Ischemic Duration is More Critical than Reperfusion Efficacy in Driving Early Neuroinflammation and Motor Deficits after Transient Middle Cerebral Artery Occlusion

Ischemic Duration is More Critical than Reperfusion Efficacy in Driving Early Neuroinflammation and Motor Deficits after Transient Middle Cerebral Artery Occlusion | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ischemic Duration is More Critical than Reperfusion Efficacy in Driving Early Neuroinflammation and Motor Deficits after Transient Middle Cerebral Artery Occlusion Laurel E Schappell, Miguel M Madeira, Claire Polizu, James DiPersio, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8712806/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Ischemic stroke remains a leading cause of long-term disability, despite more patients undergoing vessel recanalization. Ischemia/reperfusion injury (I/RI), the inflammatory cascade triggered by ischemia and reperfusion, contributes to infarct evolution and functional outcomes. Clinical stroke presentation varies due to differences in ischemic duration and reperfusion success, yet preclinical models rarely account for this physiologic contribution. This preclinical-clinical disconnect may underlie the translational failure of acute therapies targeting the myeloid cell-mediated immune response (specifically neutrophils and microglia/bone marrow-derived macrophages). To address this physiological heterogeneity, we employed variations of the transient middle cerebral artery occlusion model in myeloid-reporter mice, systematically altering ischemic duration and reperfusion success. Using longitudinal perfusion imaging and behavioral testing, we found that infarct pathology, locomotor deficits, and innate immune responses were significantly influenced by ischemic duration, and to a lesser extent, reperfusion status. Microglia/macrophage and neutrophil morphology, as well as the spatial association of these myeloid cells, were the most strongly affected cellular features. Further, neutrophil density, morphology, and spatial patterning correlated with acute locomotion and motor recovery across the entire cohort. These findings highlight the differential roles of ischemic duration and reperfusion efficacy in driving neuroinflammation and stroke outcomes, emphasizing the importance of incorporating physiologic heterogeneity into preclinical I/RI models to better guide translational strategies. Acute Stroke Animal Models Inflammation Ischemia/Reperfusion Myeloid Cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 BACKGROUND Ischemic stroke is a leading cause of morbidity and mortality worldwide [ 1 ]. Eligibility criteria for recanalization of large vessel occlusion (LVO) stroke are continuously expanding to promote cerebral reperfusion and improve outcomes, yet > 50% of patients still experience significant disability despite treatment [ 2 – 5 ]. Ischemia/reperfusion injury (I/RI), the inflammatory response induced by ischemia and continued throughout reperfusion (I/R), significantly contributes to infarct evolution and functional outcomes [ 6 ]. Stroke immunomodulatory therapies often focus on myeloid cells - polymorphonuclear neutrophils (PMN), bone-marrow derived macrophages (MΦ), and microglia (MG) - as they constitute the predominant first responders in I/RI [ 7 , 8 ]. However, preclinical trials aimed at mitigating I/RI by targeting myeloid cell recruitment have yet to translate into successful clinical trials [ 9 – 12 ]. The post-stroke inflammatory response is highly conserved between mice and humans, suggesting that this preclinical-clinical disconnect can likely be attributed to differences in the features of stroke evolution that govern the myeloid response [ 8 , 13 ]. In clinical practice, perfusion dynamics are widely variable and known to mediate disease progression, with longer ischemic duration and less complete reperfusion independently contributing to worse outcomes [ 14 , 15 ]. However, variability in these physiologic metrics of I/R remains unaccounted for in preclinical stroke models. The preclinical transient middle cerebral artery occlusion (tMCAO) model - widely adopted to simulate LVO I/RI - is often performed in homogeneous surgical cohorts with standardized ischemic duration and reperfusion status across animals. As a result, the role of I/R dynamics in mediating the myeloid cell response in I/RI, and ultimately, pathological and behavioral outcomes, has yet to be elucidated. Defining this relationship between the I/R profile and downstream inflammatory consequences will allow preclinical models to better align with the cellular processes and disease outcomes observed in the stroke patient population. We sought to address this gap by investigating the role of ischemic duration and reperfusion status in driving stroke evolution and I/RI. Using serial perfusion imaging and behavioral testing with variations of the tMCAO model in myeloid reporter mice, this study found that acute infarct topology, behavior, and myeloid cell features are more strongly modulated by ischemic duration than by reperfusion efficacy. Specifically, MG/MΦ and PMN morphology, and spatial relationships between PMNs and MG/MΦ were most profoundly affected. Based on the shared response to varied I/R dynamics, correlation analyses performed across all surgical cohorts identified an intrinsic link between locomotor function and the acute immune response. These data highlight a critical need to consider how the stroke I/R profile defines tissue pathology and establish a link between the myeloid response and functional outcomes to inform future therapeutic strategies. METHODS Mice All procedures were approved by the Institutional Animal Care and Use Committee (Animal Welfare Assurance No A3011-0) at SUNY Stony Brook School of Medicine. Mice were housed in the institutional animal facility under a 12-hour light/dark cycle, and all husbandry was performed by the Stony Brook Division of Laboratory Animal Resources. Food and water were provided ad libitum . The Ly6G-TdT x Csf1r-EGFP strain was derived by crossing Catchup (C57BL/6 Ly6G(Cre-TdTomato)) mice with MacGreen (C57BL/6-CSF1R-EGFP) mice to yield heterozygotes for each fluorophore [ 16 , 17 ]. Genotypes were confirmed via polymerase chain reaction. Male and female mice were used in equal proportion. Ly6G-TdT x Csf1r-EGFP heterozygotes aged 3 to 6 months were used for all experiments. Mice weighing 35 g were excluded from analyses. In total, 295 mice were used. Transient Middle Cerebral Artery Occlusion (tMCAO) Model Surgical procedures were performed following the IMPROVE guidelines [ 18 ]. Mice were randomized to either the Longa (complete reperfusion) or Koizumi (incomplete reperfusion) tMCAO method, as previously described, and were further randomized to 30, 60, or 90 minutes of ischemia [ 19 , 20 ]. Mice received preoperative analgesia (0.2 mg/kg s.c. bupivacaine) and fluid support (1 mL i.p. saline). The surgical site was prepared by removing all hair and sterilizing with Betadine. Lubricant was applied to both eyes to avoid corneal drying. Mice were anesthetized (isoflurane, 3.5% induction, 0.5-1.0% maintenance) under aseptic conditions with temperature maintenance (37.0°C ± 0.5°C) via rectal probe. Once anesthesia depth was confirmed by lack of response to foot pinch, a midline ventral neck incision was made on the right side. The common carotid artery (CCA) and internal carotid artery (ICA) were isolated and temporarily ligated using 7 − 0 sutures. The distal external carotid artery (ECA) was permanently ligated. For Koizumi procedures, a weight-matched silicon coated filament (Doccol 602145 for 28g; Doccol, Sharon, MA) was inserted through an incision in the CCA superior to the point of ligation. For Longa procedures, the incision was instead made in the ECA proximal to the point of ligation for insertion of the weight-matched filament (Doccol 602123 for 28 g). In both models, the ICA was unligated and the filament was advanced ~ 9 mm into the ICA to occlude the origin of the middle cerebral artery. The filament was secured with a temporary suture distal to the insertion point. The neck incision was sutured, and animals were removed from anesthesia and allowed to recover in a heated chamber. Intra-ischemic behavior was assessed just prior to re-anaesthetizing using an ordinal five-point scale as previously described [ 21 ]. Briefly, mice were allowed to move freely and then suspended by the tail to assess the degree of unilateral deficit exhibited, where 0 = no deficit, 1 = forelimb weakness and torso turning to the affected side when suspended by tail, 2 = spontaneous circling to the affected side, 3 = unable to bear weight on the affected side, and 4 = no spontaneous locomotor activity or barrel rolling. Mice with no deficits were excluded. Just prior to the end of the ischemic window, mice were re-anaesthetized and cerebral blood flow (CBF) was assessed by laser speckle contrast imaging (LSCI) before the neck incision neck was reopened. The filament was withdrawn to enable recanalization and either the CCA (Koizumi) or ECA (Longa) was permanently ligated. The CCA was opened in the Longa surgical cohorts following filament removal. Mice recovered in a heated chamber before being returned to their home cage. Mice were excluded if subarachnoid hemorrhage was detected or if inadequate occlusion was observed on LSCI. Laser Speckle Contrast Imaging (LSCI) To enable serial imaging, mice were anesthetized and the skin (~ 1 cm diameter) overlying the skull cap was removed. A layer of optically clear glue (Norland Optical Adhesive 81, Edmund Optics) was coated over the skull and cured with UV (5% UV, 10–15 seconds exposure, 5 cm height from skull) (CS 2020, ThorLabs). Skull covers were placed at least 24 hours prior to tMCAO procedure to minimize any possibility of procedure-induced vascular injury or inflammation at the time of surgery. LSCI (PeriCam PSI HR; Perimed) was conducted prior to and after UV glue coating to confirm the glue did not alter CBF patterning. LSCI was captured at skull cover placement (baseline), immediately prior to filament withdrawal (ischemia), immediately after filament withdrawal (reperfusion), and immediately prior to tissue collection (endpoint). Ischemia and reperfusion were confirmed by LSCI, acquired immediately before and after filament withdrawal, as previously described [ 22 ]. Imaging was performed for > 15 seconds and one representative frame was used for pixel quantification. CBF was represented by a color-gradated scale of arbitrary perfusion units (a.u.) according to the random motion detected at each pixel, with high random motion corresponding to healthy perfusion (red) and low random motion corresponding to hypoperfusion (blue). Images were generated as color-coded heatmaps of a.u. on a scale of 0 to 300. Matlab (24.2.0.2712019) was used to isolate the ischemic hemisphere from background signal according to the regional difference in perfusion units. Within the ischemic hemisphere, perfusion units were classified according to color (blue = 0–60, cyan = 61–120, green = 121–180, yellow = 181–240, red = 241–300 a.u.) and the number of pixels corresponding to each category was calculated. Speckle score was calculated at baseline, ischemia, reperfusion, and endpoint using a weighted scale from 1 to 5 according to the average pixel color. Specifically, each color classification was assigned an ordinal value (red = 5, yellow = 4, green = 3, cyan = 2, blue = 1). The average value of all pixels within the ischemic hemisphere was then calculated to derive the speckle score. For instance, 5 corresponded to 100% red pixels and 1 corresponded to 100% blue pixels. Behavioral Testing Behavioral testing was performed at baseline and at 24 hours (24h) and 72 hours (72h) post-reperfusion. For corner testing, mice were placed in a 30° opaque corner with their whiskers contacting the walls and allowed to escape by rearing to either side [ 23 ]. Ten trials were conducted, and laterality index was calculated as the absolute difference between left and right turns divided by total turns. For open field testing, mice were allowed to explore an open field (42 cm x 42 cm x 42 cm) for 10 minutes [ 24 ]. Behavior was recorded and processed using Handbrake (1.7.3). Anatomic landmarks were tracked via DeepLabCut-based pose estimation (0.2.1.7) using an optimized configuration of the open-source Super Animal Top View Mouse network [ 25 , 26 ]. RStudio (4.4.1) was used to calculate locomotor metrics and generate heatmaps. Tissue Collection Brains were collected at 24h or 72h post-tMCAO. Mice allocated to immunofluorescence imaging received a retro-orbital injection of 10% Tomato Lectin, Dylight 649 (Vector Laboratories) 15 minutes prior to collection to label reperfused vasculature. Mice were deeply anesthetized (isoflurane, 5%) and transcardially perfused with cold phosphate buffered saline (PBS) followed by cold 4% paraformaldehyde (PFA) if allocated to immunofluorescent imaging or PBS alone for infarct size quantification. Endogenous TdTomato signal was imaged to confirm retention of adherent PMNs in the vasculature following perfusion, as has been previously observed ( Figure S1 ) [ 9 , 27 ]. Brains were dissected, meninges removed, and brains were sectioned into 1 mm coronal slices using an adult mouse brain slicing block (5325; Zivic Instruments). Infarct Volume Quantification For infarct size quantification, brain slices were stained with triphenyl tetrazolium chloride (TTC) for 15 minutes to distinguish infarcted (white) from healthy tissue (red) [ 28 ]. Seven consecutive coronal slices were imaged using a near-infrared fluorescent imager (Azure 500; Azure Biosystems). Infarct and hemorrhage volumes were quantified by manual tracing in Fiji ImageJ, with edema correction applied using the ipsilateral-to-contralateral hemisphere ratio. Immunostaining and Confocal Imaging Brain slices for immunofluorescence were post-fixed in 4% PFA for 24 hours and cryoprotected in 15% and 30% sucrose. Slices were washed in 0.3% Triton-X in PBS and blocked in 10% goat serum for 2 hours at room temperature, and stained overnight at 4°C with NeuN primary antibody (1:1000, ABN90P; Sigma-Aldrich), followed by incubation with goat anti-guinea pig 405 secondary antibody (1:1000, SAB4600230; Sigma-Aldrich) for 2 hours at room temperature. Samples were stored in 0.02% sodium azide in PBS prior to imaging. Fluorescent imaging was performed using an Airyscan two-photon laser-scanning confocal microscope (LSM 980; Zeiss). Coronal slices at bregma − 1mm, corresponding to the region of maximal ischemia, were selected for analysis. Widefield scans of the ischemic hemisphere were acquired at 10x magnification (z-step: 10 µm, z-stack: 200–250 µm) and high-resolution z-stacks for cell counts and morphological analyses were acquired at 20x magnification (z-step: 0.48 µm, z-stack: 100–120 µm) in at least three cortical fields of view (FOV). Image stitching and reconstruction were performed in Fiji ImageJ. Imaging Analysis Cell analyses of MG/MΦ and PMNs captured in 20x FOVs were conducted using Imaris Single Full with Clear View (Imaris Bitplane v.10.2, Oxford Instruments) image analysis software. Images were pre-processed using the Gaussian Filter function to enhance cell signal relative to background. Cell and vascular reconstructions were created using the Machine Learning Segmentation Surface Creation tool with each source channel. Surfaces were filtered according to area and intensity to exclude non-cellular signal. Cell volume, sphericity, and distance to other surfaces were calculated automatically for each cell within the surface. Correlation Analysis Correlation coefficients for CBF, behavior, and immune response variables were calculated and corresponding heatmaps were generated using R Studio (4.4.1, R Foundation for Statistical Computing, Vienna, Austria) and the pheatmap package. Statistics All statistical analyses were conducted using GraphPad Prism (10.4.2). Input data for Prism was collated in Excel. For all experiments except survival analysis, statistical significance was assessed using two-way analysis of variance (ANOVA) for ischemic duration and reperfusion status with Tukey’s multiple comparison post hoc tests as indicated between cohorts with common ischemic time or reperfusion success. Survival analysis was conducted using the log-rank Mantel-Cox test. All data is reported as the mean; *p < 0.05, **p < 0.01, ***p < 0.001. Results of all statistical analyses are reported in Table S1 . RESULTS Varying stroke physiology alters infarct topology and intra-ischemic behavior Ischemic duration and reperfusion efficacy are highly variable among stroke patients. However, preclinical studies fail to account for the role of these physiologic features in mediating stroke pathology and outcomes. To mimic variable reperfusion, the Longa and Koizumi methods of the transient middle cerebral artery occlusion (tMCAO) model were employed. These methods differ in the identity of the vessel used for filament insertion and sacrificed following recanalization: the Longa method uses the ECA to mimic complete reperfusion, while the Koizumi method uses the CCA to achieve incomplete reperfusion (Fig. 1 a-b). Although both variations induce acute focal ischemia, the chronic hyporeperfusion induced by the loss of the CCA in the Koizumi technique also contributes to longitudinal infarct evolution, as is representative of patients with unsuccessful reperfusion despite recanalization therapy. To recapitulate variable ischemic time, the duration of ischemia was varied to 30, 60, and 90 minutes, corresponding to ischemic durations commonly used in preclinical studies. Through these tMCAO variations, a spectrum of stroke physiology was established, consisting of six unique conditions: 30-minute Longa (30mL), 60-minute Longa (60mL), 90-minute Longa (90mL), 30-minute Koizumi (30mK), 60-minute Koizumi (60mK), and 90-minute Koizumi (90mK). The effect of these I/R modulations on infarct topology was first investigated according to TTC staining to delineate ischemic and healthy tissue (Fig. 1 c-d). Infarct volumes tended to be larger with increasing ischemic duration (Fig. 1 e). Additionally, incomplete reperfusion was linked to larger infarct volumes specifically in the 90m cohorts. Although the 90mL group did not exhibit significantly larger infarcts than reperfusion-matched groups of shorter ischemic duration, it did demonstrate a higher frequency of hemorrhagic transformation than the 30mL cohort (Fig. 1 f). However, a difference in hemorrhage volume was only detected between the 30mL and 60mL cohorts (Fig. 1 g). To link infarct pathology to the severity of the initial ischemic insult, intra-ischemic behavior was measured using an ordinal five-point scale. More severe intra-ischemic behavior was observed with increasing ischemic duration in the Koizumi cohorts, as evidenced by an increased prevalence of high behavior scores (Fig. 1 h). Intra-ischemic behavior was also worse in the 90mK group relative to the 90mL group, similar to the trends observed in infarct volume. I/R dynamics did not influence mortality within 72 hours post-tMCAO (Fig. 1 i). Incomplete reperfusion induces a sustained drop in cerebral blood flow Given that I/R profile alters ischemic macrovascular blood flow, we investigated whether these physiologic features also mediate spatiotemporal CBF patterning following tMCAO. Serial LSCI was performed at baseline, immediately before and after filament withdrawal, and at endpoint to create spatial maps of surface CBF. LSCI was employed as it enabled spatial imaging of the superficial cortex (~ 1 mm) while minimizing unnecessary anesthesia, which would alter the inflammatory and pathological environment. First, I/R and infarct localization were confirmed for each model based on visual changes in perfusion relative to baseline (Fig. 2 a). Mirrored regions of interest (ROIs) were overlaid onto the ipsilateral and contralateral hemispheres to quantify CBF according to the scale of arbitrary perfusion units (a.u.), where higher values correspond to greater perfusion within the ROI ( Figure S2 a ). As expected, perfusion did not differ across experimental cohorts at baseline or during ischemia, but the Longa models exhibited significantly greater perfusion than their ischemic duration-matched Koizumi counterparts at reperfusion and endpoint (Fig. 2 b, S2 b-c). Perfusion was unaffected by ischemic duration within both the Longa and Koizumi cohorts across all timepoints. Although these ROIs were useful to establish a straightforward comparison of perfusion between groups, this approach did not enable assessment of CBF across the entire ischemic hemisphere or more nuanced evaluation of the perfusion dynamics driving overall differences. To compare the composition of the entire ischemic hemisphere in terms of CBF, Matlab code was generated to quantify the proportion of pixels in the ischemic hemisphere corresponding to each color-gradated quintile on the scale of a.u. (blue = 0–60, cyan = 61–120, green = 121–180, yellow = 181–240, red = 241–300 a.u.). Using this approach, a significant decrease in the proportion of red pixels and corresponding increases in yellow/green/cyan pixels were observed at reperfusion and endpoint in the Koizumi cohorts relative to their ischemia-matched Longa counterparts, indicating a sustained shift toward hyporeperfusion in the Koizumi models (Fig. 2 c, S2 d, Table S1 ). However, pixel composition of the ischemic hemisphere did not differ in response to varied ischemic duration within either reperfusion model. Pixel composition was then distilled into a quantitative summary metric, referred to as speckle score, to confirm the relevance of these perfusion dynamics in defining overall CBF across the entire ischemic hemisphere (Fig. 2 d, S2 e). Speckle scores were derived by assigning each color quintile an ordinal value (red = 5, blue = 0) and calculating the mean value across the ischemic hemisphere. Speckle score during ischemia was significantly higher in the 60mK group compared to the 60mL group. However, these CBF dynamics underwent a shift following reperfusion, as evidenced by lower speckle scores, and therefore less perfusion, in the Koizumi cohorts than their Longa counterparts across all ischemic durations. Interestingly, this decrease in perfusion observed in Koizumi models was sustained at endpoint only in the 90m group, suggesting a potential interplay between ischemic severity and longitudinal CBF dynamics. Duration of ischemia differentially alters acute locomotor function in the setting of complete and incomplete reperfusion Motor function is a primary measure of stroke severity and reflects multiple pathological features, including initial ischemia, infarct evolution, and longitudinal blood flow dynamics. We hypothesized that the variable perfusion dynamics observed between the Longa and Koizumi models may drive acute functional outcomes. Corner testing was performed at baseline, 24 hours- (24h), and 72 hours- (72h) post-tMCAO to identify functional deficits according to side preference [ 29 ]. No differences in laterality were observed across groups ( Figure S3 a ). However, corner testing assesses a single binary metric of locomotion that can be biased by starting position or repeated exposure, so laterality may not be sensitive enough to detect nuanced motor differences. To enable more robust evaluation of post-stroke locomotion, open field testing was performed at the same timepoints, and anatomic landmarks were tracked using DeepLabCut machine learning-based pose estimation ( Figure S3 b ). Representative heatmaps were generated for all cohorts at baseline, 24h, and 72h and metrics of locomotion were derived from tracking of the ‘mouse center’ landmark (Fig. 3 a-b, S3 c). Locomotion was decreased at 24h in the Koizumi model with increasing ischemic duration, as evidenced by decreases in distance traveled, average speed, border-center transitions, and percent time moving (Fig. 3 c-f). At 72h, only percent time moving was still affected by ischemic duration in the Koizumi groups. Although this response to ischemic duration was not observed in the Longa model at 24h, the Longa cohorts did exhibit a similar sensitivity at 72h to the severity of ischemia. Specifically, decreases in distance traveled, average speed, and, to a lesser extent, border-center transitions were demonstrated at 72h with increasing ischemic duration. The extent of reperfusion had a limited effect on motor function, with lower percent time moving only in the 30mL cohort relative to the 30mK group at 24h and 72h. Cumulatively, these data demonstrate that ischemic duration differentially affects locomotor function throughout acute stroke evolution according to reperfusion status. Further, this may implicate reperfusion-mediated elements of stroke, including hypoperfusion and reperfusion-induced inflammation, as time-dependent drivers of functional outcomes. Myeloid cell response to ischemia is influenced by ischemic duration and reperfusion status These data point to a role for ischemic duration and reperfusion success in mediating longitudinal stroke evolution. Given neuroinflammation, specifically through the actions of myeloid cells, has been implicated in disease progression, we utilized transgenic reporter mice with markers for MG/MΦ (Csf1r-EGFP) and PMNs (Ly6G-TdTomato) to investigate the role of I/R dynamics in mediating the acute immune response. Although the Ly6G-TdTomato reporter is somewhat promiscuous and labels a subset of neurons in addition to PMNs, these cells exhibited spatial segregation: TdTomato+ neurons were confined to the striatum (and more prominent in the contralateral hemisphere) while PMNs localized to cortical/subcortical regions of the infarct. Therefore, histological analyses were restricted to the ischemic cortex/subcortex to avoid neuronal biasing. Further, TdTomato+ neurons and PMNs were discerned according to size and morphology, as neurons were up to 2-fold larger than PMNs and exhibited branched morphology. These differentiating metrics were validated via NeuN staining, which consistently localized to cells identified as neurons based on location and morphology ( Figure S4 ). Morphological and spatial features of MG/MΦ were evaluated by creating surface reconstructions using Imaris image analysis software to link I/R dynamics with innate immune cell patterning ( Figure S5 ). Representative images were selected by mean cell counts. At 24h, the number of MG/MΦ in the ipsilateral hemisphere did not vary between cohorts (Fig. 4 a-b). MG/MΦ morphology, assessed by cell volume and sphericity, did not differ between experimental groups at 24h (Fig. 4 c-d). However, the spatial association of MG/MΦ and PMNs was affected by ischemic duration, as evidenced by a decrease in MG/MΦ distance to the nearest PMN in the 90mL cohort relative to the 30mL group (Fig. 4 e). At 72h, MG/MΦ count was lower in the 30mK group than the 30mL group (Fig. 4 f-g). The relevance of this finding, however, is unclear as it was not reflected in cohorts of longer ischemic duration. Differences in MG/MΦ volume and sphericity were also observed between the 30m and 60m groups based on reperfusion status (Fig. 4 h-i). Additionally, an increase in MG/MΦ sphericity was observed with increasing ischemic duration, but only in the Longa models. Ischemic duration also mediated MG/MΦ proximity to PMNs at 72h, but only within the Koizumi cohorts, in contrast to the trend observed at 24h (Fig. 4 j). Similar morphological analyses were also performed for PMNs. Additionally, given that PMNs have been reported to further contribute to infarct evolution through microvascular obstructions when retained in the vasculature, the proportion of intravascular PMNs was also quantified. At 24h, PMN count increased between the 30mL and 60mL cohorts (Fig. 5 a-b). However, PMN volume, sphericity, and position relative to the vasculature did not significantly differ across cohorts (Fig. 5 c-e). Given this lack of differential response at 24h, we hypothesized that this hyperacute timepoint may be too early to detect I/R-mediated changes in PMN patterning, as PMN infiltration has been demonstrated to peak around 3 days post-stroke [ 30 ]. When PMN profiles were also evaluated at 72h, a significant increase in the number of PMNs present was observed with increasing ischemic duration regardless of reperfusion status, although this effect was more pronounced in the Koizumi cohorts (Fig. 5 f-g). PMN volume was affected by reperfusion success, as evidenced by a decrease in the 60mL cohort relative to the 60mK group (Fig. 5 h). However, PMN sphericity and the proportion of PMNs retained intravascularly remained unchanged across surgical conditions (Fig. 5 i-j). Cumulatively, these data confirm unique roles for ischemic duration and reperfusion success in driving the spatial and morphological profiles of myeloid cells during the acute stages of I/RI. Cerebral blood flow and locomotor function are correlated with the innate immune response In addition to the reported role of ischemic duration and reperfusion status, a high degree of heterogeneity within each surgical cohort was observed, with subjects exhibiting more severe or mild phenotypes than were expected for their assigned physiologic profile. Given this intra-cohort variability is likely in part mediated by more nuanced differences in I/R dynamics, we questioned whether hemodynamics, myeloid cell response, and locomotion could be intrinsically linked regardless of cohort assignment, with innate immune cell features reflecting acute stroke outcomes. We thus examined the cohort as a whole and applied a correlation analysis between all metrics of CBF, behavior, and myeloid cell profile ( Figure S6 , Table S2 ). CBF dynamics demonstrated a modest association with myeloid cell features, although these correlations were inconsistent across metrics (Fig. 6 a). Specifically, a positive correlation was detected between the proportion of intravascular PMNs at 24h and the mean ROI perfusion at ischemia, but this association was not observed within any other CBF metrics and was not longitudinally sustained. Interestingly, MG/MΦ distance to the nearest PMN was the immune cell feature most robustly correlated with CBF metrics, including reperfusion and endpoint speckle scores, the proportion of red pixels at endpoint, and the proportion of green pixels at endpoint. Together, these data point to a link between CBF in the ischemic hemisphere and the spatial patterning of myeloid cells. Features of the myeloid cell response were also correlated with locomotor function. At 24h, the number of PMNs present was negatively correlated with distance traveled, average speed, and percent time moving (Fig. 6 b). These metrics of locomotion were also positively correlated with PMN volume at 24h. At 72h, the proportion of intravascular PMNs was negatively correlated laterality (Fig. 6 c). Although no other features of the myeloid response were robustly correlated with locomotion at 72h, elements of the PMN response were associated with changes in motor recovery between 24h and 72h. Specifically, both PMN sphericity and distance to the nearest vessel were correlated with recovery of percent time moving and border-center transitions. These data confirm a correlative link between elements of the PMN response in I/RI and acute locomotor function, which may serve to identify targets of future immunomodulatory strategies aimed at improving motor outcomes. DISCUSSION Despite effective recanalization therapy, persistent functional deficits remain a challenge for post-stroke management [ 5 , 31 ]. I/RI-mediated inflammation contributes significantly to infarct evolution beyond the initial ischemic insult, and therefore, drives functional outcomes [ 6 ]. PMNs have been implicated in both microvascular reperfusion failure and I/RI-induced inflammation [ 32 – 35 ]. MG/MΦ have also been linked to stroke progression, as a loss of MG/MΦ in preclinical models worsened infarct severity, inflammation, and motor function [ 36 – 40 ]. However, while preclinical trials targeting elements of the immune response to reduce I/RI burden have yielded promising results, none have translated to improved outcomes in stroke patients [ 10 – 12 , 41 – 43 ]. One factor contributing to the conflicting results in preclinical and clinical trials is the failure of preclinical models to incorporate variability in I/R dynamics and thus, recapitulate the spectrum of clinical stroke physiology. In clinical practice, recanalization via thrombectomy is the standard of care, even in patients with large ischemic core volumes, expanding the spectrum of ischemic severity among those that exhibit I/RI [ 44 ]. Patients demonstrating reperfusion injury also exhibit a range of reperfusion success according to macrovascular recanalization criteria like thrombolysis in cerebral infarction (TICI) scores [ 45 – 48 ]. Previous immunomodulatory strategies failed in clinical trials, suggesting that the target populations were not effectively modulated. This preclinical study was designed to examine how I/R variability affects the myeloid cell response and subsequent functional outcomes to inform future translational approaches that more closely resemble clinical stroke physiology. This study leverages the versatility of the tMCAO model to better represent the clinical I/R spectrum and elucidate the role of these physiologic features as drivers of the acute immune response [ 49 ]. Although previous studies have employed LSCI to demonstrate CBF heterogeneity, these blood flow dynamics have never previously been linked to the innate immune response and broader locomotor function [ 50 ]. Using pathological, functional, and inflammatory analyses, this preclinical study sought to link how nuanced I/R dynamics modulate the local response of PMN and MG/MΦ and subsequently drive pathological and functional outcomes. First, our results indicate that infarct topology, post-stroke locomotion, and myeloid cell features are more robustly mediated by ischemic severity than by reperfusion efficacy. Differences in the immune response may be due to increased damage-associated molecular pattern or cytokine release induced by the initial ischemic event, with reperfusion being a necessary therapy that also promotes open vascular channels, enabling further inflammatory cell recruitment [ 51 – 53 ]. As recanalization is now the standard of care for LVO, these data highlight the need to establish critical ischemic thresholds that trigger the myeloid response to inform future translational work. The findings presented may guide preclinical inclusion criteria for future immunomodulatory treatment strategies using imaging-based assessment of ischemic injury severity in tandem with reperfusion success. Second, this study found that ischemia and reperfusion differentially alter the profile of MG/MΦ and PMNs at 72h post-tMCAO. PMN density was more robustly affected by ischemia than PMN morphology or position but was not influenced by reperfusion status. This suggests that the magnitude of the PMN response is most susceptible to ischemic severity, while the spatial features of infiltrated PMNs are relatively insensitive to the ischemic event at these acute time points. Although PMN volume did vary at 72h according to reperfusion efficacy in the 60m cohorts, the inconsistency with cohorts of other ischemic durations and the absence of other changes in cell morphology call into question the significance of this relationship. MG/MΦ density and morphology at 72h were also sensitive to the effects of I/R variation, but in contrast to PMNs, these differences were primarily in response to varied reperfusion success. The unique sensitivities of PMNs and MG/MΦ to I/R dynamics that were observed align with the differential roles that these myeloid cell populations may play in I/RI. As PMNs are peripheral immune cells recruited to the site of ischemia to exert transient pro-inflammatory effects, PMN infiltration is triggered by the initial ischemic event, with lesser effects observed in response to infarct evolution after the initial wave of cell activation [ 30 ]. In contrast, MG/MΦ respond hyperacutely to the inciting injury and persist in the ischemic tissue to contribute longitudinally to inflammation and injury resolution. Therefore, the evolving response of MG/MΦ to hyporeperfusion, rather than their initial actions due to the ischemic insult, is captured at the acute timepoints studied [ 54 – 57 ]. These differential effects confirm that myeloid cells uniquely respond to ischemic burden and infarct evolution, potentially warranting distinct immunomodulation of PMNs and MG/MΦ according to patient-specific I/R dynamics. In addition to these individual responses, the position of MG/MΦ relative to PMNs was also influenced by ischemic duration. MG/MΦ and PMNs participate in cell-cell interactions in neuroinflammation, so proximity was measured as a surrogate for cellular association, with shorter distances suggestive of greater potential for interaction. The spatial association of MG/MΦ and PMNs in response to increasing ischemic duration is consistent with the ischemia-mediated increase in PMN density, implicating PMNs as the likely drivers of this relationship. Notably, our immunofluorescent imaging demonstrated that a subset of MG/MΦ completely overlapped with PMNs, pointing to contact-mediated interactions between these populations. Accordingly, it may be worthwhile to further investigate the changing relationship between MG/MΦ and PMNs in response to I/R to define how these interactions shape the acute tissue environment post-stroke and orchestrate subsequent neuroinflammatory patterns. Lastly, these analyses identified a correlative link between PMN features and locomotor function. These metrics correlated independently of assigned surgical cohort, establishing a critical association between stroke pathology and locomotor function across I/R profiles and highlighting the need to build on behavioral testing to evaluate outcomes in the development of I/RI therapies [ 58 ]. Importantly, the features of PMNs that were correlated to behavioral outcomes differed by timepoint. PMN density and cell volume strongly influenced locomotor outcomes at 24h, while metrics of locomotor recovery from 24h to 72h were more sensitive to PMN sphericity and proximity to the vasculature. This dynamic link between inflammation and locomotor function suggests that differential immune cell metrics may correlate to longitudinal behavior changes post-stroke, pointing to facets of the myeloid cell response that could potentially be targeted to alleviate specific motor deficits across time. These findings highlight how the acute myeloid response is mediated by I/R dynamics and emphasize the relevance of defining the longitudinal immunological response as it relates to physiology and functional outcomes. Ultimately, although reperfusion is necessary to limit clinical morbidity, this work reinforces that ischemic duration is a more robust driver of infarct topology, behavioral metrics, and PMN infiltration than reperfusion efficacy. In contrast, MG/MΦ were uniquely sensitive to the extent of reperfusion, confirming the differential responses of myeloid cell populations and emphasizing the importance of considering both ischemia and reperfusion dynamics in future translational studies. This study has several strengths, including the application of multiple I/R models, non-invasive live-animal imaging, and transgenic fluorescent reporters to explore the relationship between I/R dynamics and the post-stroke myeloid response. However, there are limitations as well. Analysis of myeloid cells did not include evaluation of cell function. Future studies will aim to link immune cell function with position and morphology, as well as extend these analyses to subacute and chronic timepoints, such as days 7- and 28-post stroke, to evaluate the longitudinal effects of I/R on neuroinflammation and stroke pathology. Lastly, this study utilized the Csf1r-EGFP reporter to label both the MG and MΦ populations in parallel with the Ly6G-TdTomato reporter as a means to investigate the spatial relationships between myeloid cell populations. Additional studies should be performed using the Cx3cr1-Cre-YFP-TdTomato lineage-tracing strain to differentiate the contributions of MG and MΦ in the absence of PMN labeling. Consideration of the consequences of I/R dynamics on neuroinflammation and behavior in future preclinical studies may lead to more translational success in developing new adjunctive therapies in the preclinical-clinical pipeline for ischemic stroke. Abbreviations 30mK: 30-minute Koizumi model 60mK: 60-minute Koizumi model 90mK: 90-minute Koizumi model 30mL: 30-minute Longa model 60mL: 60-minute Longa model 90mL: 90-minute Longa model CBF: Cerebral blood flow CCA: Common carotid artery 24h: 24 hours post-reperfusion 72h: 72 hours post-reperfusion ECA: External carotid artery ICA: Internal carotid artery I/R: Ischemia and reperfusion I/RI: Ishcemia/reperfusion injury LSCI: Laser speckle contrast imaging LVO: Large vessel occlusion MG: Microglia M Φ : Bone marrow-derived macrophages PMN: Polymorphonuclear neutrophils TICI: Thrombolysis in cerebral infarction tMCAO: Transient middle cerebral artery occlusion TTC: Triphenyl tetrazolium chloride Declarations Competing Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Author Contribution LES and NAN conceptualized this project and designed the included experiments. LES, MT, and EAZ performed experiments. LES, MMM, CP, and JD analyzed data and generated figures. LES, NAN, and SET drafted and edited the manuscript. SET provided technical expertise and assisted with experiment design. NAN provided funding for this project. All authors read and approved the final manuscript. Acknowledgement The authors would like to thank Beth Vernaleo, PhD for manuscript proofreading and members of the Tsirka lab for helpful discussions and suggestions. Data Availability The data that support the findings of this study will be openly available in Figshare at the time of publication (reference number to be provided when available). References GBD 2019 Stroke Collaborators. Global, regional, and national burden of stroke and its risk factors, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019. Lancet Neurol. 2021;20(10):795–820. Rha J-H, Saver JL. The Impact of Recanalization on Ischemic Stroke Outcome. Stroke. 2007;38(3):967–73. 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Weisenburger-Lile D, Dong Y, Yger M, Weisenburger G, Polara GF, Chaigneau T, et al. Harmful neutrophil subsets in patients with ischemic stroke. Neurol Neuroimmunol Neuroinflammation. 2019;6(4):e571. Lyden PD, Bosetti F, Diniz MA, Rogatko A, Koenig JI, Lamb J, et al. The Stroke Preclinical Assessment Network: Rationale, Design, Feasibility, and Stage 1 Results. Stroke. 2022;53(5):1802–12. Additional Declarations No competing interests reported. Supplementary Files FigS1.jpg FigS2.jpg FigS3.jpg FigS4.jpg FigS5.jpg FigS6.jpg TableS1.xlsx TableS2.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8712806","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":586674050,"identity":"561d5b09-4a5f-4490-985c-b227d6fbe7bb","order_by":0,"name":"Laurel E Schappell","email":"","orcid":"","institution":"Stony Brook University","correspondingAuthor":false,"prefix":"","firstName":"Laurel","middleName":"E","lastName":"Schappell","suffix":""},{"id":586674051,"identity":"735c4d51-1e51-4928-a1be-6db7da2d0c0a","order_by":1,"name":"Miguel M Madeira","email":"","orcid":"","institution":"Stony Brook University","correspondingAuthor":false,"prefix":"","firstName":"Miguel","middleName":"M","lastName":"Madeira","suffix":""},{"id":586674056,"identity":"86d49215-bf1a-4c9b-a73c-ffc9d4ab3182","order_by":2,"name":"Claire Polizu","email":"","orcid":"","institution":"Stony Brook University","correspondingAuthor":false,"prefix":"","firstName":"Claire","middleName":"","lastName":"Polizu","suffix":""},{"id":586674057,"identity":"78047dfb-03b4-4e2c-8e17-a5b0a794810e","order_by":3,"name":"James DiPersio","email":"","orcid":"","institution":"Stony Brook University","correspondingAuthor":false,"prefix":"","firstName":"James","middleName":"","lastName":"DiPersio","suffix":""},{"id":586674059,"identity":"eaf337a4-56dc-43a6-8e38-02d10e1a2fcf","order_by":4,"name":"Eleftherios A Zoumpanidopoulos","email":"","orcid":"","institution":"Stony Brook University","correspondingAuthor":false,"prefix":"","firstName":"Eleftherios","middleName":"A","lastName":"Zoumpanidopoulos","suffix":""},{"id":586674060,"identity":"805b260d-0167-4431-a950-3290b1a0436c","order_by":5,"name":"Meiyi Tang","email":"","orcid":"","institution":"Stony Brook University","correspondingAuthor":false,"prefix":"","firstName":"Meiyi","middleName":"","lastName":"Tang","suffix":""},{"id":586674064,"identity":"8ea8490f-360c-4a9a-b097-bd4903c7b2c1","order_by":6,"name":"Stella E Tsirka","email":"","orcid":"","institution":"Stony Brook University","correspondingAuthor":false,"prefix":"","firstName":"Stella","middleName":"E","lastName":"Tsirka","suffix":""},{"id":586674066,"identity":"37acf0eb-2416-4110-9b21-1333df652be3","order_by":7,"name":"Neil A Nadkarni","email":"data:image/png;base64,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","orcid":"","institution":"Stony Brook University","correspondingAuthor":true,"prefix":"","firstName":"Neil","middleName":"A","lastName":"Nadkarni","suffix":""}],"badges":[],"createdAt":"2026-01-27 16:39:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8712806/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8712806/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102209330,"identity":"e3116483-deda-40cd-b837-f16e3f54b110","added_by":"auto","created_at":"2026-02-09 12:12:55","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":493720,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncreased ischemic duration is associated with more severe infarct pathology and intra-ischemic behavior \u003c/strong\u003eThe degree of reperfusion achieved was varied based on the location of filament insertion: insertion via the external carotid artery induced complete reperfusion (Longa) \u003cstrong\u003e(a)\u003c/strong\u003e whereas insertion via the common carotid artery induced incomplete reperfusion (Koizumi) \u003cstrong\u003e(b)\u003c/strong\u003e. Surgical diagrams were created using BioRender, where the red area of the brain represents decreased perfusion, and the gray area denotes the region affected by initial ischemic insult. Ischemic duration was varied in each model (30m, 60m, or 90m) and infarct volume for each condition was quantified via triphenyl tetrazolium chloride (TTC) staining of seven serial tissue sections \u003cstrong\u003e(c-e)\u003c/strong\u003e. Hemorrhagic transformation was also assessed according to the presence of gross blood on TTC \u003cstrong\u003e(f)\u003c/strong\u003e, and hemorrhage volume was quantified using the same serial sections \u003cstrong\u003e(g)\u003c/strong\u003e. Intra-ischemic behavior was evaluated using an ordinal 5-point scale, ranging in severity from 0 (no deficit) to 4 (no spontaneous locomotor activity or barrel rolling) \u003cstrong\u003e(h)\u003c/strong\u003e. Mortality was also tracked over the entire study duration, up to 72 hours post-reperfusion \u003cstrong\u003e(i)\u003c/strong\u003e. N = 7 per group for infarct topology, n = 37-50 per group for intraischemic behavior and survival analysis. Data expressed as % population (f, h-i) or mean (e,g). *P \u0026lt; 0.05, **P \u0026lt; 0.01. P values were calculated using two-way ANOVA tests for ischemic duration and degree of reperfusion with Tukey’s multiple comparisons when indicated.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8712806/v1/b0f1ba0ec8012fdf72a56f0c.jpg"},{"id":102209552,"identity":"9e2805f9-ae55-4621-b385-a332148ad2ec","added_by":"auto","created_at":"2026-02-09 12:13:36","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":773671,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCerebral blood flow in the ischemic hemisphere is longitudinally affected by reperfusion efficacy\u003c/strong\u003e Cerebral blood flow patterning was mapped across the entire brain in all models at baseline, immediately before (ischemia) and after (reperfusion) filament withdrawal, and at endpoint using laser speckle contrast imaging \u003cstrong\u003e(a)\u003c/strong\u003e. Cerebral blood flow was measured according to arbitrary perfusion units (a.u.) and displayed as a color scale (red = 300 a.u., blue = 0 a.u.). Regions of interest were overlaid onto each hemisphere to quantify perfusion at each timepoint \u003cstrong\u003e(b)\u003c/strong\u003e. ROI perfusion in the ipsilateral hemisphere is reported as fold change relative to the time-matched contralateral hemisphere measurement. Perfusion dynamics were also tracked across the entire ipsilateral hemisphere according to the proportion of total pixels corresponding to each color-coded quintile of the perfusion scale \u003cstrong\u003e(c)\u003c/strong\u003e. Statistical significance of each color-coded quintile is denoted in the corresponding table for each timepoint (# = statistically significant); statistical values for each pairwise comparison are listed in the supplement. The gradient of perfusion across the ischemic hemisphere was simplified to an overall speckle score according to the average color classification of all pixels at each timepoint (red = 5, blue = 0) \u003cstrong\u003e(d)\u003c/strong\u003e. N = 15 per group. Data expressed as fold change relative to the contralateral hemisphere (b), % pixels (c), or mean (d). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001. P values were calculated using two-way ANOVA tests for ischemic duration and reperfusion status with Tukey’s multiple comparisons when indicated.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8712806/v1/cde2ba1453efcd8640deed89.jpg"},{"id":102209538,"identity":"5410daef-d8da-4535-a201-e00319b1104b","added_by":"auto","created_at":"2026-02-09 12:13:36","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":527924,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIschemic duration differentially mediates acute locomotor outcomes according to reperfusion status \u003c/strong\u003eRepresentative heatmaps of open field activity were generated for each cohort at 24h \u003cstrong\u003e(a)\u003c/strong\u003e and 72h \u003cstrong\u003e(b)\u003c/strong\u003eto enable visualization of changes in overall locomotion. Corresponding representative baseline heatmaps are included in the supplement. Tracking data from the mouse center landmark were used to quantify distance traveled \u003cstrong\u003e(c)\u003c/strong\u003e, average speed \u003cstrong\u003e(d)\u003c/strong\u003e, border-center transitions \u003cstrong\u003e(e)\u003c/strong\u003e, and percent time moving \u003cstrong\u003e(f)\u003c/strong\u003e at 24h and 72h. All open field metrics are reported as mean fold change relative to the subject’s baseline. N = 12-15 per group per timepoint. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001. P values were calculated using two-way ANOVA tests for ischemic duration and degree of reperfusion at each time point with Tukey’s multiple comparisons as appropriate.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8712806/v1/16ef44a249dd1f1834981820.jpg"},{"id":102209619,"identity":"b5006ae1-bf75-477d-a9cc-ed3bdd6a915d","added_by":"auto","created_at":"2026-02-09 12:13:42","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":736488,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroglia/macrophage cell recruitment and position are influenced by physiologic variability\u003c/strong\u003eRepresentative images of MG/ΜΦ(green) and vasculature (magenta) in the cortical region of the infarct at 24h post-tMCAO \u003cstrong\u003e(a)\u003c/strong\u003e. MG/MΦ count per FOV \u003cstrong\u003e(b)\u003c/strong\u003e, volume \u003cstrong\u003e(c)\u003c/strong\u003e, sphericity \u003cstrong\u003e(d)\u003c/strong\u003e, and position relative to the nearest PMN \u003cstrong\u003e(e)\u003c/strong\u003e were quantified at 24h using Imaris surface reconstruction. Representative images of MG/MΦ and vasculature were also captured for each experimental cohort at 72h \u003cstrong\u003e(f)\u003c/strong\u003e. MG/MΦ count \u003cstrong\u003e(g)\u003c/strong\u003e, volume \u003cstrong\u003e(h)\u003c/strong\u003e, sphericity \u003cstrong\u003e(i)\u003c/strong\u003e, and distance to nearest PMN \u003cstrong\u003e(j)\u003c/strong\u003ewere measured using the same Imaris-based approach as was employed at 24h. N = 5 per condition per time point. Scale = 100 μm. Each data point represents an average of 2-3 technical replicates for a surgical subject. *P \u0026lt; 0.05, **P \u0026lt; 0.01. P values were determined using two-way ANOVA tests for ischemic duration and degree of reperfusion at each timepoint with Tukey’s multiple comparisons as needed.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8712806/v1/06c9d1ddbe9f77dd98581137.jpg"},{"id":102209425,"identity":"b1c6f2db-fd5d-486a-8275-c525e0da1d80","added_by":"auto","created_at":"2026-02-09 12:13:15","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":608947,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeutrophil infiltration and morphology are defined in part by stroke physiology\u003c/strong\u003eRepresentative images of PMNs (red) and vasculature (magenta) in the ischemic cortex at 24h \u003cstrong\u003e(a)\u003c/strong\u003e. At 24h, the number of PMNs \u003cstrong\u003e(b)\u003c/strong\u003e, cell volume \u003cstrong\u003e(c)\u003c/strong\u003e, sphericity \u003cstrong\u003e(d)\u003c/strong\u003e, and % intravascular PMNs \u003cstrong\u003e(e)\u003c/strong\u003e, were quantified via Imaris surface reconstruction. Representative images of PMNs and vasculature in the ischemic region of the cortex at 72h \u003cstrong\u003e(f)\u003c/strong\u003e. PMN count per FOV \u003cstrong\u003e(g)\u003c/strong\u003e, volume \u003cstrong\u003e(h)\u003c/strong\u003e, sphericity \u003cstrong\u003e(i)\u003c/strong\u003e, and % intravascular PMNs \u003cstrong\u003e(j)\u003c/strong\u003ewere also measured at 72h. N = 3-5 per condition at each time point based on the presence of PMNs in samples used for MG/MΦ analysis. Scale = 100 μm. Each data point represents an average of 2-3 technical replicates for a surgical subject. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001. P values were determined using two-way ANOVA tests for ischemic duration and degree of reperfusion per timepoint with Tukey’s multiple comparisons if indicated.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8712806/v1/e30033181671415c1d60178a.jpg"},{"id":102209439,"identity":"13d7dc08-051f-4b0f-9c29-25fd6c64eb33","added_by":"auto","created_at":"2026-02-09 12:13:19","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":392811,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe profiles of microglia/macrophages and neutrophils are correlated with cerebral blood flow and locomotor function\u003c/strong\u003e Correlation matrix of cerebral blood flow metrics (ROI perfusion, proportion of pixels represented by each color classification, and overall perfusion score at baseline, ischemia, reperfusion, and endpoint) and measures of acute inflammatory response (MG/MΦ and PMN cell counts, morphology, and spatial relationships at 24h and 72h) \u003cstrong\u003e(a)\u003c/strong\u003e. Correlation matrix of 24h locomotor outcomes (raw values and delta laterality, raw values and fold change of distance traveled, average speed, percent time moving, border transitions) and metrics of MG/MΦ and PMN response (MG/MΦ and PMN cell counts, morphology, and spatial relationships at 24h and 72h) \u003cstrong\u003e(b)\u003c/strong\u003e. Correlation matrix of 72h locomotor outcomes/locomotor recovery between 24h and 72h (raw values and fold change for all metrics except laterality which is reported as a difference between timepoints) and the immune response at 72h \u003cstrong\u003e(c)\u003c/strong\u003e. Color assigned according to correlation coefficient values. Correlation coefficients and P values for all metrics are in Table S2.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8712806/v1/dd165d3fc54870372b828930.jpg"},{"id":103049802,"identity":"17f45729-88e7-4b17-8e43-b0eed737755e","added_by":"auto","created_at":"2026-02-20 07:46:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4800623,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8712806/v1/2a568771-776f-4bf9-8a9d-57ebca4aad54.pdf"},{"id":102209332,"identity":"14367254-94ca-4ef7-b05d-de8a375ace62","added_by":"auto","created_at":"2026-02-09 12:12:55","extension":"jpg","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":918156,"visible":true,"origin":"","legend":"","description":"","filename":"FigS1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8712806/v1/488358cbfd5dd1748043d5b7.jpg"},{"id":102209333,"identity":"0d10d41a-5f27-40f8-a749-c480061fd1c5","added_by":"auto","created_at":"2026-02-09 12:12:55","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":564867,"visible":true,"origin":"","legend":"","description":"","filename":"FigS2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8712806/v1/b2ce31137f5103b56154b3e8.jpg"},{"id":102209313,"identity":"245f6256-8b6e-47bf-8aa4-def8f022e857","added_by":"auto","created_at":"2026-02-09 12:12:48","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":935186,"visible":true,"origin":"","legend":"","description":"","filename":"FigS3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8712806/v1/e7e68ce6c83cbdf4e109b43c.jpg"},{"id":102209617,"identity":"bea5b525-0254-4aba-bee8-d6f4846fceb3","added_by":"auto","created_at":"2026-02-09 12:13:40","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2292768,"visible":true,"origin":"","legend":"","description":"","filename":"FigS4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8712806/v1/d8a05f0aaf9c16b1f0305ce9.jpg"},{"id":102209442,"identity":"de65cdb3-0927-4e41-9952-d98afb0dc93a","added_by":"auto","created_at":"2026-02-09 12:13:20","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":703167,"visible":true,"origin":"","legend":"","description":"","filename":"FigS5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8712806/v1/6f94cd8cdbdb49de772570cc.jpg"},{"id":102209613,"identity":"ec14a4d1-74f8-499c-8ab2-fd216bb726b8","added_by":"auto","created_at":"2026-02-09 12:13:37","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":2006694,"visible":true,"origin":"","legend":"","description":"","filename":"FigS6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8712806/v1/7c6e5ea21a35df39a33361f3.jpg"},{"id":102209618,"identity":"eae307d4-7a0a-43d0-9820-b38a2b82110f","added_by":"auto","created_at":"2026-02-09 12:13:41","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":68333,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8712806/v1/e4babcd436b7986cd4c55db0.xlsx"},{"id":102209391,"identity":"4e6a8f4f-0695-404c-8668-f63403189593","added_by":"auto","created_at":"2026-02-09 12:13:05","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":135808,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8712806/v1/55c9186b90cdb12d28fc6818.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ischemic Duration is More Critical than Reperfusion Efficacy in Driving Early Neuroinflammation and Motor Deficits after Transient Middle Cerebral Artery Occlusion","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eIschemic stroke is a leading cause of morbidity and mortality worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Eligibility criteria for recanalization of large vessel occlusion (LVO) stroke are continuously expanding to promote cerebral reperfusion and improve outcomes, yet \u0026gt;\u0026thinsp;50% of patients still experience significant disability despite treatment [\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Ischemia/reperfusion injury (I/RI), the inflammatory response induced by ischemia and continued throughout reperfusion (I/R), significantly contributes to infarct evolution and functional outcomes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Stroke immunomodulatory therapies often focus on myeloid cells - polymorphonuclear neutrophils (PMN), bone-marrow derived macrophages (MΦ), and microglia (MG) - as they constitute the predominant first responders in I/RI [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, preclinical trials aimed at mitigating I/RI by targeting myeloid cell recruitment have yet to translate into successful clinical trials [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The post-stroke inflammatory response is highly conserved between mice and humans, suggesting that this preclinical-clinical disconnect can likely be attributed to differences in the features of stroke evolution that govern the myeloid response [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn clinical practice, perfusion dynamics are widely variable and known to mediate disease progression, with longer ischemic duration and less complete reperfusion independently contributing to worse outcomes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, variability in these physiologic metrics of I/R remains unaccounted for in preclinical stroke models. The preclinical transient middle cerebral artery occlusion (tMCAO) model - widely adopted to simulate LVO I/RI - is often performed in homogeneous surgical cohorts with standardized ischemic duration and reperfusion status across animals. As a result, the role of I/R dynamics in mediating the myeloid cell response in I/RI, and ultimately, pathological and behavioral outcomes, has yet to be elucidated. Defining this relationship between the I/R profile and downstream inflammatory consequences will allow preclinical models to better align with the cellular processes and disease outcomes observed in the stroke patient population.\u003c/p\u003e \u003cp\u003eWe sought to address this gap by investigating the role of ischemic duration and reperfusion status in driving stroke evolution and I/RI. Using serial perfusion imaging and behavioral testing with variations of the tMCAO model in myeloid reporter mice, this study found that acute infarct topology, behavior, and myeloid cell features are more strongly modulated by ischemic duration than by reperfusion efficacy. Specifically, MG/MΦ and PMN morphology, and spatial relationships between PMNs and MG/MΦ were most profoundly affected. Based on the shared response to varied I/R dynamics, correlation analyses performed across all surgical cohorts identified an intrinsic link between locomotor function and the acute immune response. These data highlight a critical need to consider how the stroke I/R profile defines tissue pathology and establish a link between the myeloid response and functional outcomes to inform future therapeutic strategies.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMice\u003c/h2\u003e \u003cp\u003e All procedures were approved by the Institutional Animal Care and Use Committee (Animal Welfare Assurance No A3011-0) at SUNY Stony Brook School of Medicine. Mice were housed in the institutional animal facility under a 12-hour light/dark cycle, and all husbandry was performed by the Stony Brook Division of Laboratory Animal Resources. Food and water were provided \u003cem\u003ead libitum\u003c/em\u003e. The Ly6G-TdT x Csf1r-EGFP strain was derived by crossing Catchup (C57BL/6 Ly6G(Cre-TdTomato)) mice with MacGreen (C57BL/6-CSF1R-EGFP) mice to yield heterozygotes for each fluorophore [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Genotypes were confirmed via polymerase chain reaction. Male and female mice were used in equal proportion. Ly6G-TdT x Csf1r-EGFP heterozygotes aged 3 to 6 months were used for all experiments. Mice weighing \u0026lt;\u0026thinsp;20g or \u0026gt;\u0026thinsp;35 g were excluded from analyses. In total, 295 mice were used.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTransient Middle Cerebral Artery Occlusion (tMCAO) Model\u003c/h3\u003e\n\u003cp\u003eSurgical procedures were performed following the IMPROVE guidelines [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Mice were randomized to either the Longa (complete reperfusion) or Koizumi (incomplete reperfusion) tMCAO method, as previously described, and were further randomized to 30, 60, or 90 minutes of ischemia [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Mice received preoperative analgesia (0.2 mg/kg s.c. bupivacaine) and fluid support (1 mL i.p. saline). The surgical site was prepared by removing all hair and sterilizing with Betadine. Lubricant was applied to both eyes to avoid corneal drying. Mice were anesthetized (isoflurane, 3.5% induction, 0.5-1.0% maintenance) under aseptic conditions with temperature maintenance (37.0\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C) via rectal probe. Once anesthesia depth was confirmed by lack of response to foot pinch, a midline ventral neck incision was made on the right side. The common carotid artery (CCA) and internal carotid artery (ICA) were isolated and temporarily ligated using 7\u0026thinsp;\u0026minus;\u0026thinsp;0 sutures. The distal external carotid artery (ECA) was permanently ligated. For Koizumi procedures, a weight-matched silicon coated filament (Doccol 602145 for \u0026lt;\u0026thinsp;23 g, 602245 for 23\u0026ndash;28 g, 602345 for \u0026gt;\u0026thinsp;28g; Doccol, Sharon, MA) was inserted through an incision in the CCA superior to the point of ligation. For Longa procedures, the incision was instead made in the ECA proximal to the point of ligation for insertion of the weight-matched filament (Doccol 602123 for \u0026lt;\u0026thinsp;23 g, 602223 for 23\u0026ndash;28 g, 602323 for \u0026gt;\u0026thinsp;28 g). In both models, the ICA was unligated and the filament was advanced\u0026thinsp;~\u0026thinsp;9 mm into the ICA to occlude the origin of the middle cerebral artery. The filament was secured with a temporary suture distal to the insertion point. The neck incision was sutured, and animals were removed from anesthesia and allowed to recover in a heated chamber. Intra-ischemic behavior was assessed just prior to re-anaesthetizing using an ordinal five-point scale as previously described [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Briefly, mice were allowed to move freely and then suspended by the tail to assess the degree of unilateral deficit exhibited, where 0\u0026thinsp;=\u0026thinsp;no deficit, 1\u0026thinsp;=\u0026thinsp;forelimb weakness and torso turning to the affected side when suspended by tail, 2\u0026thinsp;=\u0026thinsp;spontaneous circling to the affected side, 3\u0026thinsp;=\u0026thinsp;unable to bear weight on the affected side, and 4\u0026thinsp;=\u0026thinsp;no spontaneous locomotor activity or barrel rolling. Mice with no deficits were excluded. Just prior to the end of the ischemic window, mice were re-anaesthetized and cerebral blood flow (CBF) was assessed by laser speckle contrast imaging (LSCI) before the neck incision neck was reopened. The filament was withdrawn to enable recanalization and either the CCA (Koizumi) or ECA (Longa) was permanently ligated. The CCA was opened in the Longa surgical cohorts following filament removal. Mice recovered in a heated chamber before being returned to their home cage. Mice were excluded if subarachnoid hemorrhage was detected or if inadequate occlusion was observed on LSCI.\u003c/p\u003e\n\u003ch3\u003eLaser Speckle Contrast Imaging (LSCI)\u003c/h3\u003e\n\u003cp\u003eTo enable serial imaging, mice were anesthetized and the skin (~\u0026thinsp;1 cm diameter) overlying the skull cap was removed. A layer of optically clear glue (Norland Optical Adhesive 81, Edmund Optics) was coated over the skull and cured with UV (5% UV, 10\u0026ndash;15 seconds exposure, 5 cm height from skull) (CS 2020, ThorLabs). Skull covers were placed at least 24 hours prior to tMCAO procedure to minimize any possibility of procedure-induced vascular injury or inflammation at the time of surgery. LSCI (PeriCam PSI HR; Perimed) was conducted prior to and after UV glue coating to confirm the glue did not alter CBF patterning. LSCI was captured at skull cover placement (baseline), immediately prior to filament withdrawal (ischemia), immediately after filament withdrawal (reperfusion), and immediately prior to tissue collection (endpoint). Ischemia and reperfusion were confirmed by LSCI, acquired immediately before and after filament withdrawal, as previously described [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Imaging was performed for \u0026gt;\u0026thinsp;15 seconds and one representative frame was used for pixel quantification. CBF was represented by a color-gradated scale of arbitrary perfusion units (a.u.) according to the random motion detected at each pixel, with high random motion corresponding to healthy perfusion (red) and low random motion corresponding to hypoperfusion (blue). Images were generated as color-coded heatmaps of a.u. on a scale of 0 to 300. Matlab (24.2.0.2712019) was used to isolate the ischemic hemisphere from background signal according to the regional difference in perfusion units. Within the ischemic hemisphere, perfusion units were classified according to color (blue\u0026thinsp;=\u0026thinsp;0\u0026ndash;60, cyan\u0026thinsp;=\u0026thinsp;61\u0026ndash;120, green\u0026thinsp;=\u0026thinsp;121\u0026ndash;180, yellow\u0026thinsp;=\u0026thinsp;181\u0026ndash;240, red\u0026thinsp;=\u0026thinsp;241\u0026ndash;300 a.u.) and the number of pixels corresponding to each category was calculated. Speckle score was calculated at baseline, ischemia, reperfusion, and endpoint using a weighted scale from 1 to 5 according to the average pixel color. Specifically, each color classification was assigned an ordinal value (red\u0026thinsp;=\u0026thinsp;5, yellow\u0026thinsp;=\u0026thinsp;4, green\u0026thinsp;=\u0026thinsp;3, cyan\u0026thinsp;=\u0026thinsp;2, blue\u0026thinsp;=\u0026thinsp;1). The average value of all pixels within the ischemic hemisphere was then calculated to derive the speckle score. For instance, 5 corresponded to 100% red pixels and 1 corresponded to 100% blue pixels.\u003c/p\u003e\n\u003ch3\u003eBehavioral Testing\u003c/h3\u003e\n\u003cp\u003eBehavioral testing was performed at baseline and at 24 hours (24h) and 72 hours (72h) post-reperfusion. For corner testing, mice were placed in a 30\u0026deg; opaque corner with their whiskers contacting the walls and allowed to escape by rearing to either side [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Ten trials were conducted, and laterality index was calculated as the absolute difference between left and right turns divided by total turns. For open field testing, mice were allowed to explore an open field (42 cm x 42 cm x 42 cm) for 10 minutes [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Behavior was recorded and processed using Handbrake (1.7.3). Anatomic landmarks were tracked via DeepLabCut-based pose estimation (0.2.1.7) using an optimized configuration of the open-source Super Animal Top View Mouse network [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. RStudio (4.4.1) was used to calculate locomotor metrics and generate heatmaps.\u003c/p\u003e\n\u003ch3\u003eTissue Collection\u003c/h3\u003e\n\u003cp\u003eBrains were collected at 24h or 72h post-tMCAO. Mice allocated to immunofluorescence imaging received a retro-orbital injection of 10% Tomato Lectin, Dylight 649 (Vector Laboratories) 15 minutes prior to collection to label reperfused vasculature. Mice were deeply anesthetized (isoflurane, 5%) and transcardially perfused with cold phosphate buffered saline (PBS) followed by cold 4% paraformaldehyde (PFA) if allocated to immunofluorescent imaging or PBS alone for infarct size quantification. Endogenous TdTomato signal was imaged to confirm retention of adherent PMNs in the vasculature following perfusion, as has been previously observed (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Brains were dissected, meninges removed, and brains were sectioned into 1 mm coronal slices using an adult mouse brain slicing block (5325; Zivic Instruments).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eInfarct Volume Quantification\u003c/h2\u003e \u003cp\u003eFor infarct size quantification, brain slices were stained with triphenyl tetrazolium chloride (TTC) for 15 minutes to distinguish infarcted (white) from healthy tissue (red) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Seven consecutive coronal slices were imaged using a near-infrared fluorescent imager (Azure 500; Azure Biosystems). Infarct and hemorrhage volumes were quantified by manual tracing in Fiji ImageJ, with edema correction applied using the ipsilateral-to-contralateral hemisphere ratio.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunostaining and Confocal Imaging\u003c/h3\u003e\n\u003cp\u003eBrain slices for immunofluorescence were post-fixed in 4% PFA for 24 hours and cryoprotected in 15% and 30% sucrose. Slices were washed in 0.3% Triton-X in PBS and blocked in 10% goat serum for 2 hours at room temperature, and stained overnight at 4\u0026deg;C with NeuN primary antibody (1:1000, ABN90P; Sigma-Aldrich), followed by incubation with goat anti-guinea pig 405 secondary antibody (1:1000, SAB4600230; Sigma-Aldrich) for 2 hours at room temperature. Samples were stored in 0.02% sodium azide in PBS prior to imaging. Fluorescent imaging was performed using an Airyscan two-photon laser-scanning confocal microscope (LSM 980; Zeiss). Coronal slices at bregma \u0026minus;\u0026thinsp;1mm, corresponding to the region of maximal ischemia, were selected for analysis. Widefield scans of the ischemic hemisphere were acquired at 10x magnification (z-step: 10 \u0026micro;m, z-stack: 200\u0026ndash;250 \u0026micro;m) and high-resolution z-stacks for cell counts and morphological analyses were acquired at 20x magnification (z-step: 0.48 \u0026micro;m, z-stack: 100\u0026ndash;120 \u0026micro;m) in at least three cortical fields of view (FOV). Image stitching and reconstruction were performed in Fiji ImageJ.\u003c/p\u003e\n\u003ch3\u003eImaging Analysis\u003c/h3\u003e\n\u003cp\u003eCell analyses of MG/MΦ and PMNs captured in 20x FOVs were conducted using Imaris Single Full with Clear View (Imaris Bitplane v.10.2, Oxford Instruments) image analysis software. Images were pre-processed using the Gaussian Filter function to enhance cell signal relative to background. Cell and vascular reconstructions were created using the Machine Learning Segmentation Surface Creation tool with each source channel. Surfaces were filtered according to area and intensity to exclude non-cellular signal. Cell volume, sphericity, and distance to other surfaces were calculated automatically for each cell within the surface.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCorrelation Analysis\u003c/h2\u003e \u003cp\u003eCorrelation coefficients for CBF, behavior, and immune response variables were calculated and corresponding heatmaps were generated using R Studio (4.4.1, R Foundation for Statistical Computing, Vienna, Austria) and the pheatmap package.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eAll statistical analyses were conducted using GraphPad Prism (10.4.2). Input data for Prism was collated in Excel. For all experiments except survival analysis, statistical significance was assessed using two-way analysis of variance (ANOVA) for ischemic duration and reperfusion status with Tukey\u0026rsquo;s multiple comparison post hoc tests as indicated between cohorts with common ischemic time or reperfusion success. Survival analysis was conducted using the log-rank Mantel-Cox test. All data is reported as the mean; *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001. Results of all statistical analyses are reported in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eVarying stroke physiology alters infarct topology and intra-ischemic behavior\u003c/h2\u003e \u003cp\u003eIschemic duration and reperfusion efficacy are highly variable among stroke patients. However, preclinical studies fail to account for the role of these physiologic features in mediating stroke pathology and outcomes. To mimic variable reperfusion, the Longa and Koizumi methods of the transient middle cerebral artery occlusion (tMCAO) model were employed. These methods differ in the identity of the vessel used for filament insertion and sacrificed following recanalization: the Longa method uses the ECA to mimic complete reperfusion, while the Koizumi method uses the CCA to achieve incomplete reperfusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-b). Although both variations induce acute focal ischemia, the chronic hyporeperfusion induced by the loss of the CCA in the Koizumi technique also contributes to longitudinal infarct evolution, as is representative of patients with unsuccessful reperfusion despite recanalization therapy. To recapitulate variable ischemic time, the duration of ischemia was varied to 30, 60, and 90 minutes, corresponding to ischemic durations commonly used in preclinical studies. Through these tMCAO variations, a spectrum of stroke physiology was established, consisting of six unique conditions: 30-minute Longa (30mL), 60-minute Longa (60mL), 90-minute Longa (90mL), 30-minute Koizumi (30mK), 60-minute Koizumi (60mK), and 90-minute Koizumi (90mK). The effect of these I/R modulations on infarct topology was first investigated according to TTC staining to delineate ischemic and healthy tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-d). Infarct volumes tended to be larger with increasing ischemic duration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Additionally, incomplete reperfusion was linked to larger infarct volumes specifically in the 90m cohorts. Although the 90mL group did not exhibit significantly larger infarcts than reperfusion-matched groups of shorter ischemic duration, it did demonstrate a higher frequency of hemorrhagic transformation than the 30mL cohort (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). However, a difference in hemorrhage volume was only detected between the 30mL and 60mL cohorts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). To link infarct pathology to the severity of the initial ischemic insult, intra-ischemic behavior was measured using an ordinal five-point scale. More severe intra-ischemic behavior was observed with increasing ischemic duration in the Koizumi cohorts, as evidenced by an increased prevalence of high behavior scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eh). Intra-ischemic behavior was also worse in the 90mK group relative to the 90mL group, similar to the trends observed in infarct volume. I/R dynamics did not influence mortality within 72 hours post-tMCAO (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ei).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIncomplete reperfusion induces a sustained drop in cerebral blood flow\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGiven that I/R profile alters ischemic macrovascular blood flow, we investigated whether these physiologic features also mediate spatiotemporal CBF patterning following tMCAO. Serial LSCI was performed at baseline, immediately before and after filament withdrawal, and at endpoint to create spatial maps of surface CBF. LSCI was employed as it enabled spatial imaging of the superficial cortex (~\u0026thinsp;1 mm) while minimizing unnecessary anesthesia, which would alter the inflammatory and pathological environment. First, I/R and infarct localization were confirmed for each model based on visual changes in perfusion relative to baseline (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Mirrored regions of interest (ROIs) were overlaid onto the ipsilateral and contralateral hemispheres to quantify CBF according to the scale of arbitrary perfusion units (a.u.), where higher values correspond to greater perfusion within the ROI (\u003cb\u003eFigure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ea\u003c/b\u003e). As expected, perfusion did not differ across experimental cohorts at baseline or during ischemia, but the Longa models exhibited significantly greater perfusion than their ischemic duration-matched Koizumi counterparts at reperfusion and endpoint (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003eS2\u003c/span\u003eb-c). Perfusion was unaffected by ischemic duration within both the Longa and Koizumi cohorts across all timepoints. Although these ROIs were useful to establish a straightforward comparison of perfusion between groups, this approach did not enable assessment of CBF across the entire ischemic hemisphere or more nuanced evaluation of the perfusion dynamics driving overall differences. To compare the composition of the entire ischemic hemisphere in terms of CBF, Matlab code was generated to quantify the proportion of pixels in the ischemic hemisphere corresponding to each color-gradated quintile on the scale of a.u. (blue\u0026thinsp;=\u0026thinsp;0\u0026ndash;60, cyan\u0026thinsp;=\u0026thinsp;61\u0026ndash;120, green\u0026thinsp;=\u0026thinsp;121\u0026ndash;180, yellow\u0026thinsp;=\u0026thinsp;181\u0026ndash;240, red\u0026thinsp;=\u0026thinsp;241\u0026ndash;300 a.u.). Using this approach, a significant decrease in the proportion of red pixels and corresponding increases in yellow/green/cyan pixels were observed at reperfusion and endpoint in the Koizumi cohorts relative to their ischemia-matched Longa counterparts, indicating a sustained shift toward hyporeperfusion in the Koizumi models (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003eS2\u003c/span\u003ed, \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). However, pixel composition of the ischemic hemisphere did not differ in response to varied ischemic duration within either reperfusion model. Pixel composition was then distilled into a quantitative summary metric, referred to as speckle score, to confirm the relevance of these perfusion dynamics in defining overall CBF across the entire ischemic hemisphere (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003eS2\u003c/span\u003ee). Speckle scores were derived by assigning each color quintile an ordinal value (red\u0026thinsp;=\u0026thinsp;5, blue\u0026thinsp;=\u0026thinsp;0) and calculating the mean value across the ischemic hemisphere. Speckle score during ischemia was significantly higher in the 60mK group compared to the 60mL group. However, these CBF dynamics underwent a shift following reperfusion, as evidenced by lower speckle scores, and therefore less perfusion, in the Koizumi cohorts than their Longa counterparts across all ischemic durations. Interestingly, this decrease in perfusion observed in Koizumi models was sustained at endpoint only in the 90m group, suggesting a potential interplay between ischemic severity and longitudinal CBF dynamics.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDuration of ischemia differentially alters acute locomotor function in the setting of complete and incomplete reperfusion\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMotor function is a primary measure of stroke severity and reflects multiple pathological features, including initial ischemia, infarct evolution, and longitudinal blood flow dynamics. We hypothesized that the variable perfusion dynamics observed between the Longa and Koizumi models may drive acute functional outcomes. Corner testing was performed at baseline, 24 hours- (24h), and 72 hours- (72h) post-tMCAO to identify functional deficits according to side preference [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. No differences in laterality were observed across groups (\u003cb\u003eFigure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003ea\u003c/b\u003e). However, corner testing assesses a single binary metric of locomotion that can be biased by starting position or repeated exposure, so laterality may not be sensitive enough to detect nuanced motor differences. To enable more robust evaluation of post-stroke locomotion, open field testing was performed at the same timepoints, and anatomic landmarks were tracked using DeepLabCut machine learning-based pose estimation (\u003cb\u003eFigure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eb\u003c/b\u003e). Representative heatmaps were generated for all cohorts at baseline, 24h, and 72h and metrics of locomotion were derived from tracking of the \u0026lsquo;mouse center\u0026rsquo; landmark (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-b, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003eS3\u003c/span\u003ec). Locomotion was decreased at 24h in the Koizumi model with increasing ischemic duration, as evidenced by decreases in distance traveled, average speed, border-center transitions, and percent time moving (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ec-f). At 72h, only percent time moving was still affected by ischemic duration in the Koizumi groups. Although this response to ischemic duration was not observed in the Longa model at 24h, the Longa cohorts did exhibit a similar sensitivity at 72h to the severity of ischemia. Specifically, decreases in distance traveled, average speed, and, to a lesser extent, border-center transitions were demonstrated at 72h with increasing ischemic duration. The extent of reperfusion had a limited effect on motor function, with lower percent time moving only in the 30mL cohort relative to the 30mK group at 24h and 72h. Cumulatively, these data demonstrate that ischemic duration differentially affects locomotor function throughout acute stroke evolution according to reperfusion status. Further, this may implicate reperfusion-mediated elements of stroke, including hypoperfusion and reperfusion-induced inflammation, as time-dependent drivers of functional outcomes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMyeloid cell response to ischemia is influenced by ischemic duration and reperfusion status\u003c/h2\u003e \u003cp\u003eThese data point to a role for ischemic duration and reperfusion success in mediating longitudinal stroke evolution. Given neuroinflammation, specifically through the actions of myeloid cells, has been implicated in disease progression, we utilized transgenic reporter mice with markers for MG/MΦ (Csf1r-EGFP) and PMNs (Ly6G-TdTomato) to investigate the role of I/R dynamics in mediating the acute immune response. Although the Ly6G-TdTomato reporter is somewhat promiscuous and labels a subset of neurons in addition to PMNs, these cells exhibited spatial segregation: TdTomato+ neurons were confined to the striatum (and more prominent in the contralateral hemisphere) while PMNs localized to cortical/subcortical regions of the infarct. Therefore, histological analyses were restricted to the ischemic cortex/subcortex to avoid neuronal biasing. Further, TdTomato+ neurons and PMNs were discerned according to size and morphology, as neurons were up to 2-fold larger than PMNs and exhibited branched morphology. These differentiating metrics were validated via NeuN staining, which consistently localized to cells identified as neurons based on location and morphology (\u003cb\u003eFigure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMorphological and spatial features of MG/MΦ were evaluated by creating surface reconstructions using Imaris image analysis software to link I/R dynamics with innate immune cell patterning (\u003cb\u003eFigure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e\u003c/b\u003e). Representative images were selected by mean cell counts. At 24h, the number of MG/MΦ in the ipsilateral hemisphere did not vary between cohorts (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-b). MG/MΦ morphology, assessed by cell volume and sphericity, did not differ between experimental groups at 24h (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003ec-d). However, the spatial association of MG/MΦ and PMNs was affected by ischemic duration, as evidenced by a decrease in MG/MΦ distance to the nearest PMN in the 90mL cohort relative to the 30mL group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). At 72h, MG/MΦ count was lower in the 30mK group than the 30mL group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003ef-g). The relevance of this finding, however, is unclear as it was not reflected in cohorts of longer ischemic duration. Differences in MG/MΦ volume and sphericity were also observed between the 30m and 60m groups based on reperfusion status (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eh-i). Additionally, an increase in MG/MΦ sphericity was observed with increasing ischemic duration, but only in the Longa models. Ischemic duration also mediated MG/MΦ proximity to PMNs at 72h, but only within the Koizumi cohorts, in contrast to the trend observed at 24h (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003ej).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimilar morphological analyses were also performed for PMNs. Additionally, given that PMNs have been reported to further contribute to infarct evolution through microvascular obstructions when retained in the vasculature, the proportion of intravascular PMNs was also quantified. At 24h, PMN count increased between the 30mL and 60mL cohorts (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-b). However, PMN volume, sphericity, and position relative to the vasculature did not significantly differ across cohorts (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-e). Given this lack of differential response at 24h, we hypothesized that this hyperacute timepoint may be too early to detect I/R-mediated changes in PMN patterning, as PMN infiltration has been demonstrated to peak around 3 days post-stroke [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. When PMN profiles were also evaluated at 72h, a significant increase in the number of PMNs present was observed with increasing ischemic duration regardless of reperfusion status, although this effect was more pronounced in the Koizumi cohorts (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003ef-g). PMN volume was affected by reperfusion success, as evidenced by a decrease in the 60mL cohort relative to the 60mK group (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eh). However, PMN sphericity and the proportion of PMNs retained intravascularly remained unchanged across surgical conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003ei-j). Cumulatively, these data confirm unique roles for ischemic duration and reperfusion success in driving the spatial and morphological profiles of myeloid cells during the acute stages of I/RI.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCerebral blood flow and locomotor function are correlated with the innate immune response\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn addition to the reported role of ischemic duration and reperfusion status, a high degree of heterogeneity within each surgical cohort was observed, with subjects exhibiting more severe or mild phenotypes than were expected for their assigned physiologic profile. Given this intra-cohort variability is likely in part mediated by more nuanced differences in I/R dynamics, we questioned whether hemodynamics, myeloid cell response, and locomotion could be intrinsically linked regardless of cohort assignment, with innate immune cell features reflecting acute stroke outcomes. We thus examined the cohort as a whole and applied a correlation analysis between all metrics of CBF, behavior, and myeloid cell profile (\u003cb\u003eFigure \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCBF dynamics demonstrated a modest association with myeloid cell features, although these correlations were inconsistent across metrics (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Specifically, a positive correlation was detected between the proportion of intravascular PMNs at 24h and the mean ROI perfusion at ischemia, but this association was not observed within any other CBF metrics and was not longitudinally sustained. Interestingly, MG/MΦ distance to the nearest PMN was the immune cell feature most robustly correlated with CBF metrics, including reperfusion and endpoint speckle scores, the proportion of red pixels at endpoint, and the proportion of green pixels at endpoint. Together, these data point to a link between CBF in the ischemic hemisphere and the spatial patterning of myeloid cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFeatures of the myeloid cell response were also correlated with locomotor function. At 24h, the number of PMNs present was negatively correlated with distance traveled, average speed, and percent time moving (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). These metrics of locomotion were also positively correlated with PMN volume at 24h. At 72h, the proportion of intravascular PMNs was negatively correlated laterality (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Although no other features of the myeloid response were robustly correlated with locomotion at 72h, elements of the PMN response were associated with changes in motor recovery between 24h and 72h. Specifically, both PMN sphericity and distance to the nearest vessel were correlated with recovery of percent time moving and border-center transitions. These data confirm a correlative link between elements of the PMN response in I/RI and acute locomotor function, which may serve to identify targets of future immunomodulatory strategies aimed at improving motor outcomes.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eDespite effective recanalization therapy, persistent functional deficits remain a challenge for post-stroke management [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. I/RI-mediated inflammation contributes significantly to infarct evolution beyond the initial ischemic insult, and therefore, drives functional outcomes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. PMNs have been implicated in both microvascular reperfusion failure and I/RI-induced inflammation [\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. MG/MΦ have also been linked to stroke progression, as a loss of MG/MΦ in preclinical models worsened infarct severity, inflammation, and motor function [\u003cspan additionalcitationids=\"CR37 CR38 CR39\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. However, while preclinical trials targeting elements of the immune response to reduce I/RI burden have yielded promising results, none have translated to improved outcomes in stroke patients [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. One factor contributing to the conflicting results in preclinical and clinical trials is the failure of preclinical models to incorporate variability in I/R dynamics and thus, recapitulate the spectrum of clinical stroke physiology. In clinical practice, recanalization via thrombectomy is the standard of care, even in patients with large ischemic core volumes, expanding the spectrum of ischemic severity among those that exhibit I/RI [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Patients demonstrating reperfusion injury also exhibit a range of reperfusion success according to macrovascular recanalization criteria like thrombolysis in cerebral infarction (TICI) scores [\u003cspan additionalcitationids=\"CR46 CR47\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Previous immunomodulatory strategies failed in clinical trials, suggesting that the target populations were not effectively modulated. This preclinical study was designed to examine how I/R variability affects the myeloid cell response and subsequent functional outcomes to inform future translational approaches that more closely resemble clinical stroke physiology.\u003c/p\u003e \u003cp\u003eThis study leverages the versatility of the tMCAO model to better represent the clinical I/R spectrum and elucidate the role of these physiologic features as drivers of the acute immune response [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Although previous studies have employed LSCI to demonstrate CBF heterogeneity, these blood flow dynamics have never previously been linked to the innate immune response and broader locomotor function [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Using pathological, functional, and inflammatory analyses, this preclinical study sought to link how nuanced I/R dynamics modulate the local response of PMN and MG/MΦ and subsequently drive pathological and functional outcomes.\u003c/p\u003e \u003cp\u003eFirst, our results indicate that infarct topology, post-stroke locomotion, and myeloid cell features are more robustly mediated by ischemic severity than by reperfusion efficacy. Differences in the immune response may be due to increased damage-associated molecular pattern or cytokine release induced by the initial ischemic event, with reperfusion being a necessary therapy that also promotes open vascular channels, enabling further inflammatory cell recruitment [\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. As recanalization is now the standard of care for LVO, these data highlight the need to establish critical ischemic thresholds that trigger the myeloid response to inform future translational work. The findings presented may guide preclinical inclusion criteria for future immunomodulatory treatment strategies using imaging-based assessment of ischemic injury severity in tandem with reperfusion success.\u003c/p\u003e \u003cp\u003eSecond, this study found that ischemia and reperfusion differentially alter the profile of MG/MΦ and PMNs at 72h post-tMCAO. PMN density was more robustly affected by ischemia than PMN morphology or position but was not influenced by reperfusion status. This suggests that the magnitude of the PMN response is most susceptible to ischemic severity, while the spatial features of infiltrated PMNs are relatively insensitive to the ischemic event at these acute time points. Although PMN volume did vary at 72h according to reperfusion efficacy in the 60m cohorts, the inconsistency with cohorts of other ischemic durations and the absence of other changes in cell morphology call into question the significance of this relationship. MG/MΦ density and morphology at 72h were also sensitive to the effects of I/R variation, but in contrast to PMNs, these differences were primarily in response to varied reperfusion success. The unique sensitivities of PMNs and MG/MΦ to I/R dynamics that were observed align with the differential roles that these myeloid cell populations may play in I/RI. As PMNs are peripheral immune cells recruited to the site of ischemia to exert transient pro-inflammatory effects, PMN infiltration is triggered by the initial ischemic event, with lesser effects observed in response to infarct evolution after the initial wave of cell activation [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In contrast, MG/MΦ respond hyperacutely to the inciting injury and persist in the ischemic tissue to contribute longitudinally to inflammation and injury resolution. Therefore, the evolving response of MG/MΦ to hyporeperfusion, rather than their initial actions due to the ischemic insult, is captured at the acute timepoints studied [\u003cspan additionalcitationids=\"CR55 CR56\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. These differential effects confirm that myeloid cells uniquely respond to ischemic burden and infarct evolution, potentially warranting distinct immunomodulation of PMNs and MG/MΦ according to patient-specific I/R dynamics.\u003c/p\u003e \u003cp\u003eIn addition to these individual responses, the position of MG/MΦ relative to PMNs was also influenced by ischemic duration. MG/MΦ and PMNs participate in cell-cell interactions in neuroinflammation, so proximity was measured as a surrogate for cellular association, with shorter distances suggestive of greater potential for interaction. The spatial association of MG/MΦ and PMNs in response to increasing ischemic duration is consistent with the ischemia-mediated increase in PMN density, implicating PMNs as the likely drivers of this relationship. Notably, our immunofluorescent imaging demonstrated that a subset of MG/MΦ completely overlapped with PMNs, pointing to contact-mediated interactions between these populations. Accordingly, it may be worthwhile to further investigate the changing relationship between MG/MΦ and PMNs in response to I/R to define how these interactions shape the acute tissue environment post-stroke and orchestrate subsequent neuroinflammatory patterns.\u003c/p\u003e \u003cp\u003eLastly, these analyses identified a correlative link between PMN features and locomotor function. These metrics correlated independently of assigned surgical cohort, establishing a critical association between stroke pathology and locomotor function across I/R profiles and highlighting the need to build on behavioral testing to evaluate outcomes in the development of I/RI therapies [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Importantly, the features of PMNs that were correlated to behavioral outcomes differed by timepoint. PMN density and cell volume strongly influenced locomotor outcomes at 24h, while metrics of locomotor recovery from 24h to 72h were more sensitive to PMN sphericity and proximity to the vasculature. This dynamic link between inflammation and locomotor function suggests that differential immune cell metrics may correlate to longitudinal behavior changes post-stroke, pointing to facets of the myeloid cell response that could potentially be targeted to alleviate specific motor deficits across time.\u003c/p\u003e \u003cp\u003eThese findings highlight how the acute myeloid response is mediated by I/R dynamics and emphasize the relevance of defining the longitudinal immunological response as it relates to physiology and functional outcomes. Ultimately, although reperfusion is necessary to limit clinical morbidity, this work reinforces that ischemic duration is a more robust driver of infarct topology, behavioral metrics, and PMN infiltration than reperfusion efficacy. In contrast, MG/MΦ were uniquely sensitive to the extent of reperfusion, confirming the differential responses of myeloid cell populations and emphasizing the importance of considering both ischemia and reperfusion dynamics in future translational studies.\u003c/p\u003e \u003cp\u003eThis study has several strengths, including the application of multiple I/R models, non-invasive live-animal imaging, and transgenic fluorescent reporters to explore the relationship between I/R dynamics and the post-stroke myeloid response. However, there are limitations as well. Analysis of myeloid cells did not include evaluation of cell function. Future studies will aim to link immune cell function with position and morphology, as well as extend these analyses to subacute and chronic timepoints, such as days 7- and 28-post stroke, to evaluate the longitudinal effects of I/R on neuroinflammation and stroke pathology. Lastly, this study utilized the Csf1r-EGFP reporter to label both the MG and MΦ populations in parallel with the Ly6G-TdTomato reporter as a means to investigate the spatial relationships between myeloid cell populations. Additional studies should be performed using the Cx3cr1-Cre-YFP-TdTomato lineage-tracing strain to differentiate the contributions of MG and MΦ in the absence of PMN labeling. Consideration of the consequences of I/R dynamics on neuroinflammation and behavior in future preclinical studies may lead to more translational success in developing new adjunctive therapies in the preclinical-clinical pipeline for ischemic stroke.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003e30mK:\u0026nbsp;\u003c/strong\u003e30-minute Koizumi model\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e60mK:\u0026nbsp;\u003c/strong\u003e60-minute Koizumi model\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e90mK:\u0026nbsp;\u003c/strong\u003e90-minute Koizumi model\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e30mL:\u0026nbsp;\u003c/strong\u003e30-minute Longa model\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e60mL:\u0026nbsp;\u003c/strong\u003e60-minute Longa model\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e90mL:\u0026nbsp;\u003c/strong\u003e90-minute Longa model\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCBF:\u0026nbsp;\u003c/strong\u003eCerebral blood flow\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCCA:\u0026nbsp;\u003c/strong\u003eCommon carotid artery\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e24h:\u0026nbsp;\u003c/strong\u003e24 hours post-reperfusion\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e72h:\u0026nbsp;\u003c/strong\u003e72 hours post-reperfusion\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eECA:\u0026nbsp;\u003c/strong\u003eExternal carotid artery\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eICA:\u0026nbsp;\u003c/strong\u003eInternal carotid artery\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI/R:\u0026nbsp;\u003c/strong\u003eIschemia and reperfusion\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI/RI:\u0026nbsp;\u003c/strong\u003eIshcemia/reperfusion injury\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLSCI:\u0026nbsp;\u003c/strong\u003eLaser speckle contrast imaging\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLVO:\u0026nbsp;\u003c/strong\u003eLarge vessel occlusion\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMG:\u0026nbsp;\u003c/strong\u003eMicroglia\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eM\u003c/strong\u003e\u003cstrong\u003eΦ\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eBone marrow-derived macrophages\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePMN:\u0026nbsp;\u003c/strong\u003ePolymorphonuclear neutrophils\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTICI:\u0026nbsp;\u003c/strong\u003eThrombolysis in cerebral infarction\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003etMCAO:\u0026nbsp;\u003c/strong\u003eTransient middle cerebral artery occlusion\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTTC:\u0026nbsp;\u003c/strong\u003eTriphenyl tetrazolium chloride\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eCompeting Interests:\u003c/strong\u003e \u003cp\u003eThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLES and NAN conceptualized this project and designed the included experiments. LES, MT, and EAZ performed experiments. LES, MMM, CP, and JD analyzed data and generated figures. LES, NAN, and SET drafted and edited the manuscript. SET provided technical expertise and assisted with experiment design. NAN provided funding for this project. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would like to thank Beth Vernaleo, PhD for manuscript proofreading and members of the Tsirka lab for helpful discussions and suggestions.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study will be openly available in Figshare at the time of publication (reference number to be provided when available).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGBD 2019 Stroke Collaborators. 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Stroke. 2022;53(5):1802\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Acute Stroke, Animal Models, Inflammation, Ischemia/Reperfusion, Myeloid Cells","lastPublishedDoi":"10.21203/rs.3.rs-8712806/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8712806/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIschemic stroke remains a leading cause of long-term disability, despite more patients undergoing vessel recanalization. Ischemia/reperfusion injury (I/RI), the inflammatory cascade triggered by ischemia and reperfusion, contributes to infarct evolution and functional outcomes. Clinical stroke presentation varies due to differences in ischemic duration and reperfusion success, yet preclinical models rarely account for this physiologic contribution. This preclinical-clinical disconnect may underlie the translational failure of acute therapies targeting the myeloid cell-mediated immune response (specifically neutrophils and microglia/bone marrow-derived macrophages). To address this physiological heterogeneity, we employed variations of the transient middle cerebral artery occlusion model in myeloid-reporter mice, systematically altering ischemic duration and reperfusion success. Using longitudinal perfusion imaging and behavioral testing, we found that infarct pathology, locomotor deficits, and innate immune responses were significantly influenced by ischemic duration, and to a lesser extent, reperfusion status. Microglia/macrophage and neutrophil morphology, as well as the spatial association of these myeloid cells, were the most strongly affected cellular features. Further, neutrophil density, morphology, and spatial patterning correlated with acute locomotion and motor recovery across the entire cohort. These findings highlight the differential roles of ischemic duration and reperfusion efficacy in driving neuroinflammation and stroke outcomes, emphasizing the importance of incorporating physiologic heterogeneity into preclinical I/RI models to better guide translational strategies.\u003c/p\u003e","manuscriptTitle":"Ischemic Duration is More Critical than Reperfusion Efficacy in Driving Early Neuroinflammation and Motor Deficits after Transient Middle Cerebral Artery Occlusion","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-09 12:11:36","doi":"10.21203/rs.3.rs-8712806/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3655a39f-898b-4acd-b010-a4d39ad84f66","owner":[],"postedDate":"February 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-18T16:09:30+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-09 12:11:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8712806","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8712806","identity":"rs-8712806","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}