Distal Middle Cerebral Artery Occlusion Research Articles

Intravenous administration of muse cells improves cerebral ischemia outcome via immunomodulation in the spleen.

Ischemic stroke is a leading cause of disability and death globally. Stem cell therapies are emerging as a frontier for enhancing post-stroke recovery, with Muse cells-a subclass of pluripotent stem cells-demonstrating considerable promise. Muse cells are notable not only for their potential in cell replacement but also for their role in modulating immune responses following cerebral infarction. In the present study, we administered Muse cells intravenously to mice after inducing a stroke via distal middle cerebral artery occlusion. We evaluated motor outcomes, splenocyte populations, cytokine profiles, and gene expression 2 weeks after inducing stroke. Additionally, comparisons were drawn between outcomes in splenectomized mice and those receiving adoptive splenocyte transfer to discern the specific influence of the spleen on treatment efficacy. Our findings revealed that Muse cell therapy facilitates motor recovery, an effect that is compromised in the absence of the spleen. Spleens in treated mice exhibited a shift in neutrophil counts, increased cytokine activity, and a notable uptick in the expression of genes related to protein folding. These insights affirm the potential therapeutic effect of Muse cells in post-stroke treatment strategies, with their efficacy attributed, at least in part, to immunomodulatory pathways involving the spleen.

Discovering novel plasma biomarkers for ischemic stroke: Lipidomic and metabolomic analyses in an aged mouse model

Ischemic stroke remains a leading cause of mortality and long-term disability worldwide, necessitating efforts to identify biomarkers for diagnosis, prognosis, and treatment monitoring. The present study aimed to identify novel plasma biomarkers of neurodegeneration and inflammation in a mouse model of stroke induced by distal middle cerebral artery occlusion. Using targeted lipidomic and global untargeted metabolomic profiling of plasma collected from aged male mice 24 h after stroke and weekly thereafter for 7 weeks, we discovered distinct acute and chronic signatures. In the acute phase, we observed elevations in myelin-associated lipids, including sphingomyelin (SM) and hexosylceramide (HCER) lipid species, indicating brain lipid catabolism. In the chronic phase, we identified 12-hydroxyeicosatetraenoic acid (12-HETE) as a putative biomarker of prolonged inflammation, consistent with our previous observation of a biphasic pro-inflammatory response to ischemia in the mouse brain. These results provide insight into the metabolic alterations detectable in the plasma after stroke and highlight the potential of myelin degradation products and arachidonic acid derivatives as biomarkers of neurodegeneration and inflammation, respectively. These discoveries lay the groundwork for further validation in human studies and may improve stroke management strategies.

Endovascular treatment of primary M3 occlusion stroke in clinical practice: analysis of the German Stroke Registry

BackgroundEndovascular treatment (ET) options for acute stroke due to distal middle cerebral artery occlusions are rapidly evolving, but data on outcome and safety are sparse. We therefore performed an analysis of patients undergoing ET for primary M3 occlusions in routine clinical practice in a nationwide registry.MethodsPatients enrolled between 01/20 and 12/21 in the prospective, multicenter German Stroke Registry-Endovascular Treatment (GSR-ET) were screened for mechanical thrombectomy performed for primary M3 occlusion. We analyzed neurological deficit as measured by the National Institute of Health Stroke Scale (NIHSS), symptomatic intracranial hemorrhage (sICH), thrombectomy technique, successful reperfusion (modified Thrombolysis in Cerebral Infarction [mTICI] score of 2b-3) and functional outcome as measured by the modified Rankin Scale (mRS) at discharge and 90 days.ResultsOut of 5574 patients, 11 patients (0.2%, median age 80 years, 54.5% female) underwent ET for primary M3 occlusion. All patients had pre-admission mRS ≤ 1, median NIHSS on admission was 8, and successful reperfusion was achieved in 6/11 patients (54.5%). While no vasospasm, dissection or perforation was reported, symptomatic intracranial hemorrhage occurred in 2 patients (18.2%). Favorable outcome (mRS ≤ 2) was achieved in 6/11 patients (54.5%) at 90-day follow-up.ConclusionsET for primary M3 occlusions is rarely performed. While technically feasible, the procedure’s potential benefits must be carefully weighed against its associated risks, including clinically relevant complications. Caution and further research is needed to optimize patient selection for this intervention.Trial RegistrationGSR-ET; ClinicalTrials.gov Identifier: NCT03356392; Trial Registration Date: 11/29/2017.

Symptomatic intracerebral hemorrhage in proximal and distal medium middle cerebral artery occlusion patients treated with mechanical thrombectomy

BackgroundAcute ischemic stroke (AIS) caused by distal medium vessel occlusions (DMVOs) represents a significant proportion of overall stroke cases. While intravenous thrombolysis (IVT) has been a primary treatment, advancements in...

A minimally invasive thrombotic stroke model to study circadian rhythm in awake mice.

Experimental stroke models in rodents are essential for mechanistic studies and therapeutic development. However, these models have several limitations negatively impacting their translational relevance. Here we aimed to develop a minimally invasive thrombotic stroke model through magnetic particle delivery that does not require craniotomy, is amenable to reperfusion therapy, can be combined with in vivo imaging modalities, and can be performed in awake mice. We found that the model results in reproducible cortical infarcts within the middle cerebral artery (MCA) with cytologic and immune changes similar to that observed with more invasive distal MCA occlusion models. Importantly, the injury produced by the model was ameliorated by tissue plasminogen activator (tPA) administration. We also show that MCA occlusion in awake animals results in bigger ischemic lesions independent of day/night cycle. Magnetic particle delivery had no overt effects on physiologic parameters and systemic immune biomarkers. In conclusion, we developed a novel stroke model in mice that fulfills many requirements for modeling human stroke.

Identifying the Sylvian Triangle on CT angiography: A technique for detecting distal middle cerebral artery occlusions.

Medium vessel occlusions (MeVOs), defined as occlusion of the M2/M3 and A2/A3 segments of the middle cerebral artery (MCA) and anterior cerebral artery, can be challenging to visualize on CT angiography (CTA) and MR angiography (MRA), given the anatomic complexity of the mid- and distal intracranial vasculature and smaller vessel caliber (Leary MC, Kidwell CS, Villablanca JP, et al. Validation of computed tomographic MCA "dot" sign: an angiographic correlation study. Stroke 2003; 34: 2636-2640; Luijten SPR, Wolff L, Duvekot MHC, et al. Diagnostic performance of an algorithm for automated large vessel occlusion (LVO) detection on CTA. J Neurointerv Surg 2022; 14: 794-798). In turn, the appearance of a sudden vessel cutoff in these vascular distributions on CTA or MRA is not always straightforward and may represent true occlusion, variant anatomy, and/or artifact (Leary MC, Kidwell CS, Villablanca JP, et al. Validation of computed tomographic MCA "dot" sign: an angiographic correlation study. Stroke 2003; 34: 2636-2640; Luijten SPR, Wolff L, Duvekot MHC, et al. Diagnostic performance of an algorithm for automated LVO detection on CTA. J Neurointerv Surg 2022; 14: 794-798). Given the importance of rapidly establishing an accurate diagnosis in the setting of stroke, combined with recent clinical trials and movements promoting the efficacy of endovascular therapeutic approaches to treat MeVOs, it remains imperative to detect such occlusions accurately and quickly on imaging. In turn, we present five imaging patterns of the Sylvian Triangle on sagittal reformatted images from CTA Head examinations, which our practice has utilized to assess patency of the M2 and M3 divisions. This approach is rapidly deployable and can be utilized by radiology and non-radiology healthcare providers alike, thus facilitating rapid and accurate diagnosis of MeVO, timely evaluation of candidacy for endovascular therapy, and ultimately supporting favorable door-to-intervention time and successful patient outcomes.

An-Gong-Niu-Huang-Wan (AGNHW) regulates cerebral blood flow by improving hypoperfusion, cerebrovascular reactivity and microcirculation disturbances after stroke

BackgroundThe restoration of cerebrovascular regulation and improvement of cerebral blood flow in ischaemic regions are crucial for improving the clinical prognosis after stroke. An-Gong-Niu-Huang-Wan (AGNHW) is a famous traditional compound Chinese medicine that has been used for over 220 years to treat acute ischaemic stroke; however, its role in the regulation of cerebral blood flow is still unclear. The aim of the present study was to investigate the regulatory effect of AGNHW on cerebral blood flow and microcirculation after ischaemic stroke and to elucidate the underlying mechanisms involved.MethodsMale C57BL/6 mice were subjected to distal middle cerebral artery occlusion (dMCAO) and randomly assigned to the sham, MCAO, or AGNHW groups. AGNHW was administered intragastrically 1 h after dMCAO. The rotarod test was utilized to evaluate behavioural function; TTC was used to determine the infarct volume; and ischaemic injury was assessed by detecting brain levels of SOD, MDA and NO. Then, cortical perfusion and acetazolamide-induced cerebrovascular reactivity were assessed using laser speckle contrast imaging, and the velocity and flux of red blood cells in cortical capillaries were detected using two-photon laser scanning microscopy. In addition, we employed RNA-Seq to identify variations in gene expression profiles and assessed endothelium-dependent changes in microcirculatory dysfunction by measuring vasoactive mediator levels.ResultsAGNHW significantly increased cerebral blood flow, reduced the infarct volume, and promoted functional recovery after cerebral ischaemia. AGNHW increased the velocity and flux of red blood cells in capillaries and improved cerebrovascular reactivity in the ischaemic cortex. Furthermore, AGNHW regulated endothelium-dependent microcirculation, as evidenced by decreases in the expression of endothelins (Edn1, Edn3 and Ednrb) and the ratios of brain and serum TXB2/6-keto-PGF1α and ET-1/CGRP.ConclusionsAGNHW improved cerebral hypoperfusion, regulated cerebrovascular reactivity and attenuated microcirculatory dysfunction within the ischaemic cortex after stroke. This outstanding effect was achieved by modulating the expression of genes related to vascular endothelial cell function and regulating endothelium-dependent vasoactive mediators.

Vascular syndrome predicts the development and course of epilepsy after perinatal stroke.

Epilepsy develops in one third of the patients after perinatal stroke. It is still unclear which vascular syndrome of ischemic stroke carries higher risk of epilepsy. The aim of the current study was to evaluate the risk of epilepsy according to the vascular syndrome of perinatal stroke. The study included 39 children with perinatal arterial ischemic stroke (13 with anterior or posterior trunk of the distal middle cerebral artery occlusion, 23 with proximal or distal M1 middle cerebral artery occlusion and three with lenticulostriate arteria infarction), and 44 children with presumed perinatal venous infarction. Magnetic resonance imaging obtained at the chronic stage was used to evaluate the vascular syndrome of stroke. The median follow-up time was 15.1 years (95% CI: 12.4-16.5 years), epilepsy developed in 19/83 (22.9%) patients. The cumulative probability to be without epilepsy at 15 years was 75.4% (95% CI: 65.8-86.4). The probability of having epilepsy was higher in the group of proximal or distal M1 artery occlusion compared to patients with periventricular venous infarction (HR 7.2, 95% CI: 2.5-26, p = .0007). Patients with periventricular venous infarction had significantly more often status epilepticus or spike-wave activation in sleep ≥85% of it compared to patients with anterior or posterior trunk of the distal middle cerebral artery occlusion (OR = 81; 95% CI: 1.3-5046, p = .029). The emphasis of this study is placed on classifying the vascular syndrome of perinatal stroke and on the targeted follow-up of patients for epilepsy until young adulthood. The risk for having epilepsy after perinatal stroke is the highest in children with proximal or distal M1 middle cerebral artery occlusion. Patients with periventricular venous infarction have a more severe course of epilepsy.

Increased sensitivity in detection of deficits following two commonly used animal models of stroke

Stroke is a leading cause of death and disability in the United States. Most strokes are ischemic, resulting in both cognitive and motor impairments. Animal models of ischemic stroke such as the distal middle cerebral artery occlusion (dMCAO) and photothrombotic stroke (PTS) procedures have become invaluable tools, with their own advantages and disadvantages. The dMCAO model is clinically relevant as it occludes the artery most affected in humans, but yields variability in the infarct location as well as the behavioral and cognitive phenotypes disrupted. The PTS model has the advantage of allowing for targeted location of infarct, but is less clinically relevant. The present study evaluates phenotype disruption over time in mice subjected to either dMCAO, PTS, or a sham surgery. Post-surgery, animals were tested over 28 days on standard motor tasks (grid walk, cylinder, tapered beam, and rotating beam), as well as a novel odor-based operant task; the 5:1 Odor Discrimination Task (ODT). Results demonstrate a significantly greater disturbance of motor control with PTS as compared with Sham and dMCAO. Disruption of the PTS group was detected up to 28 days post-stroke on the grid walk, and up to 7 days on the rotating and tapered beam tasks. PTS also led to significant short-term disruption of ODT performance (1-day post-surgery), exclusively in males, which appeared to be driven by motoric disruption of the lick response. Together, this data provides critical insights into the selection and optimization of animal models for ischemic stroke research. Notably, the PTS procedure was best suited for producing disruptions of motor behavior that can be detected with common behavioral assays and are relatively enduring, as is observed in human stroke.

Dl-3-n-butylphthalide improves stroke outcomes after focal ischemic stroke in mouse model by inhibiting the pyroptosis-regulated cell death and ameliorating neuroinflammation

Recent studies have highlighted the involvement of pyroptosis-mediated cell death and neuroinflammation in ischemic stroke (IS) pathogenesis. DL-3-n-butylphthalide (NBP), a synthesized compound based on an extract from seeds of Apium graveolens, possesses a broad range of biological effects. However, the efficacy and the underlying mechanisms of NBP in IS remain contentious. Herein, we investigated the therapeutic effects of NBP and elucidated its potential mechanisms in neuronal cell pyroptosis and microglia inflammatory responses. Adult male mice underwent permanent distal middle cerebral artery occlusion (dMCAO), followed by daily oral gavage of NBP (80 mg/kg) for 1, 7, or 21 consecutive days. Gene Expression Omnibus (GEO) dataset of IS patients peripheral blood RNA sequencing was analyzed to identify differentially expressed pyroptosis-related genes (PRGs) during the ischemic process. Our results suggested that NBP treatment effectively alleviated brain ischemic damage, resulting in decreased neurological deficit scores, reduced infarct volume, and improved neurological and behavioral functions. RNA sequence data from human unveiled upregulated PRGs in IS. Subsequently, we observed that NBP downregulated pyroptosis-associated markers at days 7 and 21 post-modeling, at both the protein and mRNA levels. Additionally, NBP suppressed the co-localization of pyroptosis markers with neuronal cells to variable degrees and simultaneously mitigated the accumulation of activated microglia. Overall, our data provide novel evidence that NBP treatment significantly attenuates ischemic brain damage and promotes recovery of neurological function in the early and recovery phases after IS, probably by negatively regulating the pyroptosis cell death of neuronal cells and inhibiting toxic neuroinflammation in the central nervous system.

ATF3 is a neuron-specific biomarker for spinal cord injury and ischaemic stroke.

Although many molecules have been investigated as biomarkers for spinal cord injury (SCI) or ischemic stroke, none of them are specifically induced in central nervous system (CNS) neurons following injuries with low baseline expression. However, neuronal injury constitutes a major pathology associated with SCI or stroke and strongly correlates with neurological outcomes. Biomarkers characterized by low baseline expression and specific induction in neurons post-injury are likely to better correlate with injury severity and recovery, demonstrating higher sensitivity and specificity for CNS injuries compared to non-neuronal markers or pan-neuronal markers with constitutive expressions. In animal studies, young adult wildtype and global Atf3 knockout mice underwent unilateral cervical 5 (C5) SCI or permanent distal middle cerebral artery occlusion (pMCAO). Gene expression was assessed using RNA-sequencing and qRT-PCR, while protein expression was detected through immunostaining. Serum ATF3 levels in animal models and clinical human samples were measured using commercially available enzyme-linked immune-sorbent assay (ELISA) kits. Activating transcription factor 3 (ATF3), a molecular marker for injured dorsal root ganglion sensory neurons in the peripheral nervous system, was not expressed in spinal cord or cortex of naïve mice but was induced specifically in neurons of the spinal cord or cortex within 1 day after SCI or ischemic stroke, respectively. Additionally, ATF3 protein levels in mouse blood significantly increased 1 day after SCI or ischemic stroke. Importantly, ATF3 protein levels in human serum were elevated in clinical patients within 24 hours after SCI or ischemic stroke. Moreover, Atf3 knockout mice, compared to the wildtype mice, exhibited worse neurological outcomes and larger damage regions after SCI or ischemic stroke, indicating that ATF3 has a neuroprotective function. ATF3 is an easily measurable, neuron-specific biomarker for clinical SCI and ischemic stroke, with neuroprotective properties. ATF3 was induced specifically in neurons of the spinal cord or cortex within 1 day after SCI or ischemic stroke, respectively. Serum ATF3 protein levels are elevated in clinical patients within 24 hours after SCI or ischemic stroke. ATF3 exhibits neuroprotective properties, as evidenced by the worse neurological outcomes and larger damage regions observed in Atf3 knockout mice compared to wildtype mice following SCI or ischemic stroke.

Abstract WP132: The Significance of Fluid-Attenuated Inversion Recovery Vascular Hyperintensity in Combination With Occlusion Site and Reperfusion Status by Mechanical Thrombectomy for Proximal and Distal Middle Cerebral Artery Occlusion

Introduction: In patients with acute ischemic stroke, FLAIR vascular hyperintensity (FVH) is a reliable indicator of large vessel occlusion and salvageable tissue requiring mechanical thrombectomy (MT). However, its significance in distal arterial occlusion has not been established. This study aimed to investigate the significance of FVH in DWI-negative areas, in combination with occlusion site and reperfusion status, by MT for regional tissue outcomes in proximal and distal middle cerebral artery (MCA) occlusion. Methods: The clinical records of patients admitted to a single institute between April 2017 and June 2022, who presented with MCA occlusion with initial NIHSS ≥ 5 and underwent MT within 24 h of onset were retrospectively analyzed. Proximal occlusion was defined as M1 segment occlusion, and distal occlusion as M2 or M3 segment based on initial MRA. Each cortical MCA area in Alberta Stroke Program Early CT Score (ASPECTS) ipsilateral to the occluded vessel was categorized into group A (DWI-, FVH-) or B (DWI-, FVH+) based on initial MRI. Regional collateral status on initial angiogram, expanded Thrombolysis in Cerebral Infarction (eTICI) grade on final angiogram, and regional unfavorable tissue outcome with the appearance of low or high density on CT 24 h after MT were recorded. Univariate and multivariate analyses using mixed-effects models for regional unfavorable tissue outcome were performed. Results: A total of 103 patients (56 with proximal and 47 with distal occlusion) were included. Categorizing cortical areas resulted in 256 areas in group A and 171 in group B. The rate of unfavorable tissue outcome in order of eTICI 0-2a, 2b50-67, and 2c-3 reperfusion status in group A were 14.3%, 11.3%, and 6.0%, respectively (p=0.06) while those in group B were 47.1%, 34.0%, and 12.9%, respectively (p<0.01). In multivariate analysis, eTICI 0-2b67 vs. eTICI 2c-3 in group B of distal occlusion was an independent predictor for unfavorable regional tissue outcome (OR 7.8, 95% CI 1.1-55.6, p=0.04), along with ASPECTS M2 or M5 and poor angiographical collaterals. Conclusions: FVH in DWI-negative areas can reveal penumbra tissue secondary to both proximal and distal MCA occlusions. However, salvage requires fairly reliable reperfusion in distal occlusion.

Abstract WP300: Ifi27l2a is a Novel Target for Dampening Microglial Inflammation Following Stroke

Microglia play a critical role in initiating and spreading inflammation and are involved in the chronic recovery phase following stroke. Microglia express multiple interferon-stimulated genes (ISGs) in response to inflammatory cues, including Ifi27l2a . However, the specific roles of individual ISGs, such as Ifi27l2a , have not been well-studied in the context of stroke. We tested the hypothesis that induction of Ifi27l2a in microglia contributes to the pro-inflammatory response following stroke. We used Ifi27l2a heterozygote (Het) mice to determine the effect of partial reduction of Ifi27l2a in primary microglia and in an experimental stroke model. Primary microglia isolated from WT and Het brains were stimulated with LPS to investigate the role of Ifi27l2a in microglial activation in vitro . Permanent distal middle cerebral artery occlusion (pdMCAO) was performed in young mice (WT vs Het, male, 3 months old) to determine whether deletion of Ifi27l2a reduces microglial activation and improves functional outcome following stroke. We found that LPS-induced expression of Il1b was markedly reduced in Het microglia compared to WT microglia (n=6, p<0.01). Moreover, other inflammation marker genes ( Il1a and Tnfa ) were significantly reduced in Het microglia compared to WT microglia in LPS-stimulated conditions (n=6, p<0.05). The levels of inducible forms of Mmp3 and Mmp9 , which are responsible for blood-brain barrier (BBB) dysfunction, were significantly reduced in Het microglia (n=3, p<0.01). However, Mmp2 expression was not altered by deletion of Ifi27l2a. These findings suggest that Ifi27l2a is involved in the process of BBB dysfunction and microglial inflammation. Lastly, gliosis (microgliosis and astrogliosis) was significantly reduced at 14 days post-stroke in the thalamus of Het brains, a known site of secondary injury in pdMCAO model (n=6, p<0.05). Foot fault tests showed improved functional outcome in Het mice (n=4-6, p<0.05). Ifi27l2a appears to be a critical molecular switch for inflammatory mechanisms in the post-stroke brain. These findings suggest that interventions targeting Ifi27l2a regulation (even partial inhibition) could reduce harmful inflammation and promote improved functional outcome following stroke.

Abstract TMP111: Selective Activation of Peritoneal TRPV1-Containing Vagal Afferents Promotes Sustained Mild Hypothermia and Neuroprotection Following Stroke

Intro: Mild hypothermia has been clinically effective at improving outcomes in cardiac arrest patients for decades. Similarly, mild hypothermia has been shown to neuroprotective in experimental stroke models. Unfortunately, hypothermia protocols in awake subjects are hampered by inconsistent temperature management due to incomplete suppression of cold defense mechanisms (e.g. shiver response). To address a need for better strategies for cooling conscious subjects, we sought to promote mild hypothermia by pharmacological activation of heat-sensing nerve fibers in the thermoregulatory system. We hypothesize that selective activation of TRPV1-containing vagal afferents in the peritoneal cavity can promote sustained hypothermia and protection following stroke. Methods: Aged (18-20 mo) and young (11 weeks) C57BL6 mice of both sexes were given a capsinoid mixture consisting of capsiate and dihydrocapsiate. Mice from both the aged and young cohorts underwent permanent distal middle cerebral artery occlusion (pdMCAO) or sham before recovering for 2hrs in warming cages to maintain normal body temperatures during post-stroke recovery. At 2 hours after stroke, mice were given capsinoids or vehicle (4 IP injections, 90 min interval) to produce 6 hours of mild hypothermia (or normothermic control). After 3 days brains were collected for histological analysis. Results: Capsinoids are particularly suited for region selective activation of TRPV1 targets, as they are degraded by ubiquitous esterases, and thus limited in biodistribution to distant targets. This regionally restricted activation was demonstrated through induction of mild hypothermia with IP capsinoid administration, but not with oral or subcutaneous delivery. We demonstrated that the hypothermic effect is TRPV1 dependent and that IP capsinoid delivery increases activity of the cervical vagus (hook electrode). We applied this capsinoid-mediated hypothermia approach to the pdMCAO model and demonstrated a significant reduction of brain infarct at 3 days post-stroke in both young and aged mice of both sexes. Conclusion: Selective activation of TRPV1-containing peritoneal vagal afferents is sufficient to promote sustained mild hypothermia and protection following ischemic stroke.

Abstract TMP112: Von Willebrand Factor (VWF) Modulates Smooth Muscle Cell Proliferation During the Outward Remodeling of Leptomeningeal Collateral Arterioles Following Stroke

Introduction: The leptomeningeal collaterals (LMCs) undergo outward remodeling after stroke to increase rescue flow toward the occluded region. We previously showed increased VWF in remodeled LMCs and others have shown that VWF can act as a smooth muscle cell (SMC) mitogen. Here we test the hypothesis that VWF modulates vascular remodeling of LMCs in the post-stroke brain. Methods: Permanent distal middle cerebral artery occlusion (pdMCAO) was performed in C57BL6 WT mice (cohort 1; 4 mos), aged (cohort 2; 24 mos) or VWF KO and WT male mice (cohort 3; 4 mos). At 3, 7, and 14 days after pdMCAO, the pial vessels were processed for VWF/Ki67/SMA immunofluorescence and morphometric analysis. Lastly, distribution of VWF was evaluated in LMCs from human and pig brains. Results: During the 3-14 days post-stroke, WT mice of cohort 1 demonstrated significantly increased VWF in ipsilateral LMCs as well as increased Ki67 positive cells. In ECs, Ki67-positive cells were 10.68 ± 1.48 and 10.73 ± 1.26 per mm of vessel at 3 and 7 days, respectively. In SMCs, Ki67-positive cells were 0.049 ± 0.22 and 4.546 ± 0.95 per mm vessel at 3 and 7 days, respectively. By 14 days, LMC no longer showed Ki67-positive ECs or SMCs. In aged cohort 2, along with attenuated VWF expression and remodeling, the Ki67-positive ECs and SMCs were significantly decreased in the aged mice compared to young mice (Ki67-positive ECs were reduced by 40% and 50% at 3 and 7 days, respectively; Ki67-positive SMCs were reduced by 75% at 7 days). In cohort 3, Ki67-positive SMCs were absent in the VWF-KOs at 3 days post-stroke. However, by 7 days, Ki67-positive SMCs were equivalent between WT and VWF-KOs (4.8-5.0 per mm vessel). Lastly, the presence of VWF was detected in the smooth muscle layer of LMCs of human and pig brains. Conclusions: Our study revealed increased VWF in the mural layer of remodeled LMCs and both EC and SMC proliferation. The remodeling response, VWF expression, and number of proliferative cells were attenuated in LMCs from aged brains. Knockout of VWF resulted in reduced LMC remodeling and delayed SMC proliferation. Our findings also provide evidence for VWF within the smooth muscle layer of pig and human brain arterioles, supporting the broader potential role of VWF as a molecular player in vascular remodeling.

Abstract 21: Similarity in the Changes of Brain Myeloid Transcriptome Signature is Shared Between Ischemic Stroke and Chronic Inflammation Caused by Diabetes Mellitus

Introduction: Ischemic stroke (IS) is a pressing global health concern. Diabetes mellitus not only heightens the risk of IS but also aggravates its outcomes. There is established evidence that neutrophil infiltration is involved in inflammation after stroke through various mechanisms, such as activating neutrophil extracellular traps. We aimed to examine the factors that contribute to the deterioration of IS in diabetic mice focusing on mechanisms underlying immune cell infiltration by single cell RNA sequencing (scRNAseq) of brain before and after stroke. Methods: Experimental stroke was induced using the distal middle cerebral artery occlusion (dMCAO) model. db/db mice were used to model type 2 diabetes and obesity, while db/+ mice served as euglycemic controls. Mononuclear cells from ipsilateral mouse brains were isolated 3 days post-dMCAO or sham surgery with the Percoll gradient following enzymatic digestion. Transcriptomic analysis was performed using scRNAseq from db/+ sham, db/db sham, db/+ stroke, and db/db stroke. Fluorescence-activated cell sorting (FACS) and immunofluorescence (IF) staining were conducted to validate the changes detected in the transcriptome results. Results: Prior to the onset of stroke, db/db mice exhibited changes in gene expression in endothelial cells and myeloid cells including macrophages, monocytes, and microglia, mirroring patterns of changes after stroke. Among the macrophage subpopulation in particular, db/db sham and db/+ stroke mice shared an overlap of 638/901 (70.8%) differentially expressed genes compared to db/+ sham. Gene enrichment analysis indicated predominant upregulation of genes linked to leukocyte migration and neutrophil chemotaxis. FACS revealed a heightened neutrophil presence in the brain parenchyma of diabetic mice before stroke, and further elevated after stroke compared to control mice. Specifically, the increased expression of anaphylatoxin receptor C3aR and complement C3 shown by IF corroborated with the transcriptomic signature, supporting a role of complement activation and myeloid cell transmigration. Conclusions: Our findings provide valuable insights into the mechanisms whereby diabetes potentially escalates neutrophil infiltration post-stroke.

Abstract WP318: Increased Synapse Elimination by Inflammatory Cells Contribute to Lon-Lasting Post-Stroke Memory Dysfunction in Aged Mice

Background and Purpose: Memory impairments are common after stroke. Memory dysfunction lasted longer in aged than in young mice, but the mechanisms are still unclear. Microglia maintain normal memory via removing excessive synapses and increase turnover of synaptic connections after stroke. Long-term memory deficits in aged mice may be associated with increased microglia in the brain. Hypothesis: Increased synapse removal by inflammatory cells contribute to the long-lasting post-stroke memory dysfunction of aged mice. Methods: Permanent distal middle cerebral artery occlusion (pMCAO) was induced in young and aged mice. The memory functions were analyzed weekly for 8 weeks by Y-maze test and at 8 weeks post stroke by novel objective recognition (NOR) test. Brain samples were collected 8 weeks after pMCAO induction. RNAseq analysis was performed to study gene expression profiles in peri-infarct area and hippocampus. Nissl staining was used to measure infarct volume and immunostaining was done to detect CD68 + and GFAP + cells and synapse in peri-infarct region and hippocampus. Results: RNAseq data shown up-regulation of CD68, GFAP, pro-inflammatory cytokines, immune response, microglia chemotaxins and phagocytosis genes; and down-regulation of synaptic assembly and transmission, synaptic plasticity, and long-term potentiation genes in aged mice at peri-infarct region and ipsilateral hippocampi. Aged mice had larger infarct volumes, more CD68 + and GFAP + cells in the peri-infarct region and ipsilateral hippocampi than young mice and had more CD68 + cells in the ipsilateral than the contralateral hippocampi. In Y-maze test, aged stroke mice made fewer spontaneous alternations from 3 to 8 weeks after pMCAO than young stroke and sham operated aged mice. In the NOR test, aged stroke mice spent less time on the novel objects than young stroke and sham aged mice. Increased synaptophysin positive CD68 + cells were detected in the hippocampi of aged stroke mice. Conclusions: Increase of synaptic elements by inflammatory cells contribute to the long-lasting post-stroke memory deficit in the aged mice, and reduction of neuroinflammation may improve their memory function.

Abstract WP298: Intracisternal Bolus Administration of Stanniocalcin-2 Promotes Post-Stroke Recovery in a Translational Aged Rodent Model

Stanniocalcin-2 (STC-2) is a glycoprotein implicated in multiple physiological pathways, including calcium and phosphate homeostasis, cell proliferation, and anti-inflammatory responses. The molecular mechanisms by which STC-2 functions in the brain and its specific roles in various neurological processes are not well-defined. We have previously shown that intraventricular administration of STC-2 post-stroke increases endogenous stem cell production and improves functional recovery in an adult rodent model of distal middle cerebral artery occlusion (dMCAO). To further investigate the translatability and preclinical efficacy of STC-2 as a protein therapeutic, we intracisternally administered recombinant STC-2 one week post-dMCAO in aged rats (18-24 months) and comprehensively assessed recovery over a 6 week period via sensorimotor, locomotor, and cognitive tasks. Our results indicate that just two bolus intracisternal injections of STC-2 improved stroke recovery in a battery of behavioral assessments in comparison to a control group (n=9 per group, unpaired t-tests with Welch's correction, see Figure 1A-D). Improved sensorimotor recovery (as assessed by vibrissae-forepaw and hind-rearing tests), locomotor recovery (as assessed by an open field test), and cognitive recovery (as assessed by a novel object recognition test) were observed. Further research is required to delve into the underlying molecular mechanisms responsible. Nevertheless, we are optimistic about the substantial translational prospects of this study. Our findings have the potential to pave the way for a novel protein therapeutic for the treatment of stroke deficits.

Targeted mRNA Nanoparticles Ameliorate Blood-Brain Barrier Disruption Postischemic Stroke by Modulating Microglia Polarization.

The ischemic stroke is a major global health concern, with high mortality and disability rates. Unfortunately, there is a dearth of effective clinical interventions for managing poststroke neuroinflammation and blood-brain barrier (BBB) disruption that are crucial for the brain injury evolving and neurological deficits. By leveraging the pathological progression of an ischemic stroke, we developed an M2 microglia-targeting lipid nanoparticle (termed MLNP) approach that can selectively deliver mRNA encoding phenotype-switching interleukin-10 (mIL-10) to the ischemic brain, creating a beneficial feedback loop that drives microglial polarization toward the protective M2 phenotypes and augments the homing of mIL-10-loaded MLNPs (mIL-10@MLNPs) to ischemic regions. In a transient middle cerebral artery occlusion (MCAO) mouse model of an ischemic stroke, our findings demonstrate that intravenously injected mIL-10@MLNPs induce IL-10 production and enhance the M2 polarization of microglia. The resulting positive loop reinforces the resolution of neuroinflammation, restores the impaired BBB, and prevents neuronal apoptosis after stroke. Using a permanent distal MCAO mouse model of an ischemic stroke, the neuroprotective effects of mIL-10@MLNPs have been further validated by the attenuation of the sensorimotor and cognitive neurological deficits. Furthermore, the developed mRNA-based targeted therapy has great potential to extend the therapeutic time window at least up to 72 h poststroke. This study depicts a simple and versatile LNP platform for selective delivery of mRNA therapeutics to cerebral lesions, showcasing a promising approach for addressing an ischemic stroke and associated brain conditions.

Gut microbiota-derived acetate promotes long-term recovery through angiogenesis guided by lymphatic ingrowth in older adults with stroke.

Ischemic stroke is a leading cause of morbidity and mortality in older adults. Therefore, in this study, we sought to understand the interplay between the microbiota, gut, and brain in the context of stroke in older adults. To determine whether gut microbiota from younger individuals promotes recovery through angiogenesis in both elderly stroke patients and aged stroke mice, we explored the changes in gut microbiota and the correlation between short-chain fatty acids (SCFAs) and angiogenesis in the aged stroke population. Then, we altered the gut microbiome in aged mice by transplanting microbiota from younger donors before inducing experimental stroke to explore the mechanism by which gut microbiota-derived SCFAs promote angiogenesis. Part I: We conducted a single-center, double-blind trial to compare gut microbiota diversity and SCFA levels in fecal samples from older stroke patients with those from younger stroke patients. Additionally, we measured levels of vascular endothelial growth factor (VEGF) and VEGFC levels in plasma to assess their correlation with SCFA levels. Part II: We performed fecal microbiota transplantation (FMT) 3 days before inducing ischemic stroke in aged male mice (16-18) via distal middle cerebral artery occlusion (dMCAO). The FMT was conducted using gut microbiomes from either young donors (2-3 months) or aged donors (16-18 months). In older stroke patients, gut microbiota diversity was significantly reduced compared to that in younger stroke patients. Furthermore, levels of acetate, a bacterially derived SCFA, were lower and positively correlated with angiogenesis markers (VEGF and VEGF-C). In aged stroke mice, transplantation of young microbiota improved stroke outcomes by promoting angiogenesis, which was facilitated by lymphatic ingrowth into the cortex. This protective effect was linked to gut microbiota-derived acetate, which enhanced lymphangiogenesis by replenishing acetyl coenzyme A. (a) Gut microbiota-derived acetate promotes angiogenesis post-stroke and (b) lymphatic ingrowth into the cerebral cortex was observed in post-dMCAO mice. These findings suggest that selectively promoting SCFA-producing bacteria, particularly acetate-producers, could be a promising therapeutic strategy to reduce functional impairments in older stroke subjects.