Ischemic stroke and factors modifying ischemic stroke responses, such as social isolation, contribute to long-term disability worldwide. Several studies demonstrated that the aberrant levels of microRNAs contribute to ischemic stroke injury. In prior studies, we established that miR-141-3p increases after ischemic stroke and post-stroke isolation. Herein, we explored two different anti-miR oligonucleotides; peptide nucleic acid (PNAs) and phosphorothioates (PS) for ischemic stroke therapy. We used US FDA approved biocompatible poly (lactic-co-glycolic acid) (PLGA)-based nanoparticle formulations for delivery. The PNA and PS anti-miRs were encapsulated in PLGA nanoparticles by double emulsion solvent evaporation technique. All the formulated nanoparticles showed uniform morphology, size, distribution, and surface charge density. Nanoparticles also exhibited a controlled nucleic acid release profile for 48 h. Further, we performed in vivo studies in the mouse model of ischemic stroke. Ischemic stroke was induced by transient (60 min) occlusion of middle cerebral artery occlusion followed by a reperfusion for 48 or 72 h. We assessed the blood-brain barrier permeability of PLGA NPs containing fluorophore (TAMRA) anti-miR probe after systemic delivery. Confocal imaging shows uptake of fluorophore tagged anti-miR in the brain parenchyma. Next, we evaluated the therapeutic efficacy after systemic delivery of nanoparticles containing PNA and PS anti-miR-141-3p in mice after stroke. Post-treatment differentially reduced both miR-141-3p levels in brain tissue and infarct injury. We noted PNA-based anti-miR showed superior efficacy compared to PS-based anti-miR. Herein, we successfully established that nanoparticles encapsulating PNA or PS-based anti-miRs-141-3p probes could be used as a potential treatment for ischemic stroke.

Background and Purpose: The ability of the brain to self-heal after stroke is limited by inadequate blood supply and acute inflammation. Hence, we hypothesized that improved blood supply via promoting neoangiogenesis will reduce acute inflammation and improve recovery after stroke. Encephalomyosynangiosis (EMS) is a neurosurgical procedure that achieves angiogenesis with low morbidity in patients with moyamoya disease, reducing risk of stroke. EMS has never been studied in ischemic stroke. Here, we developed a novel model of EMS after ischemic stroke induced by middle cerebral artery occlusion model (MCAo) in mice. We demonstrated that this novel EMS procedure can induce angiogenesis and improve recovery after stroke. Methods: After 60 minutes of MCAo, mice were randomized to MCAo or MCAo+EMS groups. After 3-4 hours, EMS was performed. In brief, EMS involves a craniotomy and placement of a vascular temporalis muscle flap on the pial surface of the ischemic brain. Mice were sacrificed at day 7 or 21 after MCAo. Viability of temporalis muscle was measured using a nicotinamide adenine dinucleotide reduced tetrazolium reductase assay. ARY015 Proteome Profiler Mouse Angiogenesis Array (R&D Systems) was used to quantify angiogenic and neuromodulating protein expression. Immunohistochemistry was used to visualize immune infiltration by measuring F4/80 positive macrophages. Results: EMS after ischemic stroke did not increase mortality despite dual invasive surgery. Grafted temporalis muscle remained viable up to 21 days after EMS. We found a significant increase in FGF-acidic (0.677±0.007 vs. 0.585±0.014, p=0.045) and decrease in osteopontin (0.692±0.007 vs. 0.758±0.014, p=0.048) protein levels in the MCAo+EMS group 21 days after stroke, suggesting improved angiogenesis and recovery respectively. Mice in the MCAo+EMS group also showed reduced F4/80+ve macrophage accumulation in the peri-lesional territory of ischemic tissue, suggesting attenuated inflammation after stroke. Conclusions: These proof-of-concept data support that our novel EMS procedure is well-tolerated and may improve angiogenesis and recovery after stroke. EMS after MCAo is a promising new method of improving angiogenesis and recovery after stroke.

Backgrounds: An acute ischemic stroke (AIS) triggers rapid infiltration of circulating immune cells in the brain. P2X4R, a receptor for adenosine triphosphate ATP, regulate activation of circulating monocytes after stroke injury. Over-stimulation of P2X4R contributes to ischemic injury. CD14 ++ CD16 – classical, CD14 ++ CD16 + intermediate, and CD14 + CD16 ++ non-classical monocytes are three primary subsets of monocytes. Alterations in activity of circulating monocyte subsets may independently predict pathogenesis of AIS, however, the role of P2X4R in the activation of these monocyte subsets is not known. Methods: Consecutive AIS patients (60-90 years) undergoing endovascular clot retrieval and healthy control subjects both young (18-45 years) and aged (60-90 years) of both sexes were recruited and informed consent obtained. Flow cytometric analysis of whole blood derived monocytes at 0-2 days (acute, n=10), 3-7 days (subacute, n=9), and 65±20 days (chronic, n=7) after stroke onset were compared with healthy subjects (n=9-10/ age group). Results: Both number of total monocyte counts and P2X4R intensity significantly increase with age when compared between healthy young and aged control (P<0.05). Total monocyte count progressively increased during recovery in AIS patient (P<0.05). No. of CD14 ++ CD16 + intermediate monocytes were significantly reduced after stroke ( p <0.05). Both CD14 ++ CD16 + intermediate, and CD14 + CD16 ++ non-classical monocytes showed a significant increased median fluorescent intensity (P<0.01) of P2X4R at subacute and chronic time after AIS. Conclusions: P2X4R expression increases with age and after stroke. Disappearance of the CD14 + CD16 ++ non-classical monocyte subpopulation from circulation during stroke recovery suggests potential migration of these cells to the site of injury, consistent with their potential role in inflammation/phagocytosis. Increased P2X4R expression in intermediate and non-classical monocytes subpopulation suggest its specific role in selective activation of these monocytes subtype. Detailed molecular characterization of P2X4R response in intermediate and non-classical monocyte subpopulations may provide novel insights into P2X4R’s therapeutic potential during AIS.

Background and Purpose: No effective treatment is available for most patients who suffer ischemic stroke. Development of novel treatment options is imperative. The brain attempts to self-heal after ischemic stroke via various mechanism mediated by restored blood circulation in affected region of brain but this process is limited by inadequate angiogenesis or neoangiogenesis. Encephalomyosynangiosis (EMS) is a neurosurgical procedure that achieves angiogenesis with low morbidity in patients with moyamoya disease, reducing risk of stroke. However, EMS, surgery has never been studied as an therapeutic option after ischemic stroke. Here we described a novel procedure and feasibility data for EMS after ischemic stroke in mice. Methods: A 60 mins of middle cerebral artery occlusion (MCAo) was used to induce ischemic stroke in mice. After 3-4 hours of MCAo onset/sham, EMS was performed. Mortality of EMS, MCAo and. MCAo+EMS mice was recorded up to 21 days after surgery. Graft tissue viability was measured using a nicotinamide adenine dinucleotide reduced tetrazolium reductase assay. Results: EMS surgery after ischemic stroke does not increase mortality compared to stroke alone. Graft muscle tissue remained viable 21 days after surgery. Conclusions: This novel protocol is effective and well-tolerated, may serve as novel platform for new angiogenesis and thus recovery after ischemic stroke. If successful in mice, EMS can a very feasible and novel treatment option for ischemic stroke in humans.

Background: Stroke and factors modifying stroke responses, such as social isolation, increases miR-141-3p. Hence, inhibiting miR-141-3p can be a promising stroke therapy. Unfortunately, commercially available miR-141-3p inhibitors cannot be used for therapeutic purpose due to several limitations; use of viral or non-viral transfection reagents for cell penetration, in vivo instability of anti-miRs and toxicity of synthetic anti-miRs. Therefore, we explored phosphorothioates (PS) and peptide nucleic acids (PNAs) based anti-miR-141-3p encapsulated in poly (lactide-co-glycolide) (PLGA)-based nanoparticles (NPs) for potential stroke therapy. Methods: PNAs and PS anti-miR 141-3p were synthesized in house using solid phase synthesis and purchased from a commercial vendor, respectively. The anti-miRs were encapsulated in PLGA NP by double emulsion solvent evaporation technique. For in vivo efficacy 8-10 weeks old C57BL/6 male mice were pair-housed (PH) for at least two weeks. After two weeks, a 60-minute right middle cerebral artery occlusion surgery (MCAO) was performed and mice were kept individually. The mice were then randomly assigned to receive NPs of either anti-miR-141-3p PNA or anti-miR-141-3p phosphorothioate (PS) or scrambled control (Sc) through lateral tail vein 4 hrs. after stroke. To measure NPs of PS were conjugated with fluorophore (TAMRA) to measure BBB permeability. The effect of inhibitor treatment was evaluated at 3 days after stroke. Results: The formulated NPs show uniform morphology with a mean dry particle size <120 nm and an average hydrodynamic diameter of 350 nm. The NPs exhibit an average polydispersity index of 0.1-0.2 indicating homogenous particle size distribution. Single dose of anti-miR 141 and (NPs containing 0.05mg/kg i.v of total either PNA or PS) Both the NPs of PNA and PS significantly reduced (P<0.05 vs. Sc) infarct injury and neurological deficit. Anti-miR of PNA and PS reduce 6 and 5-fold respectively of miR-141-3p in the brain tissue. Conclusion: Our NPs are BBB permeable. PNA based anti-miR-141 are more potent the PS based anti-miR Reduced miR-141-3p levels as well as infarct injury by both PS and PNA based anti-miR 141-3p suggest the efficacy of our novel ant-miR -141-3p for the treatment of ischemic stroke.