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Ischemic stroke is the leading cause of disability world-wide and affects over 800,000 people per year in the United States. The majority of these strokes are ischemic due to a blockage of blood flow to the brain. Damage to the brain occurs at the onset of stroke, neuronal cell death is irreversible and therefore, quick treatment to remove blockage is critical factor in the recovery from stroke. Mechanical thrombectomy as a treatment for ischemic stroke provides an ideal opportunity to collect blood distal and proximal to the cerebral thrombus to examine neurochemical changes occurring during stroke. The purpose of this dissertation was to explore the trajectory of neurochemical changes that occur in response to ischemic stroke during the first 72 hours and the physiological response from stroke patients to improve stroke outcomes. The specific aims were to: 1) to determine whether venous blood gases predict infarct volume and/or mortality in acute ischemic stroke in young male rats; 2) determine whether venous blood gases predict infarct and edema volume, and/or mortality in acute ischemic stroke in aged male and female rats; 3) compare the presence and relative concentrations of acid/base and electrolytes in static blood distal to thrombus and in peripheral blood drawn from adults who received thrombectomy for ischemic stroke and identify associations to postreperfusion functional outcomes. Specific Aim One was addressed by evaluation of young (three-month old) Sprague-Dawley rats that underwent permanent or transient middle cerebral artery occlusion (MCAO). Pre- and post-MCAO venous samples from permanent and transient models provided pH, carbon dioxide, oxygen, bicarbonate, glucose, hematocrit, hematocrit, and electrolyte values of ionized calcium, potassium and sodium. The analyses indicated that mean differences in the blood gas and electrolytes between pre- to post-MCAO and pH and iCa2+ were predictors of infarct volume in the permanent MCAO model. The second aim was addressed by evaluation of aged (18 month old) male and female rats pre-MCAO, post-MCAO, and at 72 hours of permanent MCAO venous blood gas samples (pH, carbon dioxide, oxygen, bicarbonate, glucose, hematocrit, hematocrit, and electrolyte concentrations of ionized calcium, potassium and sodium). Changes in pH (from pre-MCAO to post-MACO and post-MCAO to 72 hours) and changes in Na+ and iCa2+ (from post-MCAO to 72 hours) were predictors of infarct volume and edema volume, respectively in both sexes. Cox regression revealed there was a 3.25 times increased risk for mortality based on changes (cut-off range within -2.00 to - 7.00) in bicarbonate levels (pre- to post-MCAO). The third aim was addressed by evaluation of acid/base balance (pH, carbon dioxide, oxygen, bicarbonate, ionized calcium, potassium and sodium) of ischemic stroke patients who underwent mechanical thrombectomy. Our results suggests sex differences matter in ischemic stroke populations. Significant differences occur within proximal blood between the sexes. Additionally, females had approximately 2.5 hour increased time between stroke symptom onset to thrombectomy completion time (described as infarct time). Changes in bicarbonate and base deficit were predictors of infarct time, but only in our female population.

Background: Mechanical thrombectomy is the gold standard for treating patients with certain acute ischemic stroke (AIS) due to large vessel occlusion (LVO). However, even with major advancements and increasing procedural volumes, acute endovascular therapy remains a high-risk procedure with a considerable 90-day mortality rate, affected by a variety of factors. Purpose: To investigate various clinical and procedural factors associated with 90-day mortality in patients undergoing mechanical thrombectomy for emergent treatment of AIS and determine which of these factors made unique contributions to post-thrombectomy prognosis. Methods: We examined a prospective registry of 323 patients treated with endovascular thrombectomy for AIS between 2016 and 2019 at a high-volume comprehensive stroke center in central Massachusetts. We developed two multivariable logistic regression models adjusting for the contributions of baseline characteristics and recanalization parameters, to identify potential predictors of mortality at 90 days. Results: Among 323 AIS patients treated with mechanical thrombectomy, the overall rate of successful recanalization was 86% and the overall post-procedure mortality rate was 29% by 90 days. After univariate analysis, a baseline multivariable model comprised of: history of stroke (OR 0.28, 95% CI 0.09 – 0.68), pre-stroke modified Rankin Scale (mRS 2: OR 3.75, 95% CI), severe admission National Institutes of Health Stroke Scale (NIHSS 21–42: OR 12.36, 95% CI 1.48 – 103.27), internal carotid artery (ICA) occlusion (OR 2.77, 95% CI 1.18 – 6.55), and posterior circulation occlusion (OR 2.69, 95% CI 1.06 – 6.83) was prognostic of 90-day mortality. A second multivariable model also found the procedural factors of: clot obtained after each pass (OR 0.49, 95% CI 0.24 – 1.00), successful recanalization (OR 0.21, 95% CI 0.06 – 0.8) and symptomatic intracranial hemorrhage (sICH; OR 17.89, 95% CI 5.22 – 61.29) to be identifiable predictors of post-thrombectomy mortality. Conclusion: Death within 90 days after thrombectomy was increased among patients with higher pre-stroke disability, higher stroke severity on admission, ICA or posterior occlusion, and those with sICH complication. A history of stroke, clot extraction after each device pass, and successful recanalization are associated with decreased 90-day mortality. These identifiable contributors may inform patient selection, prognosis evolution, and shared decision-making regarding emergent thrombectomy for treatment of AIS.

Tissue engineering is an emerging field that offers novel and unmatched potential medical therapies and treatments. While the vast aim of tissue engineering endeavors is to provide clinically implantable constructs, secondary applications have been developed to utilize tissue-engineered constructs for in-vitro evaluation of devices and therapies. Specifically, in-vitro blood vessel mimics (BVM) have been developed to create a bench-top blood vessel model using human cells that can be used to test and evaluate vascular disease treatments and intravascular devices. Previous BVM work has used fat derived human microvascular endothelial cells (EC) sodded on an ePTFE scaffold. To create a more physiologically accurate model, a dual layer of large vessel endothelial and smooth muscle cells (SMC) on an ePTFE tube is investigated throughout this thesis. Human umbilical vein endothelial cells (HUVEC) and human umbilical vein smooth muscle cells (HUVSMC) were chosen as the large vessel cell types and cultivated according to standard procedures. Before dual sodding, sodding density experiments with HUVSMC were performed to determine the number of cells required to create a confluent cell layer. HUVSMC sodded by trans-luminal pressure at densities ranging from 3.5x10^5 cells/cm^2 to 1.0x10^6 cells/cm^2 were run for one day to observe luminal coverage. After determining the desirable range for HUVSMC sodding, HUVSMC experiments with 5.0x10^5 cells/cm^2 and 7.5x10^5 cells/cm^2 were run over seven days to evaluate progression of the graft over time. Histology and SEM methods were used for analysis. A HUVEC study was next conducted over 7 days to confirm that the large vessel endothelial cell could be sodded and sustained on ePTFE in-vitro. Next, dual sodding was performed by pressure sodding HUVSMC at 7.5x10^5 cells/cm^2 followed by trans-luminal flow for 30 minutes. HUVECs were subsequently trans-luminally pressure sodded at 5.0x10^5 cells/cm^2 followed by an additional 30 minutes of trans-luminal flow; perfusion flow began following the final 30 minutes of trans-luminal flow. Experiments for the dual layered grafts were run for both one and seven days to evaluate and develop the dual sodding protocol as well as observe the co-culture over time. Analysis of the dual layered grafts was performed by SEM, histology, and fluorescence microscopy. HUVECs were incubated with Cell Tracker™ prior to dual sodding and both cell types with bisbenzimide after graft harvest to attempt to distinguish between cell types. Results from the thesis illustrate that large vessel smooth muscle and endothelial cells can be sodded onto ePTFE scaffolds and sustained within the in-vitro BVM system for up to 7 days. Furthermore, cost analysis demonstrates that the addition of a smooth muscle cell layer adds minimal costs to the BVM system. In conclusion, the studies contained within this thesis culminate in a protocol for the dual sodding of smooth muscle and endothelial cells with the aim of creating a physiologically representative co-culture blood vessel mimic.

Stroke is the 5th leading cause of death in the U.S. with 130,000 deaths and around 800,000 affected annually. Currently, there is a significant disconnect between basic stroke research and clinical stroke therapeutic needs. Few animal models of stroke target the large vessels that produce cortical deficits seen in the clinical setting. Also, current routes of drug administration, intraperitoneal and intravenous, do not mimic the clinical route of intra-arterial drug administration. To bridge this divide, we have retro-engineered a mouse model of stroke from the current standard of care for emergent large vessel occlusion (ELVO) stroke, endovascular thrombectomy, to include selective intra-arterial pharmacotherapy administration. Using the tandem transient common carotid and middle cerebral artery occlusion (MCAo) model to induce stroke, we threaded micro-angio tubing into the external carotid artery (ECA) towards the bifurcation of the common carotid and internal carotid arteries (CCA/ICA) allowing for the delivery of agents to the site of acute ischemia. Our model was optimized through a flow rate and injection volume study using carbon black ink injected through the intra-arterial model at different flow rates and injection volumes. The purpose of this study was to demonstrate that our injections were arriving at the site of ischemia and to improve injection volumes for future dosing while mitigating systemic side effects by preventing or minimizing systemic distribution. We determined that a flow rate of 2.5 µl/minute and injection volume of 10 µl was optimal. Next, we tested potential neuroprotective compounds nitroglycerin, verapamil, and a combination of verapamil and lubeluzole. Compounds were chosen for drug synergy and to target specific pathways in either an acute or delayed manner. Acute treatments included nitroglycerin and/or verapamil while delayed treatment included lubeluzole. The known mechanism of action for FDA approved nitroglycerin is through vessel dilation that results in increased blood flow to the treated region. A secondary mechanism of nitroglycerin is the production of nitric oxide, which has demonstrated antioxidant and anti-apoptotic effects when processed and released from cells surrounding the blood vessels. Verapamil, a calcium channel blocker, also FDA-approved for cerebral artery vasospasm: is thought to act by blocking the L-type calcium channels on the cell membrane from opening following membrane depolarization after insult. Finally, lubeluzole, also FDA-approved, is proposed to work as an NMDA modulator inhibiting the release of glutamate and nitric oxide synthase and blocking sodium and calcium channels. Through our stroke model we were able to demonstrate that each drug(s) showed a significant decrease in infarct volume and improved functional recovery while simultaneously minimizing potential systemic side effects suggesting that our stroke model may improve the preclinical validation of potential stroke therapies and help bridge the bench to bedside divide in developing new stroke therapies.

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