Abstract TP237: Cofilin Inhibitor Protects Against Hydrogen Peroxide-induced Neuronal Cytotoxicity Via The Inhibition Of Oxidative Stress And Microglial Cell Activation

Stroke, Volume 54, Issue Suppl_1, Page ATP237-ATP237, February 1, 2023. Microglial activation & the failure of the antioxidant defense mechanisms are major hallmarks in different central nervous system injuries, mainly ischemic & hemorrhagic strokes. Cofilin is a cytoskeleton-associated protein involved in actin binding & severing. In our previous studies, we identified the potential role of cofilin in mediating microglial activation & neuronal apoptosis in ischemic & hemorrhagic conditions. Others have highlighted the involvement of cofilin in ROS production & the resultant neuronal death; however, more studies are needed to delineate the role of cofilin in oxidative stress conditions. Hence, the present study is aimed to investigate the role of cofilin in hydrogen peroxide (H2O2)-induced microglial activation & oxidative neuronal injury & its protection using a first-in-class small-molecule inhibitor of cofilin (SZ-3). An in vitro H2O2-induced cell death model was used in two different types of cells, human neuroblastoma (SH-SY5Y) & microglial (HMC3) cell lines. Cell viability was assessed by the Cell Counting Kit-8 assay. Western blotting was used to measure the expression levels of several proteins. Our results show that treatment with H2O2increases the expression of cofilin & slingshot-1 (SSH-1), an upstream regulator of cofilin, in microglial cells, which was significantly reduced in the SZ3 treated group. Cofilin inhibition significantly attenuated H2O2-induced microglial activation by reducing the release of proinflammatory mediators (HMGB1, TNF-α). Furthermore, we demonstrate that SZ3 protects against H2O2-induced cytotoxicity in SH-SY5Y cells & activates the AKT signaling pathway by increasing its phosphorylation levels. SZ3 treatment also induces the expression of Nrf2, a major transcription factor that regulates the oxidative stress response, & its related antioxidant enzymes (HO-1, SOD2, NQO1) in the SY-SY5Y cell. Together, these results suggest that cofilin inhibition plays an essential role in protecting neuronal cells under H2O2-induced oxidative stress, possibly by activating the antioxidant defense mechanisms & reducing microglial activation.

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Febbraio 2023

Abstract TMP119: Increased Stroke Severity In A Model Of Preeclampsia Is Associated With Poor Collateral Flow, Oxidative Stress, And Elevated Thrombin

Stroke, Volume 54, Issue Suppl_1, Page ATMP119-ATMP119, February 1, 2023. Background:Pregnancy is associated with an increased risk of stroke largely driven by the comorbidity of preeclampsia (PE), a hypertensive disorder that complicates 4-8% of all pregnancies. Although stroke treatment has improved in recent years, pregnant women have been excluded from stroke clinical trials and thus little is known about treatment and stroke outcome in this population. We investigated stroke outcome (infarct and hemorrhagic transformation, HT) in normal pregnancy (Preg; n=13) and in a model of preeclampsia (PE; n=6) induced by feeding pregnant Sprague Dawley rats a high cholesterol diet on gestational days 7-21. Nonpregnant (NP; n=7) rats were a control for pregnancy.Methods:Middle cerebral artery occlusion (MCAO) was used to induce focal ischemia for 3 hours with 1 hour of reperfusion. Perfusion deficit in the core MCA and collateral territories were measured using multi-site laser Doppler. Infarction and oxidative stress were determined by cresyl violet and 3-nitrotyrosine staining of fixed brain tissue, respectively. Endothelial oxidative stress and hemostatic factors were measured by ELISA for 8-isoprostane and thrombin.Results:Perfusion deficit in core MCA was similar between groups; however, collateral flow was significantly impaired in ePE vs. NP and LP: -51±8% vs. -21±15% and -25±13% (p

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Febbraio 2023

Abstract WP13: Value Of Stress Hyperglycemia Ratio In Predicting The Prognosis Of Patients With Acute Ischemic Stroke:a Subgroup Analysis Of The Rescue Bt Clinical Trial

Stroke, Volume 54, Issue Suppl_1, Page AWP13-AWP13, February 1, 2023. Objective:This study aimed to evaluate the correlation between the stress hyperglycemia and clinical outcomes after large vessel occlusion stroke (LVO) at 90 days.Methods:The RESCUE BT trial was a multicenter, randomized, double-blind, placebo-controlled clinical trial, consisting of 948 patients from 55 centers in China. In this subgroup study, 542 consecutive stroke patients with glucose or glycated hemoglobin values were included. Stress hyperglycemia was evaluated both as a tri-categorical variable (≤1.07 vs 1.08-1.29 vs ≥1.30) and a continuous variable. The primary outcome was favorable functional outcome (modified Rankin Scale [mRS] ≤ 2) at 90 days. The secondary outcome contained excellent functional outcome (modified Rankin Scale [mRS] ≤ 1) and safety outcomes such as 90-day mortality and intracranial hemorrhage.Results:542 patients were included. Compared to patients with the lowest tertiles of stress hyperglycemia, calculated as glucose/GA ratio, the highest tertiles of stress hyperglycemia was obviously associated with a higher rate of poor functional outcome at 90-day (modified Rankin Scale [mRS] score 3-6) after adjusted for potential covariates (adjusted hazard ratio, 0.44; 95% confidence interval, 0.28-0.69, P < 0.001). This result was also consistent in the excellent clinical outcome of 90-day mRS score 2-6 (adjusted hazard ratio, 0.48; 95% confidence interval, 0.29-0.79, P =0.004).Conclusion:In patients with acute ischemic stroke, lower stress hyperglycemia, as measured by glucose/GA ratio, was more likely to have favorable outcome at 90-day.Trial registrationChinese Clinical Trial Registry Identifier: ChiCTR-IOR-17014167, December 27, 2017.

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Febbraio 2023

Abstract 38: Admission Hyperglycemia In Stroke Patients Increases Oxidative Stress In Red Blood Cells

Stroke, Volume 54, Issue Suppl_1, Page A38-A38, February 1, 2023. Background:Our recent study has shown that admission hyperglycemia in stroke patients is associated with red blood cell (RBC) dysregulation, however, the underlying mechanism is still unclear. In addition to transporting oxygen via hemoglobin, RBCs also carry a robust antioxidant system to remove the reactive oxygen species (ROS) continuously produced from hemoglobin autoxidation (Fig A). It has been reported that in vitro hyperglycemia can increase oxidative stress in RBCs. We thus hypothesize that acute hyperglycemia in stroke patients may also affect the antioxidant system in RBCs.Method:Stroke patients with hyperglycemia and normoglycemia (n=12/group) were prospectively recruited in accordance with IRB protocol. Blood was collected at 2 hr, 24 hr, 72 hr and 3 mon post stroke. RBCs were precipitated from whole blood, and leucocytes were removed with depletion filter. The expression and glycosylation of RBC antioxidants, such as catalase (CAT), superoxide dismutase type 1 (SOD1), glutathione peroxidase 1 (GPX1) and peroxiredoxin 2 (PRX2), were quantified using mass spectrometry-based selected reaction monitoring (SRM) method.Result:CAT, SOD1 and GPX1 showed gradually decreased expression in hyperglycemic stroke patients, which may impair the antioxidant capacity of RBCs (Fig B). The glycosylation level of PRX2 was increased in stroke-related hyperglycemia (Fig C), and increased glycosylation was associated with decreased PRX2 activity (Fig D). Consistent with the expression and glycosylation changes in antioxidants, total RBC antioxidant capacity was reduced in hyperglycemic patients, and ROS was significantly accumulated in RBCs (Fig E).Conclusion:Acute hyperglycemia in stroke patients intensifies RBC oxidative stress by affecting the expression and post-translational modification of RBC antioxidants. Larger studies are underway to verify these findings, and explore how hyperglycemia and RBC oxidative stress influence stroke outcome.

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Febbraio 2023

Abstract WMP6: Genetic Variation In KLF5 Is Associated With Initial Stress Glucose Ratio In Acute Ischemic Stroke

Stroke, Volume 54, Issue Suppl_1, Page AWMP6-AWMP6, February 1, 2023. Introduction:Hyperglycemia has consistently been associated with worse outcome following an acute ischemic stroke (AIS). Recent studies have shown that a stress glucose ratio (SGR) may be a better predictor of critical illness than absolute hyperglycemia. An elevated initial stress glucose ratio (iSGR) was significantly associated with malignant cerebral edema and ICH after thrombectomy. We sought to identify genetic loci related to iSGR in a cohort of the GENISIS (Genetics of Early Neurological InStability after Ischemic Stroke) study.Methods:GENISIS is a multi-site international study that enrolled AIS patients within 6 hours of symptom onset. A sub-cohort with available initial glucose and HbA1c levels was selected. For all patients, Hg38 imputed genotypes were available. iSGR was calculated by dividing initial glucose by the estimated average glucose concentration (= 28.7 * A1c – 46.7) and normalized by log transformation for the GWAS. Association was adjusted by age, sex, baseline NIHSS, principal components and genotyping rounds. Samples were analyzed by country of origin and ethnicity using Plink2 and then meta-analyzed by ethnicity using METAL.Results:Three separate populations were identified (African-Americans n=299; European-Americans n=622; and Spanish n=625). Median age was 71 (IQR 60-80) with 46% female. Baseline NIHSS was 11 (IQR 5-16). A suggestive loci (chr13:rs9560146, p=9.88×10-8) was identified in the meta-analysis. Gene-based analyses suggested that the loci in chromosome 13 is driven byKLF5(p=0.002). In addition, rs9560146 is also associated withKLF5gene expression in brain (eQTL p=3.30×10-3; frontal cortex: Braineac).Conclusions:We demonstrated that a variant inKLF5may be associated with iSGR in a cohort of AIS patients.KLF5is induced under hypoxia conditions and interacts with HIF-1a to mediate glucose homeostasis. Recently, a pre-clinical study demonstrated that a micro RNA (miR-10b-3p) had neuroprotective effects against ischemia/reperfusion injury by targeting KLF5. Thus, investigating the genetic architecture of stress hyperglycemia may be informative and reveal variants, genes or pathways involved in ischemic brain injury. We plan to recruit a cohort for replication and sample size expansion.

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Febbraio 2023

Abstract WP50: Stress Hyperglycemia And Early Neurological Deterioration In Patients With Acute LVO And Low NIHSS

Stroke, Volume 54, Issue Suppl_1, Page AWP50-AWP50, February 1, 2023. Introduction:Stress hyperglycemia is an essential survival response. However, it is associated with poor prognosis after ischemic stroke, and its contribution to collateral failure is not well defined. We investigated whether stress hyperglycemia would be associated with early neurological deterioration (END) in acute large vessel occlusion (LVO) patients who present with mild neurological deficit.Methods:From a multicenter stroke registry, ischemic stroke patients with acute anterior circulation LVO and mild symptoms (NIHSS

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Febbraio 2023

Abstract TMP13: Thin Wall Regions Of Intracranial Aneurysms Are Associated With High Wall Shear Stress

Stroke, Volume 54, Issue Suppl_1, Page ATMP13-ATMP13, February 1, 2023. Background:Intra-saccular hemodynamics play a key role in the pathobiological remodeling of the intracranial aneurysm (IA) wall. Evaluating the relationship between hemodynamic forces at the aneurysm wall and wall presentation can help us understand the complex processes of dysregulated vascular remodeling that occurs during IA natural history.Hypothesis:We hypothesized that greater hemodynamic insult, i.e., high wall shear stress (WSS) and high wall shear stress divergence (WSSD), results in endothelial damage and loss of internal elastic lamina, which presents as thin, translucent regions on interoperative imaging.Methods:From digital subtraction angiography images of 15 patients with IAs, aneurysm and surrounding vessels were segmented. Computational fluid dynamics (CFD) simulations were performed using generic boundary conditions for all the patients. Aneurysm walls were identified from 2D intraoperative images of IAs taken during clipping, and wall regions were classified as thick regions (white), normal (purple-crimson), and thin/translucent (red), using a semi-supervised machine learning algorithm. 2D wall classifications were mapped onto the 3D geometries for statistical analysis of differences between average hemodynamic properties across wall regions.Results:On an average, more than 30% of IA sac was visible on the 2D intra-operative image. Of then=15 IAs,n=8,n=5 andn=2 showed pre-dominantly thick, thin, and normal wall types respectively. Average WSS and WSSD were significantly higher at thin regions of IAs as compared to both normal and thick regions. Thin wall regions also tended to co-locate with significantly lower RRT as compared to the thick and normal regions.Conclusion:Thin wall regions observed on intra-operative images of IAs are associated with greater hemodynamic insult (higher WSS and WSSD) and higher flow velocity (lower RRT).

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Febbraio 2023

Abstract TMP32: Genetic Knockout Of CD38 Is Associated With Improved Cognitive Performance And Protection Against Oxidative Stress In Aged Mice

Stroke, Volume 54, Issue Suppl_1, Page ATMP32-ATMP32, February 1, 2023. Introduction:CD38 enzymatic activity is the main determinant of the age-dependent decline in nicotinamide adenine dinucleotide (NAD+) levels and the loss of CD38 function has been associated with increased longevity in rodents. Therefore, we hypothesize that the loss of CD38 and its enzymatic function will improve cognitive performance in advanced age through the preservation of NAD+levels and the protection against oxidative stress.Methods:CD38 Knockout (CD38KO) and C57BL/6J (wild type WT) male mice were aged for at least 24 months. The cognitive performance was compared through Barnes maze, Fear conditioning and Y-maze tests. Dihydroethidium (DHE), Diaminofluorescein-2 diacetate (DAF) and nicotinamide adenine dinucleotide phosphate NAD(P)H staining were used to assess the levels of superoxide, nitric oxide (NO) and NAD(P)H in the brain, respectively.Results:5 WT and 5 CD38KO mice aged (24-30) months were included. While there were no significant differences in fear conditioning and Y-maze tests, CD38KO mice showed better memory performance in Barnes maze test including shorter distance travelled (CD38KO: 2±0.06 vs WT: 3.7±1.1 m, p=0.008), longer time in proximity of the holes (CD38KO: 51.2±11.1 vs WT: 34.6±8.4 s, p=0.027), and shorter distance from the hole (CD38KO: 0.17±0.025 vs WT: 0.23±0.04 m, p=0.038) and less errors (CD38KO: 3.5±2.3 vs WKY: 6±0.7, p=0.04). Brain tissue analysis showed 58% lower superoxide (p

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Febbraio 2023