Of note, this diagnostic test can be useful in other acantholytic dermatoses, such as pemphigus and genetic skin diseases, including Darier disease and Hailey-Hailey disease. In conclusion, the Tzanck smear test can provide an early diagnosis of mucocutaneous HSV infections and enable appropriate treatment in skilled hands. For inexperienced clinicians, artificial intelligence can be used to evaluate Tzanck smear findings with high accuracy.

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Overcoming Hepatitis B Vaccine Nonresponsiveness

Hepatitis B virus (HBV) infection continues to pose a significant global health challenge despite the availability of effective vaccines for more than 4 decades and antivirals for almost 3 decades. The World Health Organization estimates that more than 254 million people worldwide are living with chronic HBV infection; 1.2 million new infections occurred in 2022. Even though the introduction of hepatitis B vaccines has drastically reduced the incidence of new HBV infections, barriers such as limited access, low vaccine uptake, and suboptimal vaccine efficacy in specific populations such as older persons, individuals with diabetes, obesity, or current smoking, and immunocompromised persons (eg, those living with HIV or chronic kidney disease) account for the continued occurrence of new infections. These challenges necessitate public health efforts to improve access and uptake of hepatitis B vaccination and research to enhance vaccine immunogenicity.

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