Cerebrovascular injuries can cause severe edema and inflammation that adversely affect human health. Here, we observed that recanalization after successful endovascular thrombectomy for acute large vessel occlusion was associated with cerebral edema and poor clinical outcomes in patients who experienced hemorrhagic transformation. To understand this process, we developed a cerebrovascular injury model using transcranial ultrasound that enabled spatiotemporal evaluation of resident and peripheral myeloid cells. We discovered that injurious and reparative responses diverged based on time and cellular origin. Resident microglia initially stabilized damaged vessels in a purinergic receptor–dependent manner, which was followed by an influx of myelomonocytic cells that caused severe edema. Prolonged blockade of myeloid cell recruitment with anti-adhesion molecule therapy prevented severe edema but also promoted neuronal destruction and fibrosis by interfering with vascular repair subsequently orchestrated by proinflammatory monocytes and proangiogenic repair-associated microglia (RAM). These data demonstrate how temporally distinct myeloid cell responses can contain, exacerbate and ultimately repair a cerebrovascular injury.
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This research was supported by the intramural program at the NINDS, NIH. We thank A. Hoofring in the NIH Medical Arts Design Section for generating the illustration shown in Extended Data Fig. 1. We thank A. Elkahloun and W. Wu in the National Human Genome Research Institute Microarray core for their assistance with the RNA-seq experiment.
The authors declare no competing interests.
Peer review information Nature Neuroscience thanks Thiruma Arumugam, Jonathan Godbout, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Following surgical generation of a 2 mm x 2 mm x 15 µm thinned skull window, microbubbles were injected intravenously and a drop of aCSF was placed atop the thinned skull bone. Through this aCSF we applied low intensity pulse ultrasound (LIPUS) using a Mettler Sonicator 740x with a 5 cm2 planar dual frequency applicator operating at 1 MHz, ~200KPa peak negative pressure with duty cycle 10% and 1 ms burst. LIPUS induced acoustic cavitation of the microbubbles. Microbubble oscillation, inertial cavitation, and explosion caused internal injury of blood vessel walls, exposing the brain parenchyma to blood contents. This injury creates a relative column of injury in the brain tissue beneath the thinned skull window, as the ultrasound waves are not strong enough to pass through the surrounding intact bone.
a, Magnified images of the 2 mm x 2 mm x 15 µm thinned skull window pre- and post-injury depict petechial intraparenchymal hemorrhages at 10 min post-injury. b, Macroscopic depiction of a mouse brain 24 h following posterior sonication injury. c, Kaplan-Meier curve demonstrates a median survival of 2 days after posterior sonication injury. Anterior sonication injury does not result in fatalities. Cumulative data are shown from 2 independent experiments with 10 mice per group (P = 2.96e-10, Log-rank test). d, A graph showing quantification of cerebral water content demonstrates increased edema 24 h after sonication with 7.7% and 7.1% increase in water content after anterior and posterior injury, respectively, relative to uninjured control mice (**P < 0.01, anterior P = 2.9e-7, posterior p = 1.3e-6, One-way ANOVA/Tukey test). Cumulative data are shown from 2 independent experiments with 5 mice per group per experiment. e, A graph showing quantification of fluorescein extravasation into the ipsilateral versus contralateral brain hemisphere at the denoted time points post-injury (**P < 0.01, One-way ANOVA/Tukey test). Data are representative of 2 independent experiments with 4 mice per group per experiment, 2 samples were above the detection limit and not included. f-h, High parameter flow cytometric analysis of brain biopsies from mice at d1 and d6 post-injury relative to uninjured controls. A UMAP plot of concatenated live cells from each group is shown in panel F. A heatmap of Ter-119 signal on a UMAP plot reflecting the concatenated cell populations from a single experiment is shown in panel G. Panel H shows a scatter plot depicting the absolute Ter-119+ RBCs. Cumulative data are shown from 2 independent experiments (Uninjured n = 8, d1 n = 8, d6 n = 9, **P < 0.01, One-way ANOVA/Tukey test; gating strategy in Supplementary Fig. 1A). Graphs D, E, H show the mean ± SD. Source data
Extended Data Fig. 3 Microglia depletion increases BBB leakage, intraparenchymal hemorrhage, myeloid cell invasion and vascular endothelium activation.
a, Intravital microscopy of CX3CR1gfp/wt (green) mice 20 min after injury shows extensive intraparenchymal EB (red) extravasation following microglia depletion using an alternate CSF1R inhibitor, PLX5622, to that used in Fig. 2c,d. b, EB extravasation assay based on intravital microscopy time lapses depicts increased BBB leakage 20-40 min after microglia depletion using PLX5622. Graph depicts mean ± SD of cumulative data from 2 independent experiments (n = 12 mice per group, **P = 3.9e-9, two-tailed Student’s t-test). c, Confocal microscopy images of cortical brain sections from of naive and microglia depleted Cx3CR1gfp/wt (green) mice 1 h following injury. Mice received an i.v. injection of fluorescent fibrinogen (white) and tomato-lectin (red). Larger and more diffuse intraparenchymal fibrinogen is observed in mice treated with PLX3397 (CSF1R inhibitor). d, Image-based quantification of fibrin burden in the brain parenchyma. Graph depicts mean ± SD of cumulative data from 2 independent experiments (Ctrl n = 10, CSF1R inh n = 13, **P = 0.00017, Mann–Whitney U test). e, Two-photon microscopy images captured in the cerebral cortex of injured LysMgfp/wt mice treated with vehicle or PLX3397 show LysM+ myelomonocytic cell (green) invasion at 20 min post-injury. Tomato-lectin is shown in red. f, Image based quantification of myelomonocytic infiltration. Graph depicts mean ± SD of cumulative data from 2 independent experiments (Ctrl n = 8, CSF1R inh n = 12, **P = 0.0002, Mann–Whitney U test). g, Intravital microscopy images in the cerebral cortex of vehicle versus PLX3397 treated B6 mice at 24 h post-injury. Prior to imaging, mice received an i.v. injection of APC-anti-CD106 (VCAM-1; red) and PE-anti-CD54 (ICAM-1; green), which revealed increased endothelial expression in PLX3397 treated mice. Representative images are from 2 independent experiments with 3 mice per group. h, qPCR analysis of ICAM and VCAM expression in vehicle vs. PLX3397 treated B6 mice at 24 h post-injury. Graph depicts mean ± SD of cumulative data from 2 independent experiments (n = 6 mice per group, Vcam1 P = 3.58e-5, Icam1 P = 0.0072, Two-way ANOVA/Holm-Sidak test). i, Heatmap shows qPCR analysis of genes encoding for acute inflammation-related proteins in vehicle vs. PLX3397-treated B6 mice. The fold increase in gene expression was calculated relative to the uninjured contralateral hemisphere for each mouse at 24 h post-injury. Data are representative of 2 independent experiments with 5 mice per group per experiment (*P < 0.05, **P < 0.01, multiple t tests with Holm-Sidak multiple comparisons correction, source data in Supplementary Table S3). CSF1R inh refers to PLX3397. Source data
Extended Data Fig. 4 Representative images establishing a role for P2RY12 receptor and Cx43 hemichannels in microglial rosetting.
Data from these experiments are provided in Fig. 2g,h,i. a, Intravital microscopy images in the cerebral cortex of Cx3CR1gfp/wt (green) mice 20 min post-injury. Mice were treated transcranially with a vehicle or a P2RY12 inhibitor (MeSAMP). Intravenous injection of EB (red) revealed increased intraparenchymal extravasation following pre-treatment with the P2RY12 inhibitor. Images are representative of experimental data graphed in Fig. 2g. b, Intravital microscopy images in the cerebral cortex of Cx3CR1gfp/wt (green) mice 20 min post-injury. Mice were treated transcranially with a vehicle or a Cx43 inhibitor (carbenoxolone). Intravenous injection of EB (red) revealed extensive intraparenchymal extravasation following pre-treatment with the Cx43 inhibitor. Images are representative of experimental data graphed in Fig. 2g. c, Confocal microscopy images of brain sections from littermate control (GFAPCreER-Cx43f/wt and GFAPCreER-Cx43wt/wt) vs. GFAPCreER-Cx43f/f mice 1 h after injury show decreased microglia rosetting in GFAPCreER-Cx43f/f mice. Microglia rosettes were identified with Iba1 staining (green). Tomato-lectin+ blood vessels are shown in white. Images are representative of experimental data graphed in Fig. 2h. d, Intravital microscopy images from the cerebral cortex of control (GFAPCreER-Cx43f/wt, GFAPCreER-Cx43wt/wt) vs. GFAPCreER-Cx43f/f mice 20 min after injury. Intravenous injection of EB (red) revealed extensive intraparenchymal extravasation in GFAPCreER-Cx43f/f mice. Images are representative of experimental data graphed in Fig. 2i.
Extended Data Fig. 5 Immune landscape in the cerebral cortex following cerebrovascular injury in Cx3cr1gfp/wtCcr2rfp/wt mice.
Quantification of these flow cytometric experiments is provided in Fig. 3c,f, gating strategy in Supplementary Fig. 1B. The panel used for these experiments includes: Cx3CR1-GFP, Ly6C BB790, MHCII BV480, CD11b BV570, CD115 BV605, CD24 BV650, CD11c BV785, P2RY12 PE, CCR2-RFP, Ter-119 PE/Cy5, CD206 PE/Cy7, CD45 BUV395, CD4 BUV496, Ly6G BUV563, CD19 BUV661, CD44 BUV737, CD8 BUV805, F4/80 APC-R700, TCRb APC/Cy7 and live/dead fixable blue cell staining kit. Plots were pre-gated for CD45 + Ter119- live cells and subsequently analyzed using an unsupervised clustering algorithm to group data into subpopulations (PhenoGraph) and visualized using UMAP. For each experiment, the first row depicts the concatenated samples of 4 independent mice per group, and the legend shows the combined phenograph clusters corresponding to different immune cell populations. The second and third rows show six representative heatmaps of different markers used to identify the different immune cell populations. a, Immune landscape at 1 d and 6 d post-injury compared to uninjured mice. b, Immune landscape 1 d after injury in mice treated with bolus αLFA1/VLA4 or isotype control antibodies relative to uninjured mice.
Extended Data Fig. 6 Effect of myelomonocytic cell invasion on cerebral edema and survival after injury.
This figure depicts quantification of cerebral water content following anterior and posterior injury as well as survival after posterior injury in four different experimental paradigms. Left column shows quantification of cerebral water content in the ipsilateral hemisphere 1 d after anterior injury in the treatment vs control group compared to the contralateral hemisphere. The middle column shows quantification of cerebral water content after posterior injury when mice reached the survival end point or 5 d post-injury in the treatment vs. control group relative to the contralateral hemisphere (*P < 0.05, **P < 0.01, Two-way ANOVA/Holm-Sidak test). Graphs depict mean ± SD. The right column shows Kaplan-Meier survival curves after posterior injury in treatment vs. control groups (Log-rank test). a, Effect of αGr-1 vs. isotype control bolus administration 24 h prior to injury. b, Effect of αLFA1/VLA4 vs. isotype control bolus administration 24 h prior to injury. c, Water content and survival after injury in CCR2 KO mice compared to B6 mice treated with isotype or αLFA1/VLA4 24 h prior to injury. d, Effect of αLy6G or αLFA1/VLA4 vs. isotype control administration 24 h prior to injury. Data are representative of 2 independent experiments with 4 or 5 mice per group. Source data
Extended Data Fig. 7 Combined αLFA1 and αVLA4 treatment is required to prevent cerebral edema and death.
a-c, Effect of bolus treatment with αLFA1/VLA4, αLFA1, αVLA4 or isotype control on cerebral water content 24 h after anterior (A) or posterior (B, C) injury. Data compilation from 2 independent experiments. The antibodies were administered 24 h before injury. Cerebral water content was determined 1d after anterior injury (A) and 5d or when mice reached the survival endpoint after posterior injury (B). Graphs depict mean ± SD (isotype n = 8, αLV n = 8, αLFA1 n = 4 or 5, αVLA4 n = 5, *P < 0.05, **P < 0.01, Two-way ANOVA/Holm-Sidak test). Panel C demonstrates Kaplan-Meier survival curve following posterior injury (isotype n = 9, αLV n = 10, αLFA1 n = 6, αVLA4 n = 6, Logrank test P = 3.4e-5). d, Intravital microscopy images of the cerebral cortex from uninjured vs. injured B6 mice 1 h following i.v. administration of APC-anti-CD106 (VCAM-1; red) and PE-anti-CD54 (ICAM-1; green) show increased endothelial ICAM and VCAM expression 24 h after injury. Two representative images from the injured mice depict inter-sample and inter-vessel variability in VCAM-1 vs. ICAM-1 expression. Representative images are from 2 independent experiments with 3 and 5 mice per group. e, Image based quantification of vascular ICAM and VCAM expression. Graph depicts mean ± SD of cumulative data from 2 independent experiments (uninjured n = 6, d1 n = 10, **P < 0.01, Two-way ANOVA/Holm-Sidak test). f, Scatter plot of sum intensity of ICAM vs VCAM expression depicting the lack of a correlation between the two variables. Each dot represents a single mouse, and the graph is a representation of data points shown in Extended Data Fig. 7e, (R2 = 0.089, P = 0.4, Pearson’s product moment correlation test). g, Scatter plot of ICAM vs. VCAM gene expression determined by qPCR in the brain 24 h post-injury. No correlation was observed between the two variables. Each dot represents one mouse. Graphs depict cumulative data from 2 independent experiments with 5 mice per experiment, (R2 = 0.000078, P = 0.98, Pearson’s product moment correlation test). Source data
a, b, Confocal microscopy of cerebral cortex 10 days after injury shows areas of gliosis (GFAP, blue) and microglia clustering (Iba-1, red) relative to tomato-lectin+ vasculature. Mice were treated continuously with either isotype control (A) or αLFA1/VLA4 (B) antibodies. Continuous treatment with αLFA1/VLA4 results in large areas of brain tissue with blood vessels. Images are representative of 5 mice per group. c, d, Flow cytometric analysis and gating strategy of monocyte and neutrophil depletion in blood (C) and brain (D) following continuous treatment with αGR-1 and αLy6G antibodies 6 d post-injury compared to isotype treated controls. The following panel was used: Ly6C FITC, GR-1 BV421, CD11b BV570, Cx3CR1 BV711, P2RY12 PE, CD45 BUV395, Ly6G BUV563, CD44 BUV737, CD115 APC and live/dead fixable blue cell staining kit. Plots were pre-gated for single, live cells and subsequently for CD45 + CD11b + cells. In brain samples gated, microglia were identified as CD45lowCx3Cr1 + P2RY12 + CD44- cells. Monocytes were identified as CD45 + CD44 + CD115 + Ly6G-GR1low and neutrophils as CD45 + CD44 + CD115-Ly6G + GR1hi. In mice treated with αLy6G, Ly6G was not used to characterize cells flow cytometrically. Moreover, in mice treated with αGR-1, GR-1 was not used to characterize the cells. Graphs show the mean ± SD and are representative of 2 independent experiments with n = 5 (C) and n = 4 (D) mice per group (**P < 0.01, Two-way ANOVA/Holm-Sidak test). e, f, Intravital microscopy of cerebral vasculature and image-based quantification of vascular coverage in naïve B6 mice, CCR2 KO mice and CCR2 KO mice with CD115 + monocyte adoptive transfer 10 d post-injury. Adoptive transfer of CD115 + monocytes from B6 mice partially reconstitutes the angiogenic process. Graph shows mean ± SD and is representative of two independent experiments with (n = 4 mice per group, **P < 0.01, Kruskal-Wallis/Dunn’s test). Source data
qPCR analysis of genes encoding for angiogenesis related proteins. Data from these experiments are represented in Fig. 4i. a, Volcano plot of angiogenesis related gene expression between uninjured mice and mice d6 after injury. B. Bar graph of gene expression differences between injured and uninjured mice for genes with Q < 5%. c-d, Volcano plot of angiogenesis related gene expression after continuous αGr-1 (C) or αLFA1/VLA4 administration (D). e-f, Bar graphs of gene expression difference for genes with Q < 5%. Data are representative of 2 independent experiments with 4 mice per group. Statistical analysis was performed using multiple t-tests and the Benjamini, Krieger and Yekutieli method to correct for the false discovery rate, with a desired Q value of 5%. Data are representative of 2 independent experiments with 4 mice per group per experiment, source data in Supplementary Table 4.
VEGF-expressing microglia are involved in angiogenesis. a, b, Intravital microscopy of cerebral vasculature 10 d after injury (A) and image-based quantification of vascular coverage (B) in PLX5622 versus vehicle control treated mice show a lack of angiogenesis after microglia depletion. Blood vessels were labeled with EB (red) and tomato-lectin (green). Graphs depict mean ± SD of cumulative data from 3 independent experiments (n = 16 mice per group, **P = 4e-13, two-tailed Student’s t-test). c, Volcano plot of angiogenesis related gene expression in vehicle vs. PLX3397 treated (CSF1R inhibitor) mice 6 d post-injury d, Bar graph of gene expression difference for genes with Q < 5%. Data are representative of 2 independent experiments with 4 mice per group. Statistical analysis was performed using multiple t-test and the Benjamini, Krieger and Yekutieli method to correct for the false discovery rate, with a desired Q value of 5%. A heatmap of the data from panels A and B is shown in Fig. 5b, source data in Supplementary Table 4. e, Representative heatmaps used to identify different immune cell populations following high parameter flow cytometric analysis of the immune landscape in the cerebral cortex d1 and d6 following injury in Cx3cr1CreER/+ x Stopfl/+ TdTomato mice. The following panel was used: Ly6C BB790, MHCII BV480, CD11b BV570, CD115 BV605, CD24 BV650, Cx3Cr1 BV711, CD11c BV785, P2RY12 PE, Ter-119 PE/Cy5, CD206 PE/Cy7, CD45 BUV395, CD4 BUV496, Ly6G BUV563, CD19 BUV661, CD44 BUV737, CD8 BUV805, F4/80 APC-R700, TCRβ APC/Cy7, VEGF-A AF647, TdTomato and live/dead fixable blue cell staining kit. Data were pre-gated on CD45 + Ter119- live cells, subsequently analyzed using an unsupervised clustering algorithm to group data into subpopulations (PhenoGraph) and visualized using UMAP. Quantification of these experiments is provided in Fig. 5f. f, Gating strategy for flow cytometry experiment demonstrating a lack of VEGF-A + microglia in mice treated with continuous αLFA1/VLA4 after injury. The following panel was used: CD24 FITC, CD11b BV570, P2RY12 PE, TdTomato, CD45 BUV395, VEGF-A AF647 and live/dead fixable blue cell staining kit. Quantification of these experiments is provided in Fig. 5h,i and gating strategy in Supplementary Fig. 1B. Source data
Supplementary Fig. 1 and Table 1.
Demographic data, symptoms, stroke characteristics and MRI imaging data from patients who had strokes. The MRI imaging data were used to generate the graphs in Fig. 1c,d.
Gene expression data used to generate the heatmap in Extended Data Fig. 3g. ΔΔCt analysis was completed using Gapdh as the housekeeping gene and contralateral hemisphere tissue as a control. Sheets 1 and 2 depict independent experimental replicates.
Gene expression data used to generate heatmaps and bar graphs in Figs. 4i and 5b and Extended Data Figs. 9 and 10a,b. ΔΔCt analysis was completed using Gapdh as the housekeeping gene and uninjured tissue as a control. Sheets 1 and 2 depict independent experimental replicates.
RNA-seq data analysis and IPA. RNA-seq results from cerebral cortex biopsies of uninjured mice versus injured mice at day 20 treated continuously for 10 d with αLFA1/VLA4 or isotype control antibodies (data are presented in Fig. 6a–d, and methods are described in “RNA-sequencing data analysis” in the Methods).
RNA-seq. IPA of concordant dysregulated genes in αLFA1/VLA4-treated mice relative to the other two groups, uninjured and control (data are presented in Fig. 6a–d, and methods are described in “RNA-sequencing data analysis” in the Methods).
Statistical analysis for graphs in Figs. 1c,d, 2d,g–i, 3c,e–i, 4b,d,f,h, 5a,d,f,h and 6e,f and Extended Data Figs. 2c–e,h, 3b,d,f,h, 6a,b–d, 7a–c,e–g, 8c,d,f and 10b.
Vascular injury and leakage following transcranial sonication. Mice were injected with EB (red) i.v. immediately after anterior injury and were subsequently imaged for 40–60 min. The time lapse of the uninjured cortex depicts intact pial and parenchymal vessels. Following sonication, cerebral blood vessels (especially capillaries) were heavily injured, resulting in leakage of EB into the brain parenchyma. Videos are representative of five mice per group.
Cerebrovascular injury results in edema. Representative NG2-mEGFP mice were imaged before (uninjured) and after anterior injury (injured). Mice were injected i.v. with EB (red) immediately after injury and 1 min before imaging. NG2-mEGFP (green) allows for visualization of oligodendrocyte precursor cells and mural cells, thus providing a stable brain structure upon which tissue distortion (edema) could be observed. The time lapse of the injured cortex shows substantial tissue distortion resulting from edema (white dotted circle). This video represents two independent experiments with three mice each.
Destruction of glia limitans following injury. GFAP-CreER ; Stopfl/+ TdTomato mice were injected i.v. with tomato lectin DyLight 488 (green) immediately after anterior injury and 1 min before imaging. The representative time lapse of the uninjured cortex shows surface-associated astrocytes (reddish yellow) comprising the glia limitans superficialis. These cells become severely damaged and lose their fluorescence following ultrasound injury. This video is representative of two independent experiments with three mice per group.
Microglia form rosettes in response to cerebrovascular injury. Part 1, Cx3cr1GFP/+ (green) mice were injected with EB (red). Mice were imaged immediately after injury or starting 1 h after injury. The representative time lapse of the uninjured cortex (left) demonstrates resting ramified microglia. Following anterior injury, microglia immediately project processes, creating tube-like structures that compartmentalize blood vessels within ~20 min (middle). Within an hour, multiple tubular formations (rosettes) are visible (right). Part 2, a representative time lapse from a Cx3cr1GFP/+ (green) mouse initiated immediately after injury shows the formation of rosettes within 20 min. Part 3, a representative time lapse from a Cx3cr1GFP/+ (green) mouse injected i.v. with EB (red) and imaged at 24 h post-injury. At this time point, the rosette-forming microglia have transformed into phagocytic ameboid cells. Videos are representative of two experiments with five mice per group.
Extensive BBB leakage following cerebrovascular injury in microglia-depleted mice. Representative videos from Cx3cr1GFP/+ (green) mice demonstrate the EB (red) extravasation assay with and without microglia depletion (that is, PLX3397 administration). Quantification of these time lapses is shown in Fig. 2c,d. Naive and PLX3397-treated Cx3cr1GFP/+ mice were injected with EB i.v. immediately following anterior injury and were subsequently imaged for 40–60 min. Microglia depletion is associated with significantly increased BBB leakage after cerebrovascular injury. Videos are representative of three experiments with four mice per group.
Purinergic receptor signaling is responsible for microglial rosette formation. Representative time lapses from Cx3cr1GFP/+ (green) mice captured immediately after anterior injury show the effect of transcranial P2RY12 or CX43 hemichannel inhibitors on microglial rosette formation. Vehicle was applied transcranially to the control group. Quantification of these time lapses is shown in Fig. 2e,f. Both P2RY12 and CX43 inhibition impede the formation of microglia rosettes. Videos are representative of three independent experiments with three to five mice per group.
Transcranial inhibition of CX43 hemichannels increases BBB leakage after cerebrovascular injury. Representative time lapses from Cx3cr1GFP/+ (green) mice captured immediately after anterior injury show the extent of EB (red) leakage in the presence or absence of a transcranial CX43 hemichannel inhibitor. Quantification of these time lapses is shown in Fig. 2g. CX43 inhibition impedes microglia rosette formation and enhances EB extravasation. Videos are representative of two independent experiments with three to six mice per group.
Enhanced BBB leakage following cerebrovascular injury in GFAP-CreER-Cx43fl/fl mice. Part 1, representative time lapses show EB (red) leakage in WT littermate control (that is, GFAP-CreER-Cx43fl/+) versus GFAP-CreER-Cx43fl/fl mice immediately following anterior injury. Quantification of these time lapses is shown in Fig. 2i. Enhanced extravasation of EB is observed in GFAP-CreER-Cx43fl/fl mice. Part 2, representative time lapses from littermate control and GFAP-CreER-Cx43fl/fl mice after anterior injury show that EB is often confined to a clot in control mice but leaks extensively into the surrounding parenchyma in GFAP-CreER-Cx43fl/fl mice. Videos are representative of two independent experiments with four mice per group.
Cerebrovascular injury promotes massive extravasation of peripheral myelomonocytic cells. LysMGFP/+ mice were injected i.v. with tomato lectin DyLight 649 (red) immediately after anterior injury and imaged for 60–90 min. A representative time lapse shows massive infiltration of the brain parenchyma by LysM+ myelomonocytic cells (green) within 1 h of injury. Videos are representative of three experiments with two mice per experiment.
αLFA1/VLA4 antibodies prevent myelomonocytic cell infiltration into the lesion core and perimeter. Intravital imaging of the lesion core and perimeter in LysMGFP/+ (green) mice that received a bolus treatment of αLFA1/VLA4 or isotype control antibodies immediately after anterior injury. EB (red) was injected i.v. to label blood vessels. Representative time lapses from the lesion core (part 1) and perimeter (part 2) show that αLFA1/VLA4 impedes infiltration by LysM+ myelomonocytic cells. The time lapses were captured beginning at 30 min post-injury. Quantification of these time lapses is shown in Fig. 3e. Videos are representative of two independent experiments with seven to eight mice per group.
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Mastorakos, P., Mihelson, N., Luby, M. et al. Temporally distinct myeloid cell responses mediate damage and repair after cerebrovascular injury. Nat Neurosci 24, 245–258 (2021). https://doi.org/10.1038/s41593-020-00773-6