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OBJECTIVE In the absence of clear guidelines and consistent natural history data, the decision to treat unruptured intracranial aneurysms (UIAs) is a matter of some controversy. Currently, decisions are often guided by a consensus of cerebrovascular specialist teams and patient preferences. It is unclear how paradigm-shifting developments in the detection and treatment of UIAs have affected the size of the UIAs that are selected for treatment. Herein, the authors aimed to study potential changes in the average size of the UIAs that were treated over time. They hypothesized that the average size of UIAs that are treated is decreasing over time. METHODS A systematic search of the literature was performed to identify all studies describing the size of UIAs that were treated using any modality. Scatter diagrams with trend lines were used to plot the size of the aneurysms treated over time and assess for the presence of a potentially significant trend via statistical correlation tests. Subgroup analyses based on type of treatment, country of study, and specialty of the authors were performed. RESULTS A total of 240 studies including 35,150 UIAs treated between 1987 and 2021 met all eligibility criteria and were entered in the analysis. The mean age of patients was 55.5 years, and 70.7% of the patients were females. There was a significant decrease in the size of treated UIAs over time (Spearman’s r = −0.186, p < 0.001), with a 0.71-mm decrease in the average size of treated UIAs every 5 years since 1987 and an annual mean dropping below 7 mm in 2012. This decreasing trend was present in surgically and endovascularly treated UIAs (p < 0.001 for both), in more developed and developing countries (p < 0.001 for both), within neurosurgical and non-neurosurgical specialties (p < 0.001 for both), most prominently in the US (Spearman’s r = −0.482, p < 0.001), and less prominently in Europe (Spearman’s r = −0.221, p < 0.001) and was not detected in East Asia. CONCLUSIONS The present study indicates that based on the treated UIA size data published in the literature over the past 35 years, smaller UIAs are being treated over time. This trend is likely driven by safer treatments. However, future studies should elucidate the cost-effectiveness of treating smaller UIAs as well as the possible real-world contribution of this trend in preventing aneurysmal subarachnoid hemorrhage.

BACKGROUND Ischemic stroke is a prevalent, devastating disease with high morbidity and mortality. Despite extensive research using animal models, significant gaps remain in understanding the pathological processes in human stroke. We previously developed a tissue bank to analyze the blood immediately proximal and distal to an intracranial thrombus in patients undergoing mechanical thrombectomy (ClinicalTrials.gov identifier, NCT03153683). Our goal for the present project was to evaluate the blood gas changes and acid/base balance during stroke and determine how vascular collateralization affects these changes. METHODS We analyzed the blood samples and computed tomography angiography collateral scores from the first 62 patients in the BACTRAC (Blood and Clot Thrombectomy Registry and Collaboration) registry. The bicarbonate, partial pressure of oxygen, and partial pressure of carbon dioxide (pCO2) values of the intracranial (distal) and systemic (proximal) arterial blood relative to the occlusive thrombus were analyzed. Analysis of the group differences in systemic and intracranial blood gas values was also performed. RESULTS The partial pressure of oxygen, pCO2, and bicarbonate levels were all significantly higher in the systemic blood than in the intracranial blood (P < 0.001 for all) at thrombectomy. Collateralization did not significantly affect the distal blood gas values. Compared with the female patients, the male patients had had higher systemic pCO2 values (39.8 vs. 36.6 mm Hg; P = 0.0065) and lower systemic and intracranial pH values (7.351 vs. 7.392; P = 0.0047). CONCLUSIONS The arterial blood gases differed immediately proximal and distal to thrombi in large vessel occlusive stroke. Although vascular collateralization did not appear to affect the blood gas changes, some blood gas values differed between men and women. The changes in bicarbonate and pCO2 suggested a compensatory acid-base process occurring at the time of infarction.

Introduction: Stroke secondary to intracranial atherosclerosis (ICAD) results in three distinct infarct patterns: (a) border zone infarcts (BZI) due to impaired distal perfusion (b) territorial infarcts due to distal plaque/thrombus embolization, and (c) perforator infarcts due to plaque progression. Previous studies indicate higher stroke recurrence in ICAD patients with BZI. Methods: This registered systematic review (CRD42021265230) comprised Medline and Web of Science search from inception to March 2022 for keywords (Intracranial Atherosclerosis OR Intracranial Stenosis) AND (Border zone OR Infarct Pattern) to identify papers and conference abstracts reporting initial infarct patterns and recurrence rates in patients with symptomatic ICAD. Sensitivity analyses were performed for studies including any BZI vs isolated BZI and those excluding posterior circulation strokes. The study outcome included neurological deterioration and/or stroke recurrence. For all outcome events, corresponding risk ratios (RR) and 95% confidence intervals (CI) were calculated. Risk of bias assessments will be presented. Results: Literature search yielded 4478 studies,11 met inclusion criteria (n=1315 patients, 354 with BZI, weighted proportions summarized in figure). The meta-analysis of these studies with moderate heterogeneity (I 2 =38.7%) demonstrated that RR of outcomes in BZI group compared to non-BZI group was 2.10 (95% CI 1.52-2.90). Limiting analysis to studies including any BZI, RR (and 95% CI) was 2.32 (1.58-3.40), and 3.25 (2.09-5.07) for studies only including anterior circulation strokes with low heterogeneity for both (I 2 =0%). A non-significantly high outcome rate was seen with isolated BZI (RR 2.29, 95% CI 0.94-5.62) but with moderate heterogeneity across studies (I 2 =70.25%). Conclusion: We demonstrate the presence of BZI secondary to symptomatic ICAD can be imaging biomarker to predict neurological deterioration and/or stroke recurrence.

Regenerating Family Member 3 Alpha (REG3A) is a multifunctional protein with antimicrobial activity, and primarily secreted by the intestine and pancreas. Studies have shown an increased expression of REG3A in systemic inflammatory responses to acute injury and infection, but studies investigating REG3A during the pathogenesis of ischemic stroke are limited. The aims of this study were to examine the associations between arterial expression of REG3A and other arterial inflammatory proteins implicated in stroke pathogenesis, as well as associations between REG3A and markers of poor outcome for ischemic stroke. The University of Kentucky Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) protocol (clinicaltrials.gov NCT03153683) utilizes thrombectomy to isolate intracranial arterial blood (i.e. distal to thrombus) and systemic arterial blood (i.e. carotid). Samples were analyzed by Olink Proteomics for N = 42 subjects. Statistical analyses of plasma proteins included 2-sample t-tests, spearman and biserial correlations, and robust regression models to elucidate network signaling and association to clinical outcomes. Results indicated that levels of systemic REG3A were positively correlated with inflammatory proteins interleukin IL6 (R = 0.344, p = 0.030) and IL17C (R = 0.468, p = 0.002). 2-sided t- tests examining differences of systemic REG3A within quartiles of NIHSS admission score depicted significant differences between quartiles. Those with NIHSS scores corresponding to moderate and moderate-severe neurofunctional deficits had significantly higher levels of systemic REG3A compared to those with NIHSS scores corresponding to mild and mild-moderate neurofunctional deficits (p = 0.016). STRING analyses of proteins in each robust regression model demonstrated substantial networking between REG3A and other systemic proteins highly relevant to ischemic stroke. The present study provides novel data on systemic REG3A in the context of ischemic stroke. These results demonstrate the influential role of REG3A regarding surrogate functional and radiographic outcomes of stroke severity. Additionally, they provide novel insight into the role of REG3A and related proteins during the complex neuroinflammatory process of ischemic stroke. These data provide a foundation for future studies to investigate REG3A and related networking proteins as potential biomarkers with prognostic potential, as well as potential therapeutic targets.

Using standard techniques during mechanical thrombectomy, the Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) protocol (NCT03153683) isolates intracranial arterial blood distal to the thrombus and proximal systemic blood in the carotid artery. We augmented the current protocol to study leukocyte subpopulations both distal and proximal to the thrombus during human stroke (n = 16 patients), and from patients with cerebrovascular disease (CVD) undergoing angiography for unrelated conditions (e.g. carotid artery stenosis; n = 12 patients). We isolated leukocytes for flow cytometry from small volume (+ T cells and classical dendritic cells were significantly lower than CVD controls and correlated to within-patient edema volume and last known normal. This novel protocol successfully isolates leukocytes from small volume intracranial blood samples of stroke patients at time of mechanical thrombectomy and can be used to confirm preclinical results, as well as identify novel targets for immunotherapies.

BackgroundSince 2015, mechanical thrombectomy has been the standard treatment for emergent large vessel occlusion ischemic stroke.ObjectiveTo investigate, using the previously published Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) protocol (clinicaltrials.govNCT03153683), how the protein expression of a patient’s intracranial blood during ischemic stroke compares with the protein expression of their systemic arterial blood in order to better understand and treat stroke.MethodsPlasma samples from 25 subjects underwent proteomic analysis, where intracranial protein expression was compared with systemic protein levels. Data including sex, comorbidities, infarct volume, and infarct time were included for each subject.ResultsA majority of important proteins had a lower expression in intracranial blood than in systemic arterial blood. Proteins with the most significant changes in expression were: endopeptidase at −0.26 (pConclusionsEvaluation of proteomic changes in the intravascular space of a cerebral infarct in progress in human subjects suggested that changes in proteins such PLTP, fetuin-B (FETUB), and FCN2 may be involved in atherosclerotic changes, and chemokines such as CCL23 are known to play a role in the Th2 autoimmune response. These data provide a scientific springboard for identifying clinically relevant biomarkers for diagnosis/prognosis, and targets for much needed neuroprotective/neuroreparative pharmacotherapies.

Background: Emergent Large Vessel Occlusion (ELVO) strokes are devastating ischemic vascular events for which novel biomarkers and therapies are needed. The purpose of this study is to investigate the role of Body Mass Index (BMI) on protein expression and signaling at the time of ELVO intervention. Methods: The Blood And Clot Thrombectomy Registry And Collaboration (BACTRAC) is a continually enrolling tissue bank (clinicaltrials.gov NCT03153683) from stroke patients undergoing mechanical thrombectomy (MT). N=61 human carotid plasma samples were analyzed for inflammatory and cardiometabolic protein expression by Olink Proteomics. Results: The 61 subjects studied were broken into three categories: Normal weight (BMI 18.5-24.9) which contained 19 subjects, Overweight (BMI 25-30) which contained 25 subjects, and Obese (BMI ≥30) which contained 17 subjects. When compared to Normal weight and Overweight categories, the Obese category had significantly higher levels of adenosine deaminase (ADA) expression (p=0.01 and p=0.039, respectively). Elevated levels of ADA were found to have a significant positive correlation with both infarct volume and edema volume (p=0.013 and p=0.041, respectively), and were associated with a more severe stroke (NIHSS on discharge) and greater stroke-related disability (mRS on discharge) with significance of p=0.053 and p=0.032, respectively). When controlling for age and sex, increased infarct volumes were predicted by higher ADA levels in the Obese population (p=0.009), while increased ADA levels were not predictive of increased infarct volumes in the Normal weight category. Conclusions: When examined according to BMI, subjects undergoing MT for ELVO demonstrate significant differences in the expression of certain plasma proteins including ADA. The protein ADA is a deaminating enzyme that degrades adenosine, which has been shown to be neuroprotective in ischemia. Increased levels of ADA in the Obese group were predictive of increased infarct volumes. Further testing will explore the relationship of BMI and ADA on cognitive function outcomes. These data provide novel biomarker candidates as well as treatment targets while increasing the personalization of stroke prognosis and treatment.

Intrathecal baclofen (ITB) is a treatment modality used to improve the quality of life of patients with intractable spasticity and dystonia. Although it is an effective solution in patients failing oral interventions, it is associated with potential infectious complications. It is known that pediatric patients with ITB have significantly higher infection rates compared with adult patients. The cause of these higher rates in pediatric patients remains unclear. In the present study, we performed a meta-analysis focusing on the incidence of infection, and clarification of potential risk factors for infection in pediatric patients with ITB.This meta-analysis was performed in accordance with the PRISMA guidelines. An electronic database search was performed through PubMed, Web of Science, Embase, and Cochrane Library databases. Eligibility criteria and bias assessment were applied before statistical analysis.The 17 studies identified yielded 2238 pediatric patients treated with implanted ITB pumps between 1994 and 2014. Infection comprised 34% of observed complications, second only to catheter malfunction. Pediatric ITB primary infection ranged between 0% and 44% among included studies (interquartile range, 4.85%-18.85%). A linear mixed-effects regression model showed that subfascial implantation had 12% lower primary infection rates compared with subcutaneous implantations across the literature. The relative risk of infection was 56% lower in pediatric patients with subfascially implanted ITB pumps.Surgeons and clinicians should use these data to better assess patient risk-benefit when considering ITB pump implantation.

This meeting abstract was removed due to the OA licensing requirements of this journal. The full abstract is listed here : https://www.svin.org/files/SVIN_2021_Abstracts_for_Web.pdf

This meeting abstract was removed due to the OA licensing requirements of this journal. The full abstract is listed here : https://www.svin.org/files/SVIN_2021_Abstracts_for_Web.pdf

Stroke is a major cause of death and disability in the United States. Mechanical thrombectomy (MT) and tissue plasminogen activator are the current treatments for ischemic stroke, which have improved clinical outcomes. Despite these treatments, functional and cognitive deficits still occur demonstrating a need for predictive biomarkers for beneficial clinical outcomes which can be used as therapeutic targets for pharmacotherapy. The aim of this study compares the proteomic expression of systemic arterial blood collected at the time of MT to those from a matched cerebrovascular disease (CVD) control cohort.The Blood And Clot Thrombectomy Registry And Collaboration (BACTRAC) (clinicaltrials.gov NCT03153683) collects and banks arterial blood, both distal and proximal to the thrombus, from ischemic stroke subjects undergoing MT. Arterial blood from patients undergoing a diagnostic angiogram was also collected and banked as CVD controls. Changes in cardiometabolic and inflammatory proteins between stroke and CVD controls were analyzed via Olink Proteomics.Proteins including ARTN, TWEAK, HGF, CCL28, FGF-5, CXCL9, TRANCE and GDNF were found to be decreased in stroke subjects when compared to CVD controls. CXCL1, CCL5, OSM, GP1BA, IL6, MMP-1, and CXCL5 were increased in stroke subjects when compared to CVD controls. These proteins were also significantly correlated to stroke outcome metrics such as NIHSS, infarct volume and MoCA scoring.Overall, acute stroke patients had an increase in inflammatory proteins with a decrease in trophic proteins systemically compared to matched CVD controls. Using our CVD controls, proteins of interest were directly compared to stroke patients with the same cerebrovascular risk factors instead of statistically controlling for comorbidities. The novel methodology of matching an arterial blood CVD control group to a stroke group, as well as controlling for age and comorbid status add to the literature on prognostic stroke biomarkers, which are specific targets for future therapeutics.

Background Emergent large vessel occlusion (ELVO) strokes are devastating ischemic vascular events for which novel treatment options are needed. Using vascular cell adhesion molecule 1 (VCAM1) as a prototype, the objective of this study was to identify proteomic biomarkers and network signaling functions that are potential therapeutic targets for adjuvant treatment for mechanical thrombectomy. Methods The blood and clot thrombectomy and collaboration (BACTRAC) study is a continually enrolling tissue bank and registry from stroke patients undergoing mechanical thrombectomy. Plasma proteins from intracranial (distal to clot) and systemic arterial blood (carotid) were analyzed by Olink Proteomics for N=42 subjects. Statistical analysis of plasma proteomics used independent sample t tests, correlations, linear regression, and robust regression models to determine network signaling and predictors of clinical outcomes. Data and network analyses were performed using IBM SPSS Statistics, SAS v 9.4, and STRING V11. Results Increased systemic (pp=0.013) levels of VCAM1 were associated with the presence of hypertension. Intracranial VCAM1 was positively correlated to both infarct volume (p=0.032; r=0.34) and edema volume (p=0.026; r=0.35). The %∆ in NIHSS from admittance to discharge was found to be significantly correlated to both systemic (p=0.013; r = −0.409) and intracranial (p=0.011; r = −0.421) VCAM1 levels indicating elevated levels of systemic and intracranial VCAM1 are associated with reduced improvement of stroke severity based on NIHSS from admittance to discharge. STRING-generated analyses identified biologic functional descriptions as well as function-associated proteins from the predictive models of infarct and edema volume. Conclusions The current study provides novel data on systemic and intracranial VCAM1 in relation to stroke comorbidities, stroke severity, functional outcomes, and the role VCAM1 plays in complex protein-protein signaling pathways. These data will allow future studies to develop predictive biomarkers and proteomic targets for drug development to improve our ability to treat a devastating pathology.

Introduction: The University of Kentucky Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) protocol utilizes thrombectomy to isolate intracranial (i.e. distal to thrombus) arterial blood and systemic (i.e. carotid) arterial blood from thrombectomy procedures to study stroke. Here, we investigate the relationship among Vascular Cell Adhesion Molecule 1 (VCAM1), hypertension (HTN), and stroke recovery in patients undergoing mechanical thrombectomy for emergent large vessel occlusion (ELVO) stroke. Methods: Intracranial and systemic plasma samples from 25 subjects underwent cardiometabolic proteomic analysis at Olink Proteomics. Demographic data including HTN status and both admission NIHSS and discharge NIHSS were included. Linear regression analysis was run on both intracranial and systemic VCAM1 expression levels against % change in NIHSS ((Admittance NIHSS - Discharge NIHSS)/Admittance NIHSS)) and two-tailed t-tests were run assessing VCAM1 expression with HTN vs. no HTN. Results: Increased expression of intracranial VCAM1 significantly correlated with a smaller % change in NIHSS (p=0.001). Similarly, increased systemic VCAM1 expression was also found to have a significant relationship with smaller % change in NIHSS (p=0.005). Subjects with hypertension had significantly higher intracranial (p=0.03) and systemic (p=0.001) VCAM1 levels compared to those without HTN. Discussion: VCAM1 mediates leukocyte-endothelial cell adhesion and has been shown to play a role in stroke. This study takes a novel approach of sampling both intracranial and systemic arterial blood during an ELVO stroke. We found increased intracranial and systemic VCAM1 independently correlate with a smaller % change in NIHSS, an indicator of poorer initial recovery. Although preliminary, these results suggest an informative role of VCAM1 levels at the time of infarct. Additionally, those with HTN had higher levels of intracranial and systemic VCAM1. These data are in line with previous studies suggesting VCAM1 is a marker of endothelial damage due to HTN leading to negative clinical outcomes. To better understand our findings, we plan to perform subset analyses investigating VCAM1 levels in relation to dyslipidemia, infarct time and infarct volume.

Introduction: Sex differences in stroke have been apparent with premenopausal females having a lower incidence of stroke with better outcomes than postmenopausal females and males. We examined sex-specific outcomes and changes in plasma proteins following emergent large vessel occlusions. The previously published Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC), clinicaltrials.gov NCT03153683, allows for analysis of plasma proteins both systemically and distal to the thrombus. Methods: Plasma samples, processed in accordance with the BACTRAC protocol, were sent to Olink to run cardiometabolic and inflammatory panels. Demographics are reported as mean±SEM. Significance determined in Prism with Mann-Whitney, t-test, or pair mixed-effect analysis. Results: We evaluated 34 subjects, >18 yrs old (20 females, 14 males) enrolled in BACTRAC. There was no significant difference in age (68.9±2.7, 65.4±4.5 yrs, respectively) or comorbidities (hypertension, diabetes, cholesterolemia). Interestingly, males had a larger (p3 , 36,228±10,943 mm 3 , respectively). This coincided with a lower (p Conclusions: Unexpectedly, males have larger infarcts and less independence following large vessel occlusions in BACTRAC. We hypothesize this is due to fewer collaterals which leads to sex specific signaling patterns. Additional analysis of the plasma and subjects in BACTRAC are needed to target sex specific therapeutic.

Introduction Dural arteriovenous fistulae (DAVF) are intracranial vascular abnormalities encountered in neurosurgery practice. Treatment options are microsurgical disconnection, endovascular embolization and/or radiosurgery. Past studies have reported the efficacy, safety, and predictors of success of radiosurgery. In this study, we investigated the angioarchitecture of fistulae at the time of radiosurgery and how the anatomy changed in the time after treatment based on angiogram follow-ups. Methods A retrospective analysis was performed on patients with angiographic diagnosis of DAVF treated with Gamma Knife radiosurgery (GKRS) between 2013 and 2018. Data collection included demographics, symptoms, grading scores, vascular anatomy, radiation data, treatment strategy, angiographic results, and length of patient follow-up. Results Our study reports data on 10 patients with a total of 14 fistulae. On follow-up angiography, 8 (57%) had complete occlusion of the fistula with a median time to follow up of 19.5 months. The remaining 6 (43%) were deemed as near-complete occlusion of fistula with a median time to follow up of 12.0 months. Time from radiosurgery to angiogram revealing incomplete vs. angiogram revealing complete obliteration was significantly different (p=0.045). Nearly all AVFs had decreased feeders over time after treatment with only one AVF developing an additional feeder post-treatment. Arterial feeders, drainage site, sex, Borden type, lesion volume and treatment volume had no predictive value of obliteration outcome. Conclusions This study provides data on the angioarchitecture of fistulae treated with GKRS and also serves as an extension of previous studies reporting the safety and efficacy of GKRS treatment for DAVF in a specific patient population.

Introduction Since 2015, mechanical thrombectomy is the standard treatment for emergent large vessel occlusion stroke. Using standard techniques during mechanical thrombectomy, the Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) protocol (clinicaltrials.gov NCT03153683) isolates intracranial blood within the artery immediately downstream from the thrombus, systemic arterial blood proximal to the thrombus, and the thrombus itself. BACTRAC is the first protocol utilizing the thrombectomy technique to collect local whole blood samples during brain infarction. We aimed to augment the current collection protocol to reproducibly obtain and study local leukocyte populations during human stroke. Methods We started with the established BACTRAC protocol (PMID: 30064997) and modified the tissue collection protocol to isolate lymphocytes for flow cytometry and to bank the thrombus and plasma. The protocol was first established in healthy controls (n=10) using venous blood samples and then initiated for thrombectomy cases (n=13). We performed a flow cytometry panel that identifies CD3, 4, 8, 11b, 11c, 14, 19, 31, 45, 66b, 161, 183 markers to investigate populations of B-cells, T-cells, dendritic cells, NK cells, macrophages/monocytes, granulocytes, endothelial cells, and progenitors, respectively. Cells were washed twice in FACS buffer and fixed in 1% paraformaldehyde plus 0.1% EDTA on ice for 30 minutes and analyzed on a FACS Symphony the same day. All gating and event analyses were performed in FlowJo V10 (TreeStar) and all statistical analyses were performed in GraphPad Prism 7 (GraphPad Software). Results The average lymphocyte isolation yield in the intracranial blood samples of stroke patients was 2.26 × 106 cells/ml (SEM 4 × 105 cells/ml). The average viability of intracranial samples was 94.41% (SEM 2.53%). The average lymphocyte isolation from the systemic arterial blood was 4.04 × 106 cells/ml (SEM 5.1 × 105 cells/ml). The average viability of systemic arterial samples was 97.08% (SEM 0.31%). Intracranial B-cells were found to be significantly reduced in stroke patients compared to controls. Myeloid cells including monocytes and macrophages, were found to be elevated in the systemic blood of stroke patients compared to controls. Finally, T-helper cell populations were found to be diminished in the systemic compared to intracranial blood of stroke patients (paired t-tests). Conclusions This modification to the existing BACTRAC protocol provides the opportunity, for the first time, to study changes in local leukocyte populations in the arteries undergoing ischemic stroke in the human condition. Efficient processing of lymphocytes with subsequent flow cytometry analyses will provide insight into the neuroinflammatory microenvironment of the occlusion during stroke. Future studies will be aimed at investigating changes in specific leukocyte populations and how they might relate to patient demographics, patient co-morbidities, infarct volume, and functional recovery. These data will help accelerate translational stroke research to elucidate novel approaches for drug discovery and prognosis. Disclosures B. Maglinger: None. B. Shaw: None. J. Turchan: None. T. Ujas: None. S. Grupke: None. K. Pennypacker: 4; C; Cerelux, LLC. J. Fraser: 1; C; AHA, University of Kentucky. 2; C; Stream Biomedical, Medtronic, Penumbra. 4; C; Cerelux, LLC; Fawkes Biotechnology. A. Stowe: 4; C; Cerelux, LLC.

Introduction The University of Kentucky Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) protocol (clinicaltrials.gov NCT03153683) utilizes thrombectomy to isolate intracranial (i.e. distal to thrombus) arterial blood and systemic (i.e. carotid) arterial blood from thrombectomy procedures to better understand stroke. The objective of this study within that protocol was to investigate the relationship among Vascular Cell Adhesion Molecule 1 (VCAM1), hypertension (HTN), and stroke recovery in patients undergoing mechanical thrombectomy for emergent large vessel occlusion (ELVO) stroke. Here, we report how intracranial and systemic proteomic expression levels relate to HTN and the change in National Institute of Health Stroke Scale/Score (NIHSS) from admittance to discharge. Methods Intracranial and systemic plasma samples from 25 subjects underwent cardiometabolic proteomic analysis at Olink Proteomics (Olink Proteomics, Boston, MA). VCAM1 expression levels were reported as a Normalized Proteomic eXpressoin (NPX) for each subject. Demographic data were also reported including HTN and both admission NIHSS and discharge NIHSS. Linear regression analysis was run on both intracranial and systemic VCAM1 expression levels against% change in NIHSS ((Admittance NIHSS - Discharge NIHSS)/Admittance NIHSS)). Two-tailed t-tests were run assessing VCAM1 expression with hypertension (HTN) vs. no hypertension. Data analysis was performed using IBM SPSS Statistics. Results Linear regression analysis demonstrated increased expression of intracranial VCAM1 significantly correlated with a smaller% change in NIHSS (p=0.001). Similarly, increased systemic VCAM1 expression was also found to have a significant relationship with smaller% change in NIHSS (p=0.005). Subjects with hypertension had significantly higher intracranial (p=0.03) and systemic (p=0.001) VCAM1 levels compared to those without HTN. Discussion VCAM1 mediates leukocyte-endothelial cell adhesion and has been shown to play a role in atherosclerotic plaques, endothelial integrity and function, and stroke recurrence. This study takes a novel approach of sampling both intracranial and systemic arterial blood during an ELVO stroke. We found increased intracranial and systemic VCAM1 independently correlate with a smaller% change in NIHSS. Although preliminary, these results may suggest an informative role of VCAM1 levels at the time of infarct. Additionally, within our cohort, those with HTN had higher levels of intracranial and systemic VCAM1. These data are in line with previous studies suggesting VCAM1 may be a marker of endothelial damage due to HTN. To better understand our findings, we plan to perform subset analyses investigating VCAM1 levels in relation to stroke recurrence, dyslipidemia, infarct time and infarct volume. As VCAM1 plays a role in leukocyte recruitment during the inflammatory state, we also plan to investigate how the antioxidant superoxide dismutase 1 (SOD1) may modulate intracranial and systemic levels of VCAM1 and influence functional recovery based on NIHSS. Disclosures B. Maglinger: None. M. Sands: None. J. Frank: None. A. Trout: None. J. Roberts: None. S. Grupke: None. J. Turchan-Cholewo: None. A. Stowe: 4; C; Cerelux, LLC. J. Fraser: 2; C; Stream Biomedical, Medtronic, Penumbra. 4; C; Cerelux, LLC; Fawkes Biotechnology. K. Pennypacker: 4; C; Cerelux, LLC.

Introduction Ischemic stroke is a prevalent, devastating disease with high morbidity and mortality. Despite extensive research using animal models, there remains significant gaps in understanding processes of stroke in human patients. To address this, we developed a protocol to obtain and to analyze blood immediately proximal in systemic circulation and distal to a thrombus in patients undergoing mechanical thrombectomy (www.clinicaltrials.gov NCT03153683). Our goal for this project was to evaluate blood gas changes and acid/base balance during stroke and how these changes are affected by patient factors. Methods We analyzed blood samples from the first 62 patients in the BACTRAC registry. Bicarbonate, pO2, and pCO2 values of intracranial (distal) and systemic (proximal) arterial blood relative to the occlusive thrombus were analyzed. Changes were compared in patients according to vascular collateralization as measured by CTA collateral scores. Results Mean age was 68.9 years (25 – 95 years). 29 were male, 33 were female. 15 were current smokers (24%), and 47 were non-smokers (no smoking within the last 6 months; 76%). Overall, intracranial gas values differed significantly from systemic. Compared to systemic, mean intracranial pO2 was decreased (211.39 vs. 246.91, p Discussion and Conclusion A compensated metabolic acidosis is present in arterial blood gas samples immediately proximal and distal to thrombi in large vessel occlusive stroke. Vascular collateralization may not significantly affect the acid-base environment immediately distal to a large vessel occlusion. Disclosures R. Spears: None. C. McLouth: None. K. Pennypacker: None. J. Frank: None. B. Maglinger: None. A. Trout: None. J. Roberts: None. A. Stowe: None. S. Grupke: None. J. Fraser: None.

Introduction Lipocalin-2(LCN2) is a protein involved in many cellular processes, including the regulation of iron homeostasis, promotion of protective mechanisms in renal ischemia, and astrocyte activation in ischemic stroke. In some ischemic stroke models, LCN2 has been described as a ‘help me’ signal, leading to the activation of microglia, astrocytes, and drive towards cellular phenotypes favorable of recovery. While some models suggest pro-recovery effects of LCN2, other models illustrate LCN2 as having a critical role in neuroinflammation and reperfusion injury. The possibility of disease state-dependent influence of LCN2 activity in ischemic stroke has been recognized, but has yet to be further characterized. Methods Human plasma samples from thrombectomy patients were processed in the Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC clinicaltrials.gov NCT03153683), and underwent Proximity Extension Assay (PEA) via Olink (Olink Proteomics, Boston, MA). For each protein, intracranial expression distal to the stroke thrombus was compared to the same subject’s systemic arterial blood as an internal control. The percent change of protein expression was calculated as follows: (intracranial distal values- systemic proximal values).Comorbidities and sex difference were analyzed using appropriate one-way comparisons and regression. Results 25 adult patients (>18 yrs) were included in this initial analysis, of which 15 (60%) were female. Median age was 64 (24–91). 16 patients (64%) had hypertension, 15 patients (60%) had BMI >25, 10 patients (40%) had a history of smoking, 6 patients (24%) had previous stroke, 4 patients (16%) had hyperlipidemia, and 1 patient (4%) had previous MI. Mean infarct time was 513 ± 246 minutes and mean infarct volume was 58.17 ± 82.28 cc. Of 184 proteins, only 27 demonstrated an increase percent change between intracranial and systemic blood. LCN2 demonstrated a 14% increase change between intracranial and systemic blood, one of the greatest percent increases among measured proteins. The percent change of LCN2 was significantly increased in those with hypertension (p=0.024) and decreased in those with type-two diabetes (p=0.04). Conclusions Changes of LCN2 intracranially during stroke were most significant in patients with hypertension and/or diabetes. For the first time, these data provide insight into the human molecular pathology of stroke regarding this protein and its signaling cascade. Future studies will focus on the role of proteins as they relate to radiographic, functional and other clinical outcomes. Proteomic findings coupled with advanced database analysis will elucidate complex cell signaling and biomolecular interactions that occur in the blood at the site of infarct. Disclosures M. Sands: None. J. Frank: None. B. Maglinger: None. S. Grupke: None. J. Fraser: None. K. Pennypacker: None.

Introduction: Since 2015, mechanical thrombectomy is the standard treatment for emergent large vessel occlusion stroke. Using standard techniques during mechanical thrombectomy, the Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) protocol (clinicaltrials.gov NCT03153683) isolates distal blood within the artery immediately downstream from the clot, peripheral blood proximal to the clot, and the thrombus. BACTRAC is the first protocol utilizing the thrombectomy technique to collect local whole blood samples during brain infarction. We aimed to augment the current collection protocol to reproducibly obtain and study local leukocyte populations during human stroke. Methods: We started with the established BACTRAC protocol previously published (PMID: 30064997). We modified the tissue collection protocol to isolate lymphocytes for flow cytometry and to bank the clot and plasma. We performed a flow cytometry panel that identifies CD3, 4, 8, 11b, 11c, 14, 19, 31, 45, 66b, 161, 183 markers to investigate populations of B-cells, T-cells, dendritic cells, NK cells, macrophages/monocytes, granulocytes, endothelial cells, and progenitors, respectively. Results: The protocol was first established in healthy controls (n=9) by drawing venous blood and simulating a vial of distal blood and a vial of proximal blood. In healthy subjects, we were able to isolate on average 2x10 6 leukocytes/ml with a viability of 97.85%. Banked lymphocytes are also viable upon reconstitution and for flow cytometry. This protocol successfully isolates leukocytes in proximal and distal blood samples in subjects undergoing mechanical thrombectomy, with identification and analysis by flow cytometry currently ongoing. Conclusions: This modification to the existing BACTRAC protocol provides the opportunity, for the first time, to study local leukocyte populations in the arteries undergoing ischemic stroke in the human condition. Efficient processing of these samples will provide insight into the neuroinflammatory microenvironment of the occlusion and accelerate translational stroke research towards new methods of diagnosis and treatment.

Benzonatate was FDA-approved in 1958 as an antitussive. Its mechanism of action is thought to be anesthesia of vagal sensory nerve fibers that mediate cough. Vagal sensory neurons highly express the Nav1.7 subtype of voltage-gated sodium channels, and inhibition of this channel inhibits the cough reflex. Local anesthetics inhibit voltage-gated sodium channels, but there are no reports of whether benzonatate affects these channels. Our hypothesis is that benzonatate inhibits Nav1.7 voltage-gated sodium channels. We used whole cell voltage clamp recording to test the effects of benzonatate on voltage-gated sodium (Na(+)) currents in two murine cell lines, catecholamine A differentiated (CAD) cells, which express primarily Nav1.7, and N1E-115, which express primarily Nav1.3. We found that, like local anesthetics, benzonatate strongly and reversibly inhibits voltage-gated Na(+) channels. Benzonatate causes both tonic and phasic inhibition. It has greater effects on channel inactivation than on activation, and its potency is much greater at depolarized potentials, indicating inactivated-state-specific effects. Na(+) currents in CAD cells and N1E-115 cells are similarly affected, indicating that benzonatate is not Na(+) channel subtype-specific. Benzonatate is a mixture of polyethoxy esters of 4-(butylamino) benzoic acid having varying degrees of hydrophobicity. We found that Na(+) currents are inhibited most potently by a benzonatate fraction containing the 9-ethoxy component. Detectable effects of benzonatate occur at concentrations as low as 0.3 μM, which has been reported in humans. We conclude that benzonatate has local anesthetic-like effects on voltage-gated sodium channels, including Nav1.7, which is a possible mechanism for cough suppression by the drug.