Internal Carotid Artery Stenosis: A Surgical Mouse Model to Study Moyamoya Syndrome.

Moyamoya is a cerebrovascular condition that predisposes affected patients to stroke and is characterized as the progressive stenosis of the internal carotid artery (ICA) and compensatory development of collaterals at the base of the brain. Patients with moyamoya syndrome also present with comorbidities such as various autoimmune diseases and coagulopathies. We developed a surgical method using micro-coils to induce ICA-specific stenosis in mice, which induces moyamoya-like vasculopathies. An advantage of this surgical model of hypoperfusion is that it can be combined with other comorbid models to investigate pathologies associated with moyamoya syndrome.

Cannabidiol's Multifactorial Mechanisms Has Therapeutic Potential for Aneurysmal Subarachnoid Hemorrhage: a Review.

Subarachnoid hemorrhage (SAH) is a major health burden that accounts for approximately 5% of all strokes. The most common cause of a non-traumatic SAH is the rupture of a cerebral aneurysm. The most common symptom associated with SAH is a headache, often described as "the worst headache of my life." Delayed cerebral ischemia (DCI) is a major factor associated with patient mortality following SAH and is often associated with SAH-induced cerebral vasospasm (CV). Cannabidiol (CBD) is emerging as a potential drug for many therapeutic purposes, including epilepsy, anxiety, and pain relief. We aim to review the potential use of CBD as a treatment option for post-SAH critically ill patients. Through a literature review, we evaluated the known pharmacology and physiological effects of CBD and correlated those with the pathophysiological outcomes associated with cerebral vasospasm following subarachnoid hemorrhage. Although overlap exists, data were formatted into three major categories: anti-inflammatory, vascular, and neuroprotective effects. Based on the amount of information known about the actions of CBD, we hypothesize the anti-inflammatory effects are likely to be the most promising therapeutic mechanism. However, its cardiovascular effects through calcium regulation and its neuroprotective effects against cell death, excitotoxicity, and oxidative stress are all plausible mechanisms by which post-SAH critically ill patients may benefit from both early and late intervention with CBD. More research is needed to better understand if and how CBD might affect neurological and vascular functions in the brain following injury such as subarachnoid hemorrhage.

Intra-arterial combination therapy for experimental acute ischemic stroke.

Acute ischemic stroke continues to devastate millions of individuals worldwide. Current treatments work to restore blood flow but not rescue affected tissue. Our goal was to develop a combination of neuroprotective agents administered intra-arterially following recanalization to target ischemic tissue. Using C57Bl/6J male mice, we performed tandem transient ipsilateral middle cerebral/common carotid artery occlusion, followed by immediate intra-arterial pharmacotherapy administration through a standardized protocol. Two pharmacotherapy agents, verapamil and lubeluzole, were selected based on their potential to modulate different aspects of the ischemic cascade; verapamil, a calcium channel blocker, works in an acute fashion blocking L-type calcium channels, whereas lubeluzole, an N-methyl-D-aspartate modulator, works in a delayed fashion blocking intracellular glutamate trafficking. We hypothesized that combination therapy would provide complimentary and potentially synergistic benefit treating brain tissue undergoing various stages of injury. Physiological measurements for heart rate and pulse distention (blood pressure) demonstrated no detrimental effects between groups, suggesting that the combination drug administration is safe. Tissue analysis demonstrated a significant difference between combination and control (saline) groups in infarct volume, neuronal health, and astrogliosis. Although a significant difference in functional outcome was not observed, we did note that the combination treatment group had a greater percent change from baseline in forced motor movement as compared with controls. This study demonstrates the safety and feasibility of intra-arterial combination therapy following successful recanalization and warrants further study.

Smoking-Induced Sex Differences in Clinical Outcomes in Patients Undergoing Mechanical Thrombectomy for Stroke.

OBJECTIVE: Ischemic stroke is the fifth leading cause of death in the United States. Smoking accelerates the onset of stroke by 10 years. The effects of smoking status on percent change in National Institutes of Health Stroke Scale (NIHSS) score, infarct volume, and edema volume were examined following mechanical thrombectomy for large vessel occlusion in patients with acute ischemic stroke. METHODS: Subjects (N = 90; >18 years old) were divided into 3 groups based on smoking status: current smokers, previous smokers (defined as having quit >6 months before the ischemic event), and nonsmokers. Percent change in NIHSS score was defined as score at admission minus score at discharge divided by score at admission and was used as a predictor of functional outcome. Linear regression analysis was performed based on infarct or edema volume versus percent change in NIHSS score and separated by sex. RESULTS: Consistent with previous findings, smokers experienced a stroke 10 years earlier than nonsmokers (P = 0.004). Statistically significant linear regressions existed between infarct volume or edema volume in relation to worsening change in NIHSS score with female smokers only. Stroke-induced tissue damage, as measured by magnetic resonance imaging or computed tomography, was predictive of functional recovery only in female smokers. CONCLUSIONS: These findings are valuable for patient counseling, particularly for women, for smoking cessation.

Intracranial VCAM1 at time of mechanical thrombectomy predicts ischemic stroke severity.

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 (p<0.001) and intracranial (p=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.

Method of intra-arterial drug administration in a rat: Sex based optimization of infusion rate.

BACKGROUND: Endovascular thrombectomy is the process of removing a blood clot and re-establishing blood flow in patients with emergent large vessel occlusion. The technique provides an opportunity to deliver 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. Here, we adapted the IA method for use in rats, by investigating various flow rates to optimize the IA injection through the internal carotid artery (ICA). METHODS: 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 for injection into the ICA. We evaluated a range of infusion rates of carbon black ink and its vascular distribution within the brain. RESULTS: Optimal injection rates in males was 4-6 µl/min and 2-4 µl/min in females. The IA injection using these sex-specific rates resulted in appropriate limited dye delivery to only the ipsilateral region of the brain, without inducing a subarachnoid hemorrhage. CONCLUSION: Upon adapting the IA administration model to rats, it was determined that the rate of infusion varied between males and females. This variability is an important consideration for studies utilizing both sexes, such as in ischemic stroke studies.

Commentary: Use of BACTRAC Proteomic Database-Uromodulin Protein Expression During Ischemic Stroke.

INTRODUCTION: Uromodulin (UMOD) is a glycoprotein expressed by the epithelial cells of the thick ascending limb of Henle's loop in the kidney. Research has shown that increased uromodulin expression may be associated with lower risk of cardiovascular disease in adults. Utilizing the Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) (clinicaltrials.gov NCT03153683), a continuously enrolling tissue bank, we aimed to examine the associations between serum uromodulin, age, and high BMI (BMI>25) and its relationship to stroke in patients. METHODS: Arterial blood distal and proximal to the thrombus was collected during a thrombectomy procedure using the BACTRAC protocol and sent to Olink (Boston, MA) to determine proteomic expression via proximity extension assay. Uromodulin expression was recorded and analyzed using two tailed T-tests and linear regressions. RESULTS: The relationship between systemic and intracranial uromodulin, age, high BMI and hypertension were assessed. Systemic and intracranial uromodulin decreased with age (p<0.0001 and r2=0.343, p=0.0416 and r2=0.102) respectively. Systemic uromodulin expression increased with BMI>25 (p=0.014). Presence of hypertension decreased uromodulin's expression systemically (p=0.018) and intracranially (p=0.007). CONCLUSIONS: Uromodulin was increased significantly in overweight patients, decreased significantly in older patients, and decreased in patients with hypertension. The increase in uromodulin in people with high BMI could be a protective reaction of the kidney to worsening conditions that make ischemic stroke more likely, with a goal of delaying dangerous outcomes. The decreased expression of uromodulin in older adults could be associated with the decline of general kidney function that accompanies aging. Hypertension can contribute to an AKI by decreasing perfusion to the kidney, therefore decreasing kidney function and uromodulin production. Further analyses are needed to understand the role of uromodulin following ischemic stroke.

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 α2ß1 integrin, suggesting the importance of α2ß1 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 α2ß1 integrin-mediated fashion. We conclude that domain V is a clinically relevant neuroprotective and neuroreparative novel stroke therapy with a broad therapeutic window.

High Prevalence of Moyamoya Syndrome in Appalachia.

INTRODUCTION: Moyamoya is a chronic cerebrovascular condition of unclear etiology characterized by progressive occlusion of 1 or both internal carotid arteries with neovascular collateral formation. With both an idiopathic form (moya-moya disease) and congenital condition-associated form (moyamoya syndrome), it can cause ischemic and hemorrhagic stroke. Recent findings in Kentucky have challenged traditional estimates of its incidence in US populations. Using the Kentucky Appalachian Stroke Registry (KApSR), our aim was to further characterize its incidence as a cause of stroke and to understand the patient population in Appalachia. METHODS: A retrospective review of moyamoya patients was performed using the KApSR database. Data collected included demographics, county location, risk factors, comorbidities, and health-care encounters from January 1, 2012, to December 31, 2016. RESULTS: Sixty-seven patients were identified; 36 (53.7%) resided in Appalachian counties. The cohort accounted for 125 of 6,305 stroke admissions, representing an incidence of 1,983 per 100,000 stroke admissions. Patients presented with ischemic strokes rather than hemorrhagic strokes (odds ratio 5.50, 95% CI: 2.74-11.04, p < 0.01). Eleven patients (16.4%) exhibited autoimmune disorders. Compared to the general population with autoimmune disorder prevalence of 4.5%, the presence of autoimmunity within the cohort was significantly higher (p < 0.01). Compared to non-Appalachian patients, Appalachian patients tended to present with lower frequencies of tobacco use (p = 0.08), diabetes mellitus (p = 0.13), and hypertension (p = 0.16). CONCLUSIONS: Moyamoya accounts for a substantial number of stroke admissions in Kentucky; these patients were more likely to develop an ischemic stroke rather than a hemorrhagic stroke. Autoimmune disorders were more prevalent in moyamoya patients than in the general population. The reduced frequency of traditional stroke risk factors within the Appalachian group suggests an etiology distinct to the population.

The Blood And Clot Thrombectomy Registry And Collaboration (BACTRAC) protocol: novel method for evaluating human stroke.

BACKGROUND: Ischemic 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. OBJECTIVE: To develop and share a reproducible protocol to bank these specimens for future analysis. METHODS: We established a protocol approved by the institutional review board for tissue processing during thrombectomy (www.clinicaltrials.gov NCT03153683). 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. RESULTS: Our 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. CONCLUSIONS: We 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.

Bilateral carotid artery stenosis causes unexpected early changes in brain extracellular matrix and blood-brain barrier integrity in mice.

Bilateral carotid artery stenosis (BCAS) is one experimental model of vascular dementia thought to preferentially impact brain white matter. Indeed, few studies report hippocampal and cortical pathology prior to 30 days post-stenosis; though it is unclear whether those studies examined regions outside the white matter. Since changes in the blood-brain barrier (BBB) permeability precede more overt brain pathology in various diseases, we hypothesized that changes within the BBB and/or BBB-associated extracellular matrix (ECM) could occur earlier after BCAS in the hippocampus, cortex and striatum and be a precursor of longer term pathology. Here, C57Bl/6 mice underwent BCAS or sham surgeries and changes in the BBB and ECM were analyzed by collagen IV (vascular basement membrane component), α5 integrin (marker of endothelial activation), claudin-5 and occludin (tight junction proteins), Evans blue (permeability marker), Ki-67 (cell proliferation marker), and GFAP and CD11b (glial cell markers) immunohistochemistry after 14 days. Significant changes in markers of cerebrovascular integrity and glial activation were detected, not only in the striatum, but also in the hippocampus and cortex. In conclusion, this study demonstrates for the first time that changes in the BBB/ECM occur shortly after BCAS and within multiple brain regions and suggests such changes might underlie the gradual development of BCAS non-white matter pathology.

Internal carotid artery stenosis: A novel surgical model for moyamoya syndrome.

Moyamoya is a cerebrovascular disorder characterized by progressive stenosis of the intracranial internal carotid arteries. There are two forms: Disease and Syndrome, with each characterized by the sub-population it affects. Moyamoya syndrome (MMS) is more prominent in adults in their 20's-40's, and is often associated with autoimmune diseases. Currently, there are no surgical models for inducing moyamoya syndrome, so our aim was to develop a new animal model to study this relatively unknown cerebrovascular disease. Here, we demonstrate a new surgical technique termed internal carotid artery stenosis (ICAS), to mimic MMS using micro-coils on the proximal ICA. We tested for Moyamoya-like vasculopathies by fluorescently labelling the mouse cerebrovasculature with Di I for visualization and analysis of vessel diameter at the distal ICA and anastomoses on the cortical surface. Results show a significant narrowing of the distal ICA and anterior cerebral artery (ACA) in the Circle of Willis, as observed in humans. There is also a significant decrease in the number of anastomoses between the middle cerebral artery (MCA) and the ACA in the watershed region of the cortex. While further characterization is needed, this ICAS model can be applied to transgenic mice displaying co-morbidities as observed within the Moyamoya syndrome population, allowing a better understanding of the disease and development of novel treatments.

Intra-arterial nitroglycerin as directed acute treatment in experimental ischemic stroke.

BACKGROUND: Nitroglycerin (also known as glyceryl trinitrate (GTN)), a vasodilator best known for treatment of ischemic heart disease, has also been investigated for its potential therapeutic benefit in ischemic stroke. The completed Efficacy of Nitric Oxide in Stroke trial suggested that GTN has therapeutic benefit with acute (within 6 hours) transdermal systemic sustained release therapy. OBJECTIVE: To examine an alternative use of GTN as an acute therapy for ischemic stroke following successful recanalization. METHODS: We administered GTN IA following transient middle cerebral artery occlusion in mice. Because no standard dose of GTN is available following emergent large vessel occlusion, we performed a dose-response (3.12, 6.25, 12.5, and 25 µg/µL) analysis. Next, we looked at blood perfusion (flow) through the middle cerebral artery using laser Doppler flowmetry. Functional outcomes, including forced motor movement rotor rod, were assessed in the 3.12, 6.25, and 12.5 µg/µL groups. Histological analysis was performed using cresyl violet for infarct volume, and glial fibrillary activating protein (GFAP) and NeuN immunohistochemistry for astrocyte activation and mature neuron survival, respectively. RESULTS: Overall, we found that acute post-stroke IA GTN had little effect on vessel dilatation after 15 min. Functional analysis showed a significant difference between GTN (3.12 and 6.25 µg/µL) and control at post-stroke day 1. Histological measures showed a significant reduction in infarct volume and GFAP immunoreactivity and a significant increase in NeuN. CONCLUSIONS: These results demonstrate that acute IA GTN is neuroprotective in experimental ischemic stroke and warrants further study as a potentially new stroke therapy.

Stroke neuroprotection revisited: Intra-arterial verapamil is profoundly neuroprotective in experimental acute ischemic stroke.

While clinical trials have now solidified the role of thrombectomy in emergent large vessel occlusive stroke, additional therapies are needed to optimize patient outcome. Using our previously described experimental ischemic stroke model for evaluating adjunctive intra-arterial drug therapy after vessel recanalization, we studied the potential neuroprotective effects of verapamil. A calcium channel blocker, verapamil is often infused intra-arterially by neurointerventionalists to treat cerebral vasospasm. Such a direct route of administration allows for both focused targeting of stroke-impacted brain tissue and minimizes potential systemic side effects. Intra-arterial administration of verapamil at a flow rate of 2.5 µl/min and injection volume of 10 µl immediately after middle cerebral artery recanalization in C57/Bl6 mice was shown to be profoundly neuroprotective as compared to intra-arterial vehicle-treated stroke controls. Specifically, we noted a significant (P ≤ 0.05) decrease in infarct volume, astrogliosis, and cellular apoptosis as well as a significant increase in neuronal survival and functional outcome over seven days. Furthermore, intra-arterial administration of verapamil was well tolerated with no hemorrhage, systemic side effects, or increased mortality. Thus, verapamil administered intra-arterially immediately following recanalization in experimental ischemic stroke is both safe and neuroprotective and merits further study as a potential therapeutic adjunct to thrombectomy.

Juvenile traumatic brain injury induces long-term perivascular matrix changes alongside amyloid-beta accumulation.

In our juvenile traumatic brain injury (jTBI) model, emergence of cognitive dysfunctions was observed up to 6 months after trauma. Here we hypothesize that early brain injury induces changes in the neurovascular unit (NVU) that would be associated with amyloid-beta (Aß) accumulation. We investigated NVU changes for up to 6 months in a rat jTBI model, with a focus on the efflux protein P-glycoprotein (P-gp) and on the basement membrane proteins perlecan and fibronectin, all known to be involved in Aß clearance. Rodent-Aß staining is present and increased after jTBI around cerebral blood microvessels, and the diameter of those is decreased by 25% and 34% at 2 and 6 months, respectively, without significant angiogenesis. P-glycoprotein staining in endothelium is decreased by 22% and parallels an increase of perlecan and fibronectin staining around cerebral blood vessels. Altogether, these results strongly suggest that the emergence of long-term behavioral dysfunctions observed in rodent jTBI may be related to endothelial remodeling at the blood-brain barrier alongside vascular dysfunction and altered Aß trafficking. This study shows that it is important to consider jTBI as a vascular disorder with long-term consequences on cognitive functions.