Extended Middle Cerebral Artery Occlusion (MCAO) Model to Mirror Stroke Patients Undergoing Thrombectomy.

Stroke remains a leading global cause of death and disability. In the last decade, the therapeutic window for mechanical thrombectomy has increased from a maximum of 6 to 24 h and beyond. While endovascular advancements have improved rates of recanalization, no post-stroke pharmacotherapeutics have been effective in enhancing neurorepair and recovery. New experimental models are needed to closer mimic the human patient. Our group has developed a model of transient 5-h occlusion in rats to mimic stroke patients undergoing thrombectomy. Our procedure was designed specifically in aged rats and was optimized based on sex in order to keep mortality and extent of injury consistent between aged male and female rats. This model uses a neurological assessment modeled after the NIH Stroke Scale. Finally, the potential for translation between our rat model of stroke and humans was assessed using comparative gene expression for key inflammatory genes. This model will be useful in the evaluation of therapeutic targets to develop adjuvant treatments for large vessel occlusion during the thrombectomy procedure.

Integrin α5ß1 inhibition by ATN-161 reduces neuroinflammation and is neuroprotective in ischemic stroke.

Stroke remains a leading cause of death and disability with limited therapeutic options. Endothelial cell ß1 integrin receptors play a direct role in blood-brain barrier (BBB) dysfunction through regulation of tight junction proteins and infiltrating leukocytes, potentially mediated by ß1 integrins. Following tandem transient common carotid artery/middle cerebral artery occlusion on wild-type mice, we administered the integrin a5b1 inhibitor, ATN-161, intraperitoneal (IP) injection at 1 mg/kg acutely after reperfusion, on post-stroke day (PSD)1 and PSD2. Systemic changes (heart rate, pulse distension, and body temperature) were determined. Additionally, infarct volume and edema were determined by 2,3-triphenyltetrazolium chloride and magnetic resonance imaging, while neurological changes were evaluated using an 11-point Neuroscore. Brain immunohistochemistry was performed for claudin-5, α5ß1, IgG, and CD45 + cells, and quantitative polymerase chain reaction (qPCR) was performed for matrix metalloproteinase-9 (MMP-9), interleukin (IL)-1ß, collagen IV, and CXCL12. ATN-161 significantly reduced integrin α5ß1 expression in the surrounding peri-infarct region with no systemic changes. Infarct volume, edema, and functional deficit were significantly reduced in ATN-161-treated mice. Furthermore, ATN-161 treatment reduced IgG extravasation into the parenchyma through conserved claudin-5, collagen IV, CXCL12 while reducing MMP-9 transcription. Additionally, IL-1ß and CD45 + cells were reduced in the ipsilateral cortex following ATN-161 administration. Collectively, ATN-161 may be a promising novel stroke therapy by reducing post-stroke inflammation and BBB permeability.

Interleukin 1 alpha administration is neuroprotective and neuro-restorative following experimental ischemic stroke.

BACKGROUND: Stroke remains a leading cause of death and disability worldwide despite recent treatment breakthroughs. A primary event in stroke pathogenesis is the development of a potent and deleterious local and peripheral inflammatory response regulated by the pro-inflammatory cytokine interleukin-1 (IL-1). While the role of IL-1ß (main released isoform) has been well studied in stroke, the role of the IL-1α isoform remains largely unknown. With increasing utilization of intravenous tissue plasminogen activator (t-PA) or thrombectomy to pharmacologically or mechanically remove ischemic stroke causing blood clots, respectively, there is interest in pairing successful cerebrovascular recanalization with neurotherapeutic pharmacological interventions (Fraser et al., J Cereb Blood Flow Metab 37:3531-3543, 2017; Hill et al., Lancet Neurol 11:942-950, 2012; Amaro et al., Stroke 47:2874-2876, 2016). METHODS: Transient stroke was induced in mice via one of two methods. One group of mice were subjected to tandem ipsilateral common carotid artery and middle cerebral artery occlusion, while another group underwent the filament-based middle cerebral artery occlusion. We have recently developed an animal model of intra-arterial (IA) drug administration after recanalization (Maniskas et al., J Neurosci Met 240:22-27, 2015). Sub groups of the mice were treated with either saline or Il-1α, wherein the drug was administered either acutely (immediately after surgery) or subacutely (on the third day after stroke). This was followed by behavioral and histological analyses. RESULTS: We now show in the above-mentioned mouse stroke models (transient tandem ipsilateral common carotid artery (CCA) and middle cerebral artery occlusion (MCA) occlusion, MCA suture occlusion) that IL-1α is neuroprotective when acutely given either intravenously (IV) or IA at low sub-pathologic doses. Furthermore, while IV administration induces transient hemodynamic side effects without affecting systemic markers of inflammation, IA delivery further improves overall outcomes while eliminating these side effects. Additionally, we show that delayed/subacute IV IL-1α administration ameliorates functional deficit and promotes neurorepair. CONCLUSIONS: Taken together, our present study suggests for the first time that IL-1α could, unexpectedly, be an effective ischemic stroke therapy with a broad therapeutic window.

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.

IL-1alpha induces angiogenesis in brain endothelial cells in vitro: implications for brain angiogenesis after acute injury.

Inflammation is a major contributor to neuronal injury and is associated with poor outcome after acute brain injury such as stroke. The pro-inflammatory cytokine interleukin (IL)-1 is a critical regulator of cerebrovascular inflammation after ischemic injury, mainly through action of both of its isoforms, IL-1α and IL-1ß, at the brain endothelium. In contrast, the differential action of these ligands on endothelial activation and post-stroke angiogenesis is largely unknown. Here, we demonstrate that IL-1α is chronically elevated in the brain after experimental stroke suggesting that it is present during post-stroke angiogenic periods. Furthermore, we demonstrate that IL-1α is a potent mediator of endothelial activation and inducer of angiogenic markers in endothelial cells in vitro. Using brain endothelial cell lines, we found that IL-1α was significantly more potent than IL-1ß at inducing endothelial cell activation, as measured by expression of the pro-angiogenic chemokine CXCL-1. IL-1α also induced strong expression of the angiogenic mediator IL-6 in a concentration-dependent manner. Furthermore, IL-1α induced significant proliferation and migration of endothelial cells, and promoted formation of tube-like structures that are established key hallmarks of angiogenesis in vitro. Finally, all of those responses were blocked by the IL-1 receptor antagonist (IL-1RA). In conclusion, our data highlights a potential new role for IL-1 in brain repair mechanisms and identifies IL-1α as a potential new therapy to promote post-stroke angiogenesis. Inflammation is a major contributor to neuronal injury and is associated with poor outcome after neurotrauma. We demonstrate that cytokine IL-1α is chronically elevated in the brain after experimental stroke suggesting that it is present chronically post-stroke. We demonstrate that IL-1α is a potent mediator of endothelial activation and inducer of angiogenic markers in endothelial cells. Our data highlights a new role for IL-1 in brain repair mechanisms and identifies IL-1α as a potential therapy to promote post-stroke angiogenesis.