Human Stroke Research Articles

Blood-based biomarkers and stem cell therapy in human stroke: a systematic review.

Stroke is one of the main causes of death and disability worldwide. Cell therapy represents a promising therapeutic approach to improve stroke outcome. Measurement of blood-based biomarkers might serve as a proof-of-concept to monitor the mechanisms undergirding these treatments, and such compounds could be used as surrogate biomarkers to monitor the safety and efficacy of cell therapies in the future. Additionally, the measurement of biomarkers that correlate with circulating stem cells in observational studies might be of interest to improve the understanding of how these cells are spontaneously mobilized and carry out their action after stroke. Thus, a systematic review has been herein performed on blood-based biomarkers assessed in stroke patients treated with cell therapy or in observational studies in which circulating stem cells have been measured after stroke.

Show Me Your Friends and I Tell You Who You Are: The Many Facets of Prion Protein in Stroke.

Ischemic stroke belongs to the leading causes of mortality and disability worldwide. Although treatments for the acute phase of stroke are available, not all patients are eligible. There is a need to search for therapeutic options to promote neurological recovery after stroke. The cellular prion protein (PrPC) has been consistently linked to a neuroprotective role after ischemic damage: it is upregulated in the penumbra area following stroke in humans, and animal models of stroke have shown that lack of PrPC aggravates the ischemic damage and lessens the functional outcome. Mechanistically, these effects can be linked to numerous functions attributed to PrPC: (1) as a signaling partner of the PI3K/Akt and MAPK pathways, (2) as a regulator of glutamate receptors, and (3) promoting stem cell homing mechanisms, leading to angio- and neurogenesis. PrPC can be cleaved at different sites and the proteolytic fragments can account for the manifold functions. Moreover, PrPC is present on extracellular vesicles (EVs), released membrane particles originating from all types of cells that have drawn attention as potential therapeutic tools in stroke and many other diseases. Thus, identification of the many mechanisms underlying PrPC-induced neuroprotection will not only provide further understanding of the physiological functions of PrPC but also new ideas for possible treatment options after ischemic stroke.

Multi-frequency symmetry difference electrical impedance tomography with machine learning for human stroke diagnosis

Objective: Multi-frequency symmetry difference electrical impedance tomography (MFSD-EIT) can robustly detect and identify unilateral perturbations in symmetric scenes. Here, an investigation is performed to assess if the algorithm can be successfully applied to identify the aetiology of stroke with the aid of machine learning. Methods: Anatomically realistic four-layer finite element method models of the head based on stroke patient images are developed and used to generate EIT data over a 5 Hz–100 Hz frequency range with and without bleed and clot lesions present. Reconstruction generates conductivity maps of each head at each frequency. Application of a quantitative metric assessing changes in symmetry across the sagittal plane of the reconstructed image and over the frequency range allows lesion detection and identification. The algorithm is applied to both simulated and human (n = 34 subjects) data. A classification algorithm is applied to the metric value in order to differentiate between normal, haemorrhage and clot values. Main results: An average accuracy of 85% is achieved when MFSD-EIT with support vector machines (SVM) classification is used to identify and differentiate bleed from clot in human data, with 77% accuracy when differentiating normal from stroke in human data. Conclusion: Applying a classification algorithm to metrics derived from MFSD-EIT images is a novel and promising technique for detection and identification of perturbations in static scenes. Significance: The MFSD-EIT algorithm used with machine learning gives promising results of lesion detection and identification in challenging conditions like stroke. The results imply feasible translation to human patients.

Acute Neurofilament Light Chain Plasma Levels Correlate With Stroke Severity and Clinical Outcome in Ischemic Stroke Patients.

Background: Ischemic stroke causes increased blood–brain barrier permeability and release of markers of axonal damage and inflammation. To investigate diagnostic and prognostic roles of neurofilament light chain (NF-L), we assessed levels of NF-L, S100B, interleukin-6 (IL-6), E-selectin, vascular endothelial growth factor-A (VEGF-A), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) in patients with acute ischemic stroke or transient ischemic attack (TIA) and healthy controls.Methods: We studied neurofilament (NF) expression in 2 cases of human postmortem ischemic stroke, representing infarcts aged 3- to >7-days. In a prospective study, we measured plasma NF-L and inflammatory markers <8 h of symptom onset and at 72 h in acute ischemic stroke (n = 31), TIA (n = 9), and healthy controls (n = 29). We assessed whether NF-L, S100B, and IL-6 were associated with clinical severity on admission (Scandinavian Stroke Scale, SSS), diagnosis of ischemic stroke vs. TIA, and functional outcome at 3 months (modified Rankin Scale, mRS).Results: NF expression increased in ischemic neurons and in the infarcted brain parenchyma after stroke. Plasma NF-L levels were higher in stroke patients than in TIA patients and healthy controls, but IL-6 levels were similar. Higher acute NF-L levels were associated with lower SSS scores at admission and higher mRS scores at 3 months. No correlation was observed between NF-L and S100B, NF-L and IL-6, nor between S100B or IL-6 and SSS or mRS. Compared to controls, stroke patients had significantly higher VEGF-A and VCAM-1 at <8 h that remained elevated at 72 h, with significantly higher VEGF-A at <8 h; ICAM-1 was significantly increased at <8 h, while S100B and E-selectin were unchanged.Conclusions: Plasma NF-L levels, but not IL-6 and S100B, were significant predictors of clinical severity on admission and functional outcome at 3 months. Plasma NF-L is a promising biomarker of functional outcome after ischemic stroke.

Characterization of the TNF and IL-1 systems in human brain and blood after ischemic stroke

Preclinical and clinical proof-of-concept studies have suggested the effectiveness of pharmacological modulation of inflammatory cytokines in ischemic stroke. Experimental evidence shows that targeting tumor necrosis factor (TNF) and interleukin (IL)-1 holds promise, and these cytokines are considered prime targets in the development of new stroke therapies. So far, however, information on the cellular expression of TNF and IL-1 in the human ischemic brain is sparse.We studied 14 cases of human post-mortem ischemic stroke, representing 21 specimens of infarcts aged 1 to > 8 days. We characterized glial and leukocyte reactions in the infarct/peri-infarct (I/PI) and normal-appearing tissue (NAT) and the cellular location of TNF, TNF receptor (TNFR)1 and TNFR2, IL-1α, IL-1β, and IL-1 receptor antagonist (IL-1Ra). The immunohistochemically stained tissue sections received a score reflecting the number of immunoreactive cells and the intensity of the immunoreactivity (IR) in individual cells where 0 = no immunoreactive cells, 1 = many intermediately to strongly immunoreactive cells, and 2 = numerous and intensively immunoreactive cells. Additionally, we measured blood TNF, TNFR, and IL-1 levels in surviving ischemic stroke patients within the first 8 h and again at 72 h after symptom onset and compared levels to healthy controls.We observed IL-1α and IL-1β IR in neurons, glia, and macrophages in all specimens. IL-1Ra IR was found in glia, in addition to macrophages. TNF IR was initially found in neurons located in I/PI and NAT but increased in glia in older infarcts. TNF IR increased in macrophages in all specimens. TNFR1 IR was found in neurons and glia and macrophages, while TNFR2 was expressed only by glia in I/PI and NAT, and by macrophages in I/PI. Our results suggest that TNF and IL-1 are expressed by subsets of cells and that TNFR2 is expressed in areas with increased astrocytic reactivity. In ischemic stroke patients, we demonstrate that plasma TNFR1 and TNFR2 levels increased in the acute phase after symptom onset compared to healthy controls, whereas TNF, IL-1α, IL-1β, and IL-1Ra did not change.Our findings of increased brain cytokines and plasma TNFR1 and TNFR2 support the hypothesis that targeting post-stroke inflammation could be a promising add-on therapy in ischemic stroke patients.

Imaging Ischemic and Hemorrhagic Disease of the Brain in Dogs

Strokes, both ischemic and hemorrhagic, are the most common underlying cause of acute, non-progressive encephalopathy in dogs. In effect, substantial information detailing the underlying causes and predisposing factors, affected vessels, imaging features, and outcomes based on location and extent of injury is available. The features of canine strokes on both computed tomography (CT) and magnetic resonance imaging (MRI) have been described in numerous studies. This summary article serves as a compilation of these various descriptions. Drawing from the established and emerging stroke evaluation sequences used in the investigation of strokes in humans, this summary describes all theoretically available sequences. Particular detail is given to logistics of image acquisition, description of imaging findings, and each sequence's advantages and disadvantages. As the imaging features of both forms of strokes are highly representative of the underlying pathophysiologic stages in the hours to months following stroke onset, the descriptions of strokes at various stages are also discussed. It is unlikely that canine strokes can be diagnosed within the same rapid time frame as human strokes, and therefore the opportunity for thrombolytic intervention in ischemic strokes is unattainable. However, a thorough understanding of the appearance of strokes at various stages can aid the clinician when presented with a patient that has developed a stroke in the days or weeks prior to evaluation. Additionally, investigation into new imaging techniques may increase the sensitivity and specificity of stroke diagnosis, as well as provide new ways to monitor strokes over time.

Immune responses to stroke: mechanisms, modulation, and therapeutic potential.

Stroke is the second leading cause of death worldwide and a leading cause of disability. Most strokes are caused by occlusion of a major cerebral artery, and substantial advances have been made in elucidating how ischemia damages the brain. In particular, increasing evidence points to a double-edged role of the immune system in stroke pathophysiology. In the acute phase, innate immune cells invade brain and meninges and contribute to ischemic damage, but may also be protective. At the same time, danger signals released into the circulation by damaged brain cells lead to activation of systemic immunity, followed by profound immunodepression that promotes life-threatening infections. In the chronic phase, antigen presentation initiates an adaptive immune response targeted to the brain, which may underlie neuropsychiatric sequelae, a considerable cause of poststroke morbidity. Here, we briefly review these pathogenic processes and assess the potential therapeutic value of targeting immunity in human stroke.

Targeting GSNOR for functional recovery in a middle-aged mouse model of stroke

The nitric oxide (NO) metabolome and the NO metabolite-based neurovascular protective pathways are dysregulated after stroke. The major NO metabolite S-nitrosoglutahione (GSNO) is essential for S-nitrosylation-based signaling events and the inhibition of S-nitrosoglutahione (GSNO)-metabolizing enzyme GSNO reductase (GSNOR) provides protective effects following cardiac ischemia. However, the role of GSNOR and GSNOR inhibition-mediated increased GSNO/S-nitrosylation is not understood in neurovascular diseases such as stroke. Because age is the major risk factor of stroke and recovery in aged stroke patients is low and slow, we investigated the efficacy of GSNOR inhibition using a GSNOR selective inhibitor N6022 in a clinically relevant middle-aged cerebral ischemia and reperfusion (IR) mouse model of stroke. N6022 (5 mg/kg; iv) treatment of IR mice at 2 h after reperfusion followed by the treatment of the same dose daily for 3 days reduced the infarct volume and decreased the neurological score. Daily treatment of IR animals with N6022 for 2 weeks significantly improved neurological score, brain infarctions/atrophy, survival rate, motor (measured by cylinder test) and cognitive (evaluated by novel object recognition test) functions which paralleled the decreased activity of GSNOR, reduced levels of peroxynitrite and decreased neurological score. These results are the first evidence of a new pathway for the treatment of stroke via the inhibition of GSNOR. Based on the efficacy of N6022 in the stroke animal model and its use in human therapeutic studies without toxicity, we submit that GSNOR is a druggable target, and N6022 is a promising drug candidate for human stroke therapy.

PCN202 PREVALENCE OF PRE-EXISTING MULTIMORBIDITY AMONG OLDER PATIENTS WITH LATE-STAGE MELANOMA

Presence of multimorbidity can affect prognosis, treatment, and outcomes of patients with cancer. We estimated the prevalence of any and type of pre-existing multimorbidity (autoimmune (AD), physical health (PHC), and mental health conditions (MHC)) among older adults with late-stage melanoma in the United States. We derived data on older fee-for-service Medicare beneficiaries (age >65 years) diagnosed with late-stage (stage III/IV) melanoma between 2011 and 2015 (N = 4,519) from the linked SEER cancer registry and Medicare claims. We defined multimorbidity as the prevalence of two or more chronic conditions prior to the diagnosis of melanoma. Further, PHC consisted of pre-existing AD, arthritis, asthma, coronary artery disease, cardiac arrhythmias, diabetes, congestive heart failure, chronic kidney disease, chronic obstructive pulmonary disease, hepatitis, hyperlipidemia, hypertension, human immunodeficiency virus, obesity, osteoporosis, and stroke while MHC consisted of anxiety, bipolar disorder, depression, psychoses, schizophrenia, and substance abuse. We further explored any multimorbidity using combinations of dyads and triads. Chi-square test was used to identify significant unadjusted associations of individual characteristics to multimorbidity. An overwhelming majority (85%) of older patients with late-stage melanoma had multimorbidity. Pre-existing PHC multimorbidity (84%) was the most prevalent, followed by AD (12%), and MHC (6%) multimorbidity. Among those with any multimorbidity, the most prevalent dyad combination was endocrine and musculoskeletal disorder combination (21.7%) followed by cardiovascular (CVD) and endocrine disorder combination (20.3%). Among triads, CVD, endocrine, and musculoskeletal disorder combination (22.6%) was the most prevalent followed by endocrine, MHC, and musculoskeletal disorder combination (7%). Although oncologists and other healthcare providers are aware of high prevalence of multimorbidity among older patients, evidence on prevalence in melanoma patients is lacking. This is the first real-world study to confirm oncologists’ suspicions and helps guide them to develop systematic approaches to optimize care of older patients with late-stage melanoma.

Hypothesis and Theory: A Pathophysiological Concept of Stroke-Induced Acute Phase Response and Increased Intestinal Permeability Leading to Secondary Brain Damage.

Gut integrity impairment leading to increased intestinal permeability (IP) is hypothesized to be a trigger of critically illness. Approximately 15–20% of human ischemic stroke (IS) victims require intensive care, including patients with impaired level of consciousness or a high risk for developing life-threatening cerebral edema. Local and systemic inflammatory reactions are a major component of the IS pathophysiology and can significantly aggravate brain tissue damage. Intracerebral inflammatory processes following IS have been well studied. Until now, less is known about systemic inflammatory responses and IS consequences apart from a frequently observed post-IS immunosuppression. Here, we provide a hypothesis of a crosstalk between systemic acute phase response (APR), IP and potential secondary brain damage during acute and subacute IS stages supported by preliminary experimental data. Alterations of the acute phase proteins (APPs) C-reactive protein and lipopolysaccharide-binding protein and serum level changes of antibodies directed against Escherichia coli-cell extract antigen (IgA-, IgM-, and IgG-anti-E. coli) were investigated at 1, 2, and 7 days following IS in ten male sheep. We found an increase of both APPs as well as a decrease of all anti-E. coli antibodies within 48 h following IS. This may indicate an early systemic APR and increased IP, and underlines the importance of the increasingly recognized gut-brain axis and of intestinal antigen release for systemic immune responses in acute and subacute stroke stages.

Optimized mouse model of embolic MCAO: From cerebral blood flow to neurological outcomes

The embolic middle cerebral artery occlusion (eMCAO) model mimics ischemic stroke due to large vessel occlusion in humans and is amenable to thrombolytic therapy with rtPA. However, two major obstacles, the difficulty of the eMCAO surgery and unpredictable occurrence of clot autolysis, had impeded its application in mice. In this study, we modified catheters to produce suitable fibrin-rich embolus and optimized the eMCAO model using cerebral blood flow (CBF) monitored by both laser Doppler flowmetry (LDF) and 2D laser speckle contrast imaging (LSCI) to confirm occlusion of MCA. The results showed that longer embolus resulted in higher mortality. There was a compensatory increase in MCA territory perfusion after eMCAO associated with decreased infarct volume; however, this was only partly dependent on recanalization as clot autolysis was only observed in ∼30% of mice. Cortical CBF monitoring with LSCI showed that the size of peri-core area at 3 h displayed the best correlation with infarct volume that is attributed to compensatory collateral blood flow. The peri-core area best predicted functional outcome after eMCAO. In summary, we developed a reliable eMCAO mouse model that better mimics embolic ischemic stroke in humans, which will increase the potential for successful translation of stroke neuroprotective therapies.

Bone Marrow-Derived NCS-01 Cells Advance a Novel Cell-Based Therapy for Stroke.

Human mesenchymal stem cells have been explored for their application in cell-based therapies targeting stroke. Identifying cell lines that stand as safe, accessible, and effective for transplantation, while optimizing dosage, timing, and method of delivery remain critical translational steps towards clinical trials. Preclinical studies using bone marrow-derived NCS-01 cells show the cells’ ability to confer functional recovery in ischemic stroke. Coculturing primary rat cortical cells or human neural progenitor cells with NCS-01 cells protects against oxygen-glucose deprivation. In the rodent middle cerebral artery occlusion model, intracarotid artery administration of NCS-01 cells demonstrate greater efficacy than other mesenchymal stem cells (MSCs) at improving motor and neurological function, as well as reducing infarct volume and peri-infarct cell loss. NCS-01 cells secrete therapeutic factors, including basic fibroblast growth factor and interleukin-6, while also demonstrating a potentially novel mechanism of extending filopodia towards the site of injury. In this review, we discuss recent preclinical advancements using in vitro and in vivo ischemia models that support the transplantation of NCS-01 in human stroke trials. These results, coupled with the recommendations put forth by the consortium of Stem cell Therapeutics as an Emerging Paradigm for Stroke (STEPS), highlight a framework for conducting preclinical research with the ultimate goal of initiating clinical trials.

Mechanisms in blood-brain barrier opening and metabolism-challenged cerebrovascular ischemia with emphasis on ischemic stroke.

Stroke is the leading cause of disability among adults as well as the 2nd leading cause of death globally. Ischemic stroke accounts for about 85% of strokes, and currently, tissue plasminogen activator (tPA), whose therapeutic window is limited to up to 4.5 h for the appropriate population, is the only FDA approved drug in practice and medicine. After a stroke, a cascade of pathophysiological events results in the opening of the blood-brain barrier (BBB) through which further complications, disabilities, and mortality are likely to threaten the patient's health. Strikingly, tPA administration in eligible patients might cause hemorrhagic transformation and sustained damage to BBB integrity. One must, therefore, delineate upon stroke onset which cellular and molecular factors mediate BBB permeability as well as what key roles BBB rupture plays in the pathophysiology of stroke. In this review article, given our past findings of mechanisms underlying BBB opening in stroke animal models, we elucidate cellular, subcellular, and molecular factors involved in BBB permeability after ischemic stroke. The contribution of each factor to stroke severity and outcome is further discussed. Determinant factors in BBB permeability and stroke include mitochondria, miRNAs, matrix metalloproteinases (MMPs), immune cells, cytokines, chemokines, and adhesion proteins. Once these factors are interrogated and their roles in the pathophysiology of stroke are determined, novel targets for drug discovery and development can be uncovered in addition to novel therapeutic avenues for human stroke management.

Perlecan Domain-V Enhances Neurogenic Brain Repair After Stroke in Mice

The extracellular matrix fragment perlecan domain V is neuroprotective and functionally restorative following experimental stroke. As neurogenesis is an important component of chronic post-stroke repair, and previous studies have implicated perlecan in developmental neurogenesis, we hypothesized that domain V could have a broad therapeutic window by enhancing neurogenesis after stroke. We demonstrated that domain V is chronically increased in the brains of human stroke patients, suggesting that it is present during post-stroke neurogenic periods. Furthermore, perlecan deficient mice had significantly less neuroblast precursor cells after experimental stroke. Seven-day delayed domain V administration enhanced neurogenesis and restored peri-infarct excitatory synaptic drive to neocortical layer 2/3 pyramidal neurons after experimental stroke. Domain V’s effects were inhibited by blockade of α2β1 integrin, suggesting the importance of α2β1 integrin to neurogenesis and domain V neurogenic effects. Our results demonstrate that perlecan plays a previously unrecognized role in post-stroke neurogenesis and that delayed DV administration after experimental stroke enhances neurogenesis and improves recovery in an α2β1 integrin-mediated fashion. We conclude that domain V is a clinically relevant neuroprotective and neuroreparative novel stroke therapy with a broad therapeutic window.

Proteomic Changes in Intracranial Blood During Human Ischemic Stroke

IntroductionSince 2015, mechanical thrombectomy is the standard treatment for emergent large vessel occlusion stroke. The previously published Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) protocol (clinicaltrials.gov NCT03153683) utilizes thrombectomy to isolate distal blood within the artery immediately downstream from the clot, peripheral blood proximal to the clot, and the thrombus.MethodsTissue samples were obtained in accordance with the established BACTRAC protocol and immediately frozen at −80°C. For this study, we compared the intracranial intraluminal blood distal to the thrombus, using each subject’s systemic arterial blood as an internal comparative control. 40μl aliquots of plasma were thawed and randomized to 96‐well plates and shipped on dry ice to Olink Proteomics (Olink Proteomics, Boston, MA). Cardiometabolic and inflammatory panels were run on 1μl aliquots using a proximity extension assay (PEA) with proximal blood plasma as an internal control for each subject and distal blood plasma as the injury site sample. Protein data were analyzed as average protein expression levels in distal plasma as compared to average protein expression levels in proximal plasma.Results25 adult subjects (&gt;18yrs) were included in the study, of which 15 (60%) were female. Median age was 64 (24–91). 16 subjects (64%) had hypertension, 15 (60%) had BMI &gt; 25, 10 (40%) were active smokers, 6 (24%) had previous stroke, 4 (16%) had type II diabetes, and 4 (16%) had hyperlipidemia. Mean infarct time was 513 ± 246 minutes and mean infarct volume was 58,172 ± 82,284 mm3. Proteins with the greatest change in expression in the cardiometabolic panel were superoxide dismutase 1 (SOD1), CD59, lipocalin‐2 (LCN2), and defensin alpha 1 (DEFA1). Proteins with greatest change in expression in the inflammatory panel were interleukin 4 (IL‐4), AXIN1, interleukin 1 alpha (IL‐1a), sirtuin 2 (SIRT2), interleukin 5 (IL‐5) and leukemia inhibitory factor (LIF). There was a significant sex difference in expression levels of interleukin 20 receptor subunit alpha (IL‐20RA) (p=0.04) and IL‐4 (p=0.03).ConclusionsThese protein data show a complex set of intraluminal responses occurring distal to the thrombus in acute ischemic stroke. Changes in SOD1, LIF, and SIRT2 demonstrate neuroprotective and anti‐inflammatory activity, LCN2 and DEFA1 suggest a response similar to antimicrobial activity, and proteins such as IL‐4 and IL‐5 suggest eosinophil activation and a Th2 phenotype. Future studies will focus on the relationships of proteins with infarct time, infarct volume, and functional outcomes. Such analyses will provide insight into the initial molecular and cell signaling in response to ischemic stroke in the human condition.Support or Funding InformationThe project described was supported by the National Center for Advancing Translational Sciences, through Grant UL1TR001998 and UKHealthCare. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Grant through Center for Clinical and Translational Science

Method of Intra‐Arterial Administration in a transient MCAO Rat model to mimic the treatment of the human patient

Endovascular thrombectomy is routinely performed in patients undergoing a large vessel occlusion. This process not only allows for the removal of a blood clot but also for the selective delivery of potential therapeutics directly to the site of injury. The intra‐arterial (IA) route of drug administration in the mouse was developed to bridge the gap between animal stroke treatments and clinical stroke therapy. We have previously shown this delivery method targets the site of injury while blunting systemic effects that have proven problematic with intraperitoneal routes. Here, we adapted the IA method for use in rats, combining it with a clinically relevant large vessel occlusion stroke model (MCAO). Our goal was to characterize variances in the model in order to optimize potential therapeutic delivery methods to the ipsilaterally affected hemisphere. Male and female Sprague‐Dawley rats (4 months of age) were subjected to placement of micro‐angio tubing at the bifurcation of the common carotid artery (CCA), with delivery through the internal carotid artery (ICA). We then determined the optimal infusion rate and volume using injection of India ink and evaluation of vascular distribution within the brain and the liver. Importantly, we found the infusion rate and volume varied depending on sex and body weight. India ink was selectively delivered to the ipsilateral side of the brain in males (370–460 g) at 4 μl/min with a max volume of 25 μl and in females (250–300 g) at 2.5 μl/min with a max volume of 25 μl. Following these guidelines, no ink was observed in the liver of these animals, indicating reduced systemic circulation of administered compounds. We then performed a 5‐hour transient MCAO, on male and female Sprague‐Dawley rats (4 months of age), to mimic human stroke, since 5 hours is the average time from the clinical presentation of stroke to the thrombectomy procedure. A silicone coated monofilament was advanced till resistance was felt, occluding the MCA territory. At the 5 hour time point, we removed the monofilament and inserted the mico‐angio tubing at the same entry point on the bifurcation of the CCA. We performed variant injections using different volumes and rates, measuring delivery of dye (India Ink) and rate of induced subarachnoid hemorrhage. A rate of 4 ul/min with a max volume of 25 ul was optimal in males, and a rate of 2.5 ul/min with a max volume of 25 ul was optimal in females. The results showed that, even with a large vessel occlusion, and removal of the monofilament (recanalization), the IA injection using these sex‐specific rates and volumes resulted in appropriate limited dye delivery without ruptured subarachnoid hemorrhage. This IA method is ideally suited for combination with the MCAO stroke model and mirrors the human patient undergoing an endovascular thrombectomy. This model provides an investigative opportunity to test neuroprotective drugs and other pharmacotherapies.Support or Funding InformationUniversity of Kentucky Center for Advanced Translational Stroke Science, Surgery Core

Human Immunodeficiency Virus and Stroke

Human Immunodeficiency Virus and Stroke

C3a receptor antagonist therapy is protective with or without thrombolysis in murine thromboembolic stroke.

Intravenous thrombolysis (IVT) after stroke enhances C3a generation, which may abrogate the benefits of reperfusion. The C3aR antagonist SB290157 is neuroprotective following transient but not permanent middle cerebral artery occlusion (MCAo). SB290157 remains untested in thromboembolic (TE) models, which better approximate human stroke and also facilitate testing in combination with IVT. We hypothesized SB290157 would confer neuroprotection in TE stroke with and without "late" IVT. We used two different models of TE stroke to examine the efficacy of SB290157 alone and in combination with late IVT. We evaluated the benefit of SB290157 in attenuating post-ischaemic behavioural deficits, infarction, brain oedema and haemorrhage. Plasma C3a was elevated 6 hr after TE stroke alongside increased cerebrovascular C3aR expression, which was sustained to 4 weeks. Increased C3aR expression also was visualized in human ischaemic brain. In a photothrombotic (PT) stroke model, which exhibits rapid spontaneous reperfusion, SB290157 given at 1 hr post-PT significantly improved neurofunction and reduced infarction at 48 hr. In an embolic (eMCAo) model, SB290157 administered at 2 hr improved histological and functional outcomes. Conversely, late IVT administered 4.5 hr post-eMCAo was ineffective likely due to increased haemorrhage and brain oedema. However, SB290157 administered prior to late IVT ameliorated haemorrhage and oedema and improved outcomes. We conclude that SB290157 is safe and effective with and without late IVT following TE stroke. Therefore, C3a receptor antagonist therapy represents a promising candidate for clinical translation in stroke, particularly as an adjuvant to IVT.

RNA expression studies in stroke: what can they tell us about stroke mechanism?

Diagnosis of stroke and understanding the mechanism of stroke is critical to implement optimal treatment. RNA expressed in peripheral blood cells is emerging as a precision biomarker to aid in stroke diagnosis and prediction of stroke cause. In this review, we summarize available data regarding the role of RNA to predict stroke, the rationale for these changes, and a discussion of novel mechanistic insight and clinical applications. Differences in RNA gene expression in blood have been identified in patients with stroke, including differences to distinguish ischemic from hemorrhagic stroke, and differences between cardioembolic, large vessel atherosclerotic, and small vessel lacunar stroke cause. Gene expression differences show promise as novel stroke biomarkers to predict stroke of unclear cause (cryptogenic stroke). The differences in RNA expression provide novel insight to stroke mechanism, including the role of immune response and thrombosis in human stroke. Important insight to regulation of gene expression in stroke and its causes are being acquired, including alternative splicing, noncoding RNA, and microRNA. Improved diagnosis of stroke and determination of stroke cause will improve stroke treatment and prevention. RNA biomarkers show promise to aid in the diagnosis of stroke and cause determination, as well as providing novel insight to mechanism of stroke in patients. While further study is required, an RNA profile may one day be part of the stroke armamentarium with utility to guide acute stroke therapy and prevention strategies and refine stroke phenotype.

Abstract WMP116: Thromboembolism in Atrial Fibrillation: Relationship to Leukocyte Gene Expression

Background: Atrial fibrillation (AF) is an important cause of stroke, for which anticoagulation provides substantial benefit. However, not all patients with AF will have a stroke. There remains uncertainty regarding factors that promote thromboembolism and stroke in patients with AF. In this study we examined differences in blood cell gene expression unique to AF in acute stroke to better understand factors important to atrial fibrillation thromboembolism in human stroke. Methods: Gene expression in blood was compared in acute stroke patients with AF to non-AF stroke and to controls without stroke. Blood was collected in PAXgene tubes, and leukocyte/platelet gene expression was measured by Affymetrix microarray. Differentially expressed genes were identified using ANOVA adjusted for age, sex and batch. Results: In the 184 patients studied, 40 were acute strokes with AF, 143 had non-AF acute stroke, and 116 were non-stroke controls. There were 43 genes unique to AF in patients with stroke, and 69 genes associated AF that were shared between AF stroke and controls (FDR&lt;0.05, fold change&gt;|1.5|). Functional analysis indicate acute stroke AF genes are associated with changes in the hematological system including blood cell rheology and leukocyte activation. In contrast non-stroke AF genes are associated cardiac hypertrophy and blood vessel injury. Conclusions: AF has differences in blood cell gene expression in acute stroke that may relate to risk of thromboembolism. Acute stroke patients with AF display changes in blood cell rheology and leukocyte activation; whereas non-stroke AF patients have changes in cardiac hypertrophy and vascular injury. These differences are important to understanding blood cell contribution to thrombus formation and stroke risk in patients with AF. Further study is required to assess the relationship of these gene changes to stroke risk and response to anticoagulation in patients with AF.