Search

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.

OBJECTIVE: The aim of this study was to identify a signature lipid profile from cerebral thrombi in acute ischemic stroke (AIS) patients at the time of ictus. METHODS: We performed untargeted lipidomics analysis using liquid chromatography-mass spectrometry on cerebral thrombi taken from a nonprobability, convenience sampling of adult subjects (≥18 years old, n = 5) who underwent thrombectomy for acute cerebrovascular occlusion. The data were classified using random forest, a machine learning algorithm. RESULTS: The top 10 metabolites identified from the random forest analysis were of the glycerophospholipid species and fatty acids. CONCLUSION: Preliminary analysis demonstrates feasibility of identification of lipid metabolomic profiling in cerebral thrombi retrieved from AIS patients. Recent advances in omic methodologies enable lipidomic profiling, which may provide insight into the cellular metabolic pathophysiology caused by AIS. Understanding of lipidomic changes in AIS may illuminate specific metabolite and lipid pathways involved and further the potential to develop personalized preventive strategies.

Background and Purpose: The University of Washington Clot Bank (UWCB) has established a tissue banking protocol optimized for “omic” analysis for cerebral thrombi retrieved during standard-of-care thrombectomy. Thrombi from cerebral arteries of acute ischemic stroke (AIS) patients are a novel resource that allow for the evaluation of lipid biological markers present at the time of ictus. The pathophysiological processes following AIS are complex, and involve ischemia, inflammation, and disturbances of lipid metabolism. The purpose of this study was to characterize the lipid profile from cerebral thrombi in AIS patients. Methods: The UWCB study is a non-probability, convenience sampling of adult subjects (> 18 years of age) who undergo thrombectomy for vascular occlusion. Lipidomics analysis was performed using LC-MS of untargeted lipids on thrombi taken from cerebral arteries of AIS subjects who underwent thrombectomy. Random forest, a supervised machine learning method, was employed to identify lipids associated with cerebral thrombi. Results: Five subjects (age = 53 ± 26.4, 3 males) were included from the UWCB registry. The most common comorbid conditions were atrial fibrillation (40%) and hypertension (40%). The top 10 metabolites identified from the random forest analysis, were of phosphatidylethanolamine molecular species. Phospholipids are involved in structural and functional integrity of cellular membranes and mediate host inflammatory responses. They are a key component of bacterial membranes. Changes in the structure or function of membrane phospholipids of host or relevant human microbiota may lead to alterations in membrane permeability and contribute to pathology of diseases such as neurodegeneration. Conclusion: An untargeted LC-MS lipidomics approach was used to identify lipids in cerebral thrombi at the time of mechanical thrombectomy. The top 10 lipids present in cerebral thrombi were identified, which included significant concentrations of phosphatidylethanolamine species. Because phospholipids play a key regulatory role in complex downstream cellular function, these findings may be of great significance in understanding AIS pathophysiology that has the potential to inform personalized preventive strategies.

Objective: To compare differences using a metabolomics approach in older adults (≥55) with mild traumatic brain injury (mTBI) to control adults and to identify a signature profile related to functional outcome 3–6 months post-injury. Methods: We performed metabolomics analysis using LC-MS of untargeted aqueous metabolites on plasma samples taken from a parent prospective cohort study. Older adults with mTBI (n = 14) were purposively sampled to include participants with worsening (decrease in GOS-E of at least 1 level) and improved (increase in GOS-E of at least 1 level) outcomes from 3 to 6 months. The data were analyzed using PLS-DA with VIP scores, Random Forest, and spline fit between the different groups as a function of time for exposure on outcome. Results: Separation of comparisons were seen at 24 hours (negative ionization) and 6 months (positive ionization), revealing two metabolites of interest, phosphatidylcholine and phosphatidylethanolamine. Phosphatidylcholine levels were higher in those with mTBI compared to controls ( p < 0.05), while lower concentration of phosphatidylethanolamine was seen in those with mTBI compared to controls ( p < 0.05). Phosphatidylinositol-3,4,5-trisphosphate was significant in those with mTBI compared to controls (n = 10) based on improved (n = 6) versus worsened (n = 8) outcomes from 3 to 6 months. Conclusion: We identified plasma metabolites related to phospholipid metabolism in older adults following mTBI and associated with long-term functional outcome. These findings may underly pathological mechanisms of outcome differences in older adults who experience mTBI.

INTRODUCTION: Primary brain tumors are the leading cause of cancer mortality in the United States affecting approximately 90,000 Americans each year. A major complication for brain tumor survivors is acute ischemic stroke (AIS). Currently, there are limited research to provide guidelines for AIS prevention and management in adult brain tumor survivors. The purpose of this review is to discuss the most common risk factors for AIS in adult brain tumor survivors along with best evidence for assessment, screening, and strategies to prevent AIS in this population. METHODS: Relevant literature was identified by searching CINAHL and PubMed databases using the following keywords: "brain tumor survivors," "adults," "stroke," "risk factors," "guidelines," "prevention," and "management". Articles not pertaining to adult brain tumor survivors and AIS were excluded. RESULTS: The location of the tumor, dose, extent, and type of radiation contribute to the development of vascular injury and subsequent carotid stenosis among brain tumor survivors. Endothelial growth factor inhibitor and chemotherapy drugs induces vascular remodeling. Other symptoms such as neurological impairments and co-morbidities are also present among brain tumor survivors. Furthermore, AIS increases from the time of primary brain tumor diagnosis and incidence further increases among patients who were diagnosed with a brain tumor as a child. CONCLUSION: Nurses play a key role in the assessment, prevention, and identifying individuals who are at risk of AIS during brain tumor survivorship. Engaging patients and their caregivers on minimizing their risks of AIS is crucial in the outpatient setting. Annual surveillance visits that include intracranial artery imaging should be used to identify individuals considered most at risk for developing AIS symptoms.

Background and Purpose: The ability to predict ischemic stroke outcomes in the first day of admission could be vital for patient counseling, rehabilitation, and care planning. The Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC; clinicaltrials.gov NCT03153683) collects blood samples distal and proximal to the intracranial thrombus during mechanical thrombectomy. These samples are a novel resource in evaluating acute gene expression changes at the time of ischemic stroke. The purpose of this study was to identify inflammatory genes and patient demographics that are predictive of stroke outcomes (infarct and/or edema volume) in acute ischemic stroke patients. Methods: The BACTRAC study is a non-probability, convenience sampling of subjects (≥ 18 year olds) treated with mechanical thrombectomy for emergent large vessel occlusion. We evaluated relative concentrations of mRNA for gene expression in 84 inflammatory molecules in static blood distal and proximal to the intracranial thrombus from adults who underwent thrombectomy. We employed a machine learning method, Random Forest, utilizing the first set of enrolled subjects, to predict which inflammatory genes and patient demographics were important features for infarct and edema volumes. Results: We analyzed the first 28 subjects (age = 66 ± 15.48, 11 males) in the BACTRAC registry. Results from machine learning analyses demonstrate that the genes CCR4, IFNA2, IL9, CXCL3, Age, DM, IL7, CCL4, BMI, IL5, CCR3, TNF, and IL27 predict infarct volume. The genes IFNA2, IL5, CCL11, IL17C, CCR4, IL9, IL7, CCR3, IL27, DM, and CSF2 predict edema volume. There is an intersection of genes CCR4, IFNA2, IL9, IL7, IL5, CCR3 to both infarct and edema volumes. Overall, these genes depicts a microenvironment for chemoattraction and proliferation of autoimmune cells, particularly Th2 cells and neutrophils. Conclusions: Machine learning algorithms can be employed to develop predictive biomarker signatures for stroke outcomes in ischemic stroke patients, particularly in regard to identifying acute gene expression changes that occur during stroke.

Proprioception of limbs and joints is a basic sensory function throughout most of the animal kingdom. It is important to understand how proprioceptive organs and the associated sensory neurons function with altered environments such as increased potassium ion concentrations ([K+]) from diseased states, ionic imbalances, and damaged tissues. These factors can drastically alter neuronal activity. To assess this matter, we used the chordotonal organ in a walking leg of a blue crab (Callinectes sapidus) and the muscle receptor organ of the crayfish (Procambarus clarkii). These organs serve as tractable models for the analysis of proprioception. The preparations can help serve as translational models for these effects, which may be observed in other invertebrate species as well as mammalian species (including humans). When extracellular potassium concentration ([K+]o) is increased to 20 mM in both preparations, mixed results are observed with activity increasing in some preparations and decreasing in others after mechanical displacement. However, when [K+]o is increased to 40 mM, activity drastically decreases in all preparations. Additionally, proprioceptor sensory activity declines upon exposure to a diluted muscle homogenate, which contains a host of intracellular constituents. The robust effects of altered [K+] on proprioception in these models illuminate the potential detriments on neuronal function in cases of severe tissue damage as well as altered [K+]o.

Introduction: Ischemic stroke remains one of the most debilitating diseases and is the fifth leading cause of death in the US. The ability to predict stroke outcomes within the acute period of stroke would be essential for care planning and rehabilitation. The Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC; clinicaltrials.gov NCT03153683) study collects arterial blood immediately distal and proximal to the intracranial thrombus at the time of mechanical thrombectomy. These blood samples are an innovative resource in evaluating acute gene expression changes at the time of ischemic stroke. The purpose of this study was to identify inflammatory genes and important immune factors during mechanical thrombectomy for emergent large vessel occlusion (ELVO) and which patient demographics were predictors for stroke outcomes (infarct and/or edema volume) in acute ischemic stroke patients. Methods: The BACTRAC study is a non-probability sampling of male and female subjects (≥18 year old) treated with mechanical thrombectomy for ELVO. We evaluated 28 subjects (66 ± 15.48 years) relative concentrations of mRNA for gene expression in 84 inflammatory molecules in arterial blood distal and proximal to the intracranial thrombus who underwent thrombectomy. We used the machine learning method, Random Forest to predict which inflammatory genes and patient demographics were important features for infarct and edema volumes. To validate the overlapping genes with outcomes, we perform ordinary least squares regression analysis. Results: Machine learning analyses demonstrated that the genes and subject factors CCR4, IFNA2, IL-9, CXCL3, Age, T2DM, IL-7, CCL4, BMI, IL-5, CCR3, TNFα, and IL-27 predicted infarct volume. The genes and subject factor IFNA2, IL-5, CCL11, IL-17C, CCR4, IL-9, IL-7, CCR3, IL-27, T2DM, and CSF2 predicted edema volume. The overlap of genes CCR4, IFNA2, IL-9, IL-7, IL-5, CCR3, and IL-27 with T2DM predicted both infarct and edema volumes. These genes relate to a microenvironment for chemoattraction and proliferation of autoimmune cells, particularly Th2 cells and neutrophils. Conclusions: Machine learning algorithms can be employed to develop prognostic predictive biomarkers for stroke outcomes in ischemic stroke patients, particularly in regard to identifying acute gene expression changes that occur during stroke.

Background and purpose Mechanical thrombectomy (MT) is the standard of care for emergent large vessel occlusion (ELVO), one of the most severe subtypes of ischemic stroke, which accounts for 30–40% of all cases. Through MT, we can isolate distal blood within the artery immediately downstream from the clot and compare it to systemic arterial blood to provide insight into local intraluminal changes during ischemia. CXCL9 is in interferon gamma-inducible chemokine that binds CXCR3, degrading endothelial tight junctions, attracting T cells, and facilitating immune cell extravasation into brain parenchyma. We aimed to study local CXCL9 expression distal to the intracranial thrombus during large vessel occlusion in human patients. Methods Tissue samples of distal and proximal blood were collected as part of the BACTRAC tissue bank (www.clinicaltrials.gov NCT03153683). Adult subjects with ELVO were prospectively enrolled, and arterial blood distal (intracranial) and proximal (cervical) were collected and processed to optimize RNA quality. RNA were isolated and used to evaluate gene expression in both samples for each subject; proximal systemic blood was used as an internal control for each subject. Results 22 subjects were included in this preliminary analysis. 15 (68.2%) were female. 54.5% of subjects had a CTA collateral score of 1 (18.2% had a score of 0). 4.5% (1 subject) did not attain TICI 2B or 3 recanalization. Infarct time (last known normal to thrombectomy recanalization) was 491 ± 243 minutes. Mean change in NIHSS from admission to discharge was -8 ± 8. CXCL9 expression in distal blood was upregulated an average of 106-fold with a maximum upregulation of 805-fold in one subject. In plotting CXCL9 expression against infarct time, there was a clear negative correlation (Spearman coefficient -0.43, p=0.05). Conclusion For the first time, we evaluate chemokine alterations in human stroke patients in distal stagnant blood during ELVO. There is a significant variance in CXCL9 expression in distal blood in relationship to infarct time, which mirrors the known timing of blood-brain barrier disruption. Disclosures J. Fraser: None. S. Martha: None. L. Collier: None. S. Davis: None. A. Alhajeri: None. S. Grupke: None. K. Pennypacker: None.

Introduction Ischemic stroke is the one of the most severe and debilitating diseases, and despite animal models, there is much to learn about the neuropathology in humans in a way that could inform the development of therapies. We have developed a protocol to collect and evaluate arterial blood immediately distal and proximal from the removed intracranial thrombus during mechanical thrombectomy. These samples provide a unique resource in evaluating acute changes in acid/base and electrolyte concentrations at the time of ischemic stroke. The purpose of this study was to compare acid/base and electrolytes obtained proximal and distal to the occluded intracranial thrombi between male and female acute ischemic stroke subjects at the time of thrombectomy; and to determine whether arterial blood gas values predict outcomes in male and female subjects. Methods We analyzed the first 49 subjects (age = 67 ± 15.0, 21 males) in the BACTRAC registry. We compared arterial blood gas of blood distal versus proximal to the thrombus during thrombectomy which provided acid/base levels (pH, pCO2, pO2, BD, HCO3−) and electrolyte values (iCa2+, K+, and Na+). Comparisons were evaluated by one-way repeated measures ANOVA (p Results In general, distal intracranial luminal blood sample showed a compensated metabolic acidosis with an elevated oxygen concentration in both blood samples. Analysis indicated several significant differences in the proximal blood samples between sexes (pH, pCO2, and K+). Bicarbonate and base deficit were predictors of infarct time specifically in female subjects. Discussion and conclusion Acid/base and electrolyte response to ischemic conditions differ between men and women, and these early changes could be used to predict local acid/base changes and how they develop differently in men and women during ischemia. These findings provide a novel insight into the pathology of large vessel stroke in humans, particularly potential variations based on sex.

BACKGROUND: Early changes in acid/base and electrolyte concentrations could provide insights into the development of neuropathology at the onset of stroke. We evaluated associations between acid/base and electrolyte concentrations, and outcomes in permanent middle cerebral artery occlusion (pMCAO) model. METHODS: 18-month old male and female Sprague-Dawley rats underwent pMCAO. Pre-, post- (7 minutes after occlusion), and at 72 hours of pMCAO venous blood samples provided pH, carbon dioxide, oxygen, glucose, hematocrit, hemoglobin, and electrolyte values of ionized calcium, potassium and sodium. Multiple linear regression determined predictors of infarct and edema volumes from these values, Kaplan-Meier curve analyzed morality between males and females at 72 hours, and a Cox regression model was used to determine predictors for mortality. RESULTS: Analysis indicated significant differences in acid/base balance and electrolyte levels in aged rats not dependent on sex between the three time points in the pMCAO model. Changes in pH (from pre- to post and post- to 72 hours) and changes in sodium and ionized calcium (from post- to 72 hours) were predictors of infarct volume and edema volume, respectively. Cox Regression revealed there is a 3.25 times increased risk for mortality based on changes in bicarbonate (pre- to post-MCAO). CONCLUSIONS: These early venous blood changes in acid/base balance and electrolytes can be used to predict stroke outcomes in our rat model of stroke. This study provides potential biomarkers to be examined in the human condition that could provide profound prognostic tools for stroke patients.

Background and Purpose: The Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) tissue bank collects blood proximal and distal to the intracranial thrombus during the process of mechanical thrombectomy in human stroke patients. These samples provide a unique resource in evaluating acute gene expression changes at the time of ischemic stroke. The purpose of this study was to evaluate gene expression changes occurring within the thrombus and across the occlusion in the intravascular space in acute ischemic stroke patients. Methods: We developed the BACTRAC protocol: an IRB-approved tissue banking approach for ELVO (clinicaltrials.gov NCT03153683). We evaluated relative concentrations of gene expression in 84 inflammatory molecules in thrombi removed from adults who received thrombectomy for ischemic stroke, in static blood distal to thrombus, and in peripheral circulation. Results: We analyzed the first 46 subjects (age = 67 ± 14.23, 20 males) in the BACTRAC registry. Results from qPCR gene expression analyses demonstrate that 21 genes (CCL1, CCL11, CCL13, CCL17, CCL26, CCL8, CSF3, CX3CL1, CXCL1, CXCL9, IFNA2, IL13, IL17C, IL17F, IL1A, IL27, IL3, IL33, LTA, TNFSF11, and TNFSF13) had at least 25 mean fold change in the distal blood compared to the peripheral blood. Fourteen genes (AIMP1, CCL11, CCL13, CCL15, CCL16, CCL23, CCR2, CCR4, CCR8, CD40LG, CXCL10, IL10RA, IL15, and TNFSF13B) had at least 10 mean fold change in the thrombus compared to the peripheral blood. Overall, these genes are associated with chemo-attraction and cell proliferation of monocytes, neutrophils, and T cells. Conclusions: These findings provide a novel insight into the initial pathology of large vessel stroke in humans, particularly in regard to identifying acute gene expression changes that occur during stroke.

Background and Purpose: In the setting of mechanical thrombectomy for emergent large vessel occlusion (ELVO), we have developed a protocol to collect and evaluate blood immediately distal and proximal from the removed intracranial thrombus. These samples provide a unique resource in evaluating acute changes in acid/base and electrolyte concentrations at the time of ischemic stroke. The purpose of this study is to compare acid/base and electrolyte differences obtained proximal and distal to the occluded intracranial thrombus in acute ischemic stroke patients. Methods: We developed the Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) protocol: an IRB-approved tissue banking strategy for ELVO (clinicaltrials.gov NCT03153683). We compared arterial blood gases (ABG) of blood distal versus proximal to the thrombus during thrombectomy. Comparisons were evaluated by Paired Samples T-Tests (p < 0.05). Results: We analyzed the first 46 subjects (age = 67 ± 14.23, 20 males) in the BACTRAC registry. Preliminary results demonstrate that, while pH is nonsignificant (p = 0.513), distal blood in relation to proximal blood showed significantly lower oxygen (p < 0.001), carbon dioxide (p < 0.001), bicarbonate (p < 0.001), ionized calcium (p < 0.001), and potassium (p < 0.001). Sodium concentration was significantly higher (p < 0.001) in distal blood. These results suggest alterations occurring intravascularly during ischemia. Conclusions: These preliminary findings provide a novel insight into the pathology of large vessel stroke in humans, particularly in regard to identifying acute changes in acid/base balance and electrolyte concentrations that occur during stroke.

Background Mechanical thrombectomy offers a unique opportunity to examine neurophysiological changes occurring during stroke. We developed a tissue banking protocol to capture intracranial thrombi as well as the blood immediately proximal and distal. These tissues provide a rare resource to correlate changes occurring at the time of stroke to patient outcomes. The purpose of this study is to develop a tissue banking protocol in order to provide high-quality yields of RNA for gene expression analysis and protein for proteomics. Methods We instituted an IRB-approved protocol for tissue processing during mechanical thrombectomy (www.clinicaltrials.gov NCT03153683). The protocol is a joint clinical/basic science effort among multiple laboratories and the NeuroInterventional Radiology service line. We developed a step-by-step process for specimen retrieval, initial processing, and storage, which begins in a designated workspace constructed for the study in the angiography suite. Further processing and analysis is performed in our basic science laboratories, and includes extraction of RNA from thrombi with distal and proximal blood samples to assess RNA integrity number (RIN) values. To demonstrate viability of the collected plasma, proteomics of distal and proximal blood was evaluated through an outside vendor (Olink Proteomics). Results We have enrolled 34 subjects (age=66±13.3, 14 males) in the BACTRAC registry thusfar. In our preliminary dataset, the process yielded adequate RNA for gene expression analysis and protein for proteomics. RNA integrity analysis reveal mean RIN values (±SD) for distal arterial blood (n=8) were 8.89±0.41 (range from 8.30 to 9.60) and mean RIN values (±SD) for proximal arterial blood (n=8) were 6.98±0.29 (range from 6.60 to 7.50), indicating moderate to high quality RNA with minimal degradation. Mean RIN values for the thrombi (n=7) were undetectable. The first subject samples of distal and proximal blood were analyzed for proteomics. Duplicates of these samples passed quality control, and were run on all 11 of their proteomic panels (92 proteins/panel) for a total of 1012 proteins. 100% of proteins were detected on cardiovascular 3 and neurology panels. The organ damage panel detected the fewest, with 61% of this panel. The cardiometabolic and cardiovascular 3 panels each showed the most proteins with the greatest differences in expression. Conclusion We have developed a novel tissue banking protocol utilizing mechanical thrombectomy to capture thrombus along with arterial blood distal and proximal to it. The protocol provides high-quality specimens facilitating analysis of the initial molecular response to ischemic stroke in the human condition for the first time. In addition, preliminary integrity analyses demonstrated high-quality yields for RNA and protein in distal and proximal blood samples. Disclosures S. Martha: None. L. Collier: None. A. Gorman: None. K. Salmeron: None. A. Trout: None. D. Edwards: None. S. Davis: None. A. Alhajeri: None. S. Grupke: None. J. Roberts: None. G. Bix: None. K. Pennypacker: None. J. Fraser: None.

Introduction In the setting of mechanical thrombectomy for emergent large vessel occlusion (ELVO), we have for the first time developed a protocol to collect and evaluate blood immediately distal and proximal from the removed intracranial thrombus. These samples provide a unique resource in evaluating acute changes in acid/base and electrolyte concentrations at the time of ischemic stroke. The purpose of this study is to compare acid/base and electrolyte differences obtained proximal and distal to the occluded intracranial thrombus in acute ischemic stroke patients. Methods We developed the BACTRAC protocol: an IRB-approved tissue banking strategy for ELVO (clinicaltrials.gov NCT03153683). We compared arterial blood gases (ABG) of blood distal versus proximal to the thrombus during thrombectomy. Comparisons were evaluated by Paired Samples T-Tests (p Results We analyzed the first 24 subjects (age=65±0.54, 11 males) in the BACTRAC registry. Preliminary results demonstrate that, while pH is nonsignificant, distal blood in relation to proximal blood showed significantly lower oxygen (p=0.010), carbon dioxide (p=0.001), bicarbonate (p=0.002), ionized calcium (p=0.001), and potassium (p=0.001). Sodium concentration was significantly higher (p=0.002) in distal blood. These results suggest alterations occurring intravascularly during ischemia. Conclusion These findings provide a novel insight into the pathology of large vessel stroke in humans, particularly in regard to identifying acute changes in acid/base balance and electrolyte concentrations that occur during stroke. Disclosures J. Fraser: None. S. Martha: None. L. Collier: None. S. Davis: None. A. Abdulnasser: None. S. Grupke: None. K. Pennypacker: None.

BackgroundIschemic stroke research faces difficulties in translating pathology between animal models and human patients to develop treatments. Mechanical thrombectomy, for the first time, offers a momentary window into the changes occurring in ischemia. We developed a tissue banking protocol to capture intracranial thrombi and the blood immediately proximal and distal to it.ObjectiveTo develop and share a reproducible protocol to bank these specimens for future analysis.MethodsWe established a protocol approved by the institutional review board for tissue processing during thrombectomy (www.clinicaltrials.govNCT03153683). The protocol was a joint clinical/basic science effort among multiple laboratories and the NeuroInterventional Radiology service line. We constructed a workspace in the angiography suite, and developed a step-by-step process for specimen retrieval and processing.ResultsOur protocol successfully yielded samples for analysis in all but one case. In our preliminary dataset, the process produced adequate amounts of tissue from distal blood, proximal blood, and thrombi for gene expression and proteomics analyses. We describe the tissue banking protocol, and highlight training protocols and mechanics of on-call research staffing. In addition, preliminary integrity analyses demonstrated high-quality yields for RNA and protein.ConclusionsWe have developed a novel tissue banking protocol using mechanical thrombectomy to capture thrombus along with arterial blood proximal and distal to it. The protocol provides high-quality specimens, facilitating analysis of the initial molecular response to ischemic stroke in the human condition for the first time. This approach will permit reverse translation to animal models for treatment development.

Background and Purpose: Monitoring for hypotension, hyperglycemia, hypoxia, and dehydration is key to reducing early death in stroke patients. Evaluation of physiological predictors of infarct volume and mortality may provide opportunities for effective interventions to improve outcomes. The purpose of this study is: 1) to describe and compare the predictive effects of venous blood gas (VBG) on infarct volume and mortality in acute stroke in rats; 2) and we have begun to collect arterial blood gas (ABG) to compare differences obtained proximal and distal to the occluded intracranial thrombi in acute ischemic stroke patients. Methods: 3-month old Sprague-Dawey rats (n = 9) underwent permanent or transient middle cerebral artery occlusion (MCAO). Pre- and post-MCAO venous samples provided pH, pCO 2 , pO 2 , and electrolyte values (iCa 2+ , K + , and Na + ). Linear regression determined predictors of infarct volume from these values, and Cox regression analyzed VBG changes between tMCAO (n = 28) and pMCAO (n = 29) to determine predictors of mortality. We compared mean proximal and distal pH, pCO 2 , pO 2 , and electrolytes (iCa 2+ , K + , and Na + ) in stroke patients (n = 7) arterial samples using Wilcoxon Signed Ranks test. Results: Animal studies demonstrated pH and iCa 2+ are predictors of infarct volume, but not mortality. After pMCAO (n = 9), change in pH or iCa 2+ significantly predicted infarct volume [F(1,7) = 7.351, β = -0.716, p = 0.03] and [F(1, 7) = 6.782, β = -0.701, p = 0.035]; as pH and calcium decreased, infarct volume increased. These variables explained 44% and 42% of the total variance in these models. In human patients (n = 7), there were significant differences in blood samples proximal and distal to the intracranial thrombus for pCO 2 (p = 0.018), HCO 3- (p = 0.028), iCa 2+ (p = 0.043), K + (p = 0.028), and Na + (p = 0.044). Conclusions: In conclusion, there are acute changes in acid/base balance and electrolytes during stroke in rodent models and humans. In cross-species comparison ionized calcium changes were significant in both, with iCa 2+ changes predicting stroke volume in the rat model. These preliminary findings are novel, and warrant further exploration in human patients.

Objective: To determine whether the anti-inflammatory effects of leukemia inhibitory factor (LIF) are altered in aged male and female rats. Methods: Focal ischemia was induced in 3 month old male and 18 month old male/female Sprague-Dawley using the middle cerebral artery occlusion (MCAO) procedure. Animals were treated with PBS or LIF at 6, 24, and 48 h after MCAO (125 μg/kg). Infarct volume in aged animals was quantified with T2-weighted MRI. Functional motor skills were assessed immediately prior to euthanization at 72 h post-MCAO. Protein levels of CD11b and IL-12 p40 in brain and spleen tissue were measured using immunoblotting. Results: At 72 h, there was a trend towards decreased infarct volume in aged male (189 ± 45OD; n=4) and female LIF-treated rats (104 ± 71 OD; n=4) compared to PBS-treated aged male (188 ± 45; n=4) and female rats (179 ± 72; n=5). LIF-treated aged female rats showed significant decreases in circling (p=0.0209) and body swing bias (p=0.05) compared to the PBS-treated aged females. Normalized CD11b levels were significantly in LIF-treated young males (1.1 ± 0.3 OD; n=5) compared to PBS-treated young males (2.5 ± 0.5 OD; n=6; p Conclusions: LIF is more effective at decreasing splenic IL-12 expression and immune cell infiltration in young rats compared to aged rats. However, LIF counteracts the post-stroke splenic response in aged females but not aged male rats.

Background and Purpose Acid/base and electrolytes could provide clinically valuable information about cerebral infarct core and penumbra. We evaluated associations between acid/base and electrolyte changes and outcomes in 2 rat models of stroke, permanent, and transient middle cerebral artery occlusion. Methods Three-month old Sprague-Dawley rats underwent permanent or transient middle cerebral artery occlusion. Pre- and post-middle cerebral artery occlusion venous samples for permanent and transient models provided pH, carbon dioxide, oxygen, glucose, and electrolyte values of ionized calcium, potassium, and sodium. Multiple regression determined predictors of infarct volume from these values, and Kaplan-Meier curve analyzed morality between permanent and transient middle cerebral artery occlusion models. Results Analysis indicated significant differences in the blood gas and electrolytes between pre- to post-middle cerebral artery occlusion. A decrease in pH and sodium with increases in carbon dioxide, potassium, ionized calcium, and glucose changes were found in both middle cerebral artery occlusion models; while hematocrit and hemoglobin were significant in the transient model. pH and ionized calcium were predictors of infarct volume in the permanent model, as changes in pH and ionized calcium decreased, infarct volume increased. Conclusions There are acute changes in acid/base balance and electrolytes during stroke in transient and permanent rodent models. Additionally, we found pH and ionized calcium changes predicted stroke volume in the permanent middle cerebral artery occlusion model. These preliminary findings are novel, and warrant further exploration in human conditions.

Proprioceptive neurons monitor the movements of limbs and joints to transduce the movements into electrical signals. These neurons function similarly in species from arthropods to humans. These neurons can be compromised in disease states and in adverse environmental conditions such as with changes in external and internal pH. We used two model preparations (the crayfish muscle receptor organ and a chordotonal organ in the limb of a crab) to characterize the responses of these proprioceptors to external and internal pH changes as well as raised CO2. The results demonstrate the proprioceptive organs are not highly sensitive to changes in extracellular pH, when reduced to 5.0 from 7.4. However, if intracellular pH is decreased by exposure to propionic acid or saline containing CO2, there is a rapid decrease in firing rate in response to joint movements. The responses recover quickly upon reintroduction of normal pH (7.4) or saline not tainted with CO2. These basic understandings may help to address the mechanistic properties of mechanosensitive receptors in other organisms, such as muscle spindles in skeletal muscles of mammals and tactile as well as pressure (i.e., blood pressure) sensory receptors. KEYWORDS: Proprioception; Sensory; Invertebrate; Carbon Dioxide; Protons; Mechanosensory; Intracellular pH; Extracellular pH

Background Mutations that impair mitochondrial functioning are associated with a variety of metabolic and age-related disorders. A barrier to rigorous tests of the role of mitochondrial dysfunction in aging processes has been the lack of model systems with relevant, naturally occurring mitochondrial genetic variation. Toward the goal of developing such a model system, we studied natural variation in life history, metabolic, and aging phenotypes as it relates to levels of a naturally-occurring heteroplasmic mitochondrial ND5 deletion recently discovered to segregate among wild populations of the soil nematode, Caenorhabditis briggsae. The normal product of ND5 is a central component of the mitochondrial electron transport chain and integral to cellular energy metabolism. Results We quantified significant variation among C. briggsae isolates for all phenotypes measured, only some of which was statistically associated with isolate-specific ND5 deletion frequency. We found that fecundity-related traits and pharyngeal pumping rate were strongly inversely related to ND5 deletion level and that C. briggsae isolates with high ND5 deletion levels experienced a tradeoff between early fecundity and lifespan. Conversely, oxidative stress resistance was only weakly associated with ND5 deletion level while ATP content was unrelated to deletion level. Finally, mean levels of reactive oxygen species measured in vivo showed a significant non-linear relationship with ND5 deletion level, a pattern that may be driven by among-isolate variation in antioxidant or other compensatory mechanisms. Conclusions Our findings suggest that the ND5 deletion may adversely affect fitness and mitochondrial functioning while promoting aging in natural populations, and help to further establish this species as a useful model for explicit tests of hypotheses in aging biology and mitochondrial genetics.