Temporally distinct myeloid cell responses mediate damage and repair after cerebrovascular injury


Temporally distinct myeloid cell responses mediate damage and repair after cerebrovascular injury

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ABSTRACT 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. Access through your institution Buy or subscribe This is a


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SINGLE VESSEL OCCLUSION AND RECANALIZATION Article Open access 19 August 2022 REPAIR-ASSOCIATED MACROPHAGES INCREASE AFTER EARLY-PHASE MICROGLIA ATTENUATION TO PROMOTE ISCHEMIC STROKE


RECOVERY Article Open access 31 March 2025 STROKE INDUCES DISEASE-SPECIFIC MYELOID CELLS IN THE BRAIN PARENCHYMA AND PIA Article Open access 17 February 2022 DATA AVAILABILITY The data that


support the findings of this study are available from the corresponding author upon request. There are no restrictions on data availability. Bulk RNA-seq data are available in the NCBI Gene


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PubMed  PubMed Central  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS 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. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Viral Immunology & Intravital Imaging Section, National Institute of Neurological Disorders


and Stroke, National Institutes of Health, Bethesda, MD, USA Panagiotis Mastorakos, Nicole Mihelson & Dorian B. McGavern * Department of Surgical Neurology, National Institute of


Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA Panagiotis Mastorakos * Acute Cerebrovascular Diagnostics Unit, National Institute of Neurological


Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA Marie Luby, Amie W. Hsia & Lawrence Latour * Frank Laboratory, Radiology and Imaging Sciences, Clinical Center,


National Institutes of Health, Bethesda, MD, USA Scott R. Burks, Jaclyn Witko & Joseph A. Frank * National Institute of Neurological Disorders and Stroke, National Institutes of Health,


Bethesda, MD, USA Kory Johnson * MedStar Washington Hospital Center Comprehensive Stroke Center, Washington, DC, USA Amie W. Hsia * National Institute of Biomedical Imaging and


Bioengineering, National Institutes of Health, Bethesda, MD, USA Joseph A. Frank Authors * Panagiotis Mastorakos View author publications You can also search for this author inPubMed Google


Scholar * Nicole Mihelson View author publications You can also search for this author inPubMed Google Scholar * Marie Luby View author publications You can also search for this author


inPubMed Google Scholar * Scott R. Burks View author publications You can also search for this author inPubMed Google Scholar * Kory Johnson View author publications You can also search for


this author inPubMed Google Scholar * Amie W. Hsia View author publications You can also search for this author inPubMed Google Scholar * Jaclyn Witko View author publications You can also


search for this author inPubMed Google Scholar * Joseph A. Frank View author publications You can also search for this author inPubMed Google Scholar * Lawrence Latour View author


publications You can also search for this author inPubMed Google Scholar * Dorian B. McGavern View author publications You can also search for this author inPubMed Google Scholar


CONTRIBUTIONS P.M. and N.M. performed the data acquisition and analysis. P.M., M.L., A.W.H. and L.L. contributed to the design, acquisition and analysis of clinical data. S.R.B., J.W. and


J.A.F. contributed to optimization of the ultrasound model and performed the mouse MRI studies. K.J. conducted computation analyses of RNA-seq data. P.M. and D.B.M. wrote and edited the


manuscript. D.B.M. supervised and directed the project and participated in data acquisition and analysis. CORRESPONDING AUTHOR Correspondence to Dorian B. McGavern. ETHICS DECLARATIONS


COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION 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. EXTENDED DATA EXTENDED DATA FIG. 1 MODEL OF ULTRASOUND-INDUCED INJURY. 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. EXTENDED DATA FIG. 2


CHARACTERIZATION OF THE CEREBROVASCULAR INJURY MODEL. 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 EXTENDED DATA FIG. 8 EFFECT OF MYELOMONOCYTIC CELLS ON CEREBRAL REPAIR AND ANGIOGENESIS. 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 EXTENDED DATA FIG. 9 QPCR ANALYSIS OF GENES ENCODING FOR ANGIOGENESIS RELATED PROTEINS. 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. EXTENDED DATA FIG. 10 VEGF-EXPRESSING MICROGLIA ARE INVOLVED IN ANGIOGENESIS.


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 INFORMATION SUPPLEMENTARY INFORMATION Supplementary Fig. 1 and Table 1. REPORTING SUMMARY SUPPLEMENTARY TABLE 2 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. SUPPLEMENTARY TABLE 3 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. SUPPLEMENTARY TABLE 4 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. SUPPLEMENTARY TABLE 5 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). SUPPLEMENTARY TABLE 6 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).


SUPPLEMENTARY TABLE 7 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.


SUPPLEMENTARY VIDEO 1 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. SUPPLEMENTARY VIDEO 2 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. SUPPLEMENTARY


VIDEO 3 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. SUPPLEMENTARY VIDEO 4


Microglia form rosettes in response to cerebrovascular injury. Part 1, _Cx3cr1_GFP/+ (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 _Cx3cr1_GFP/+ (green) mouse initiated immediately after injury shows the formation of rosettes within 20 min. Part 3, a representative time lapse from a _Cx3cr1_GFP/+ (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. SUPPLEMENTARY VIDEO 5 Extensive BBB leakage following cerebrovascular injury in microglia-depleted mice. Representative videos from _Cx3cr1_GFP/+


(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 _Cx3cr1_GFP/+ 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. SUPPLEMENTARY VIDEO 6 Purinergic receptor


signaling is responsible for microglial rosette formation. Representative time lapses from _Cx3cr1_GFP/+ (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.


SUPPLEMENTARY VIDEO 7 Transcranial inhibition of CX43 hemichannels increases BBB leakage after cerebrovascular injury. Representative time lapses from _Cx3cr1_GFP/+ (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.


SUPPLEMENTARY VIDEO 8 Enhanced BBB leakage following cerebrovascular injury in GFAP-CreER-_Cx43_fl/fl mice. Part 1, representative time lapses show EB (red) leakage in WT littermate control


(that is, GFAP-CreER-_Cx43_fl/+) versus GFAP-CreER-_Cx43_fl/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-_Cx43_fl/fl mice. Part 2, representative time lapses from littermate control and GFAP-CreER-_Cx43_fl/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-_Cx43_fl/fl mice. Videos are representative of two independent experiments with four mice per


group. SUPPLEMENTARY VIDEO 9 Cerebrovascular injury promotes massive extravasation of peripheral myelomonocytic cells. _LysM_GFP/+ 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. SUPPLEMENTARY VIDEO 10 αLFA1/VLA4 antibodies prevent myelomonocytic cell infiltration into the


lesion core and perimeter. Intravital imaging of the lesion core and perimeter in _LysM_GFP/+ (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. SOURCE DATA SOURCE DATA FIG. 1 Source data for graphs SOURCE DATA FIG. 2 Source data for graphs SOURCE DATA


FIG. 3 Source data for graphs SOURCE DATA FIG. 4 Source data for graphs SOURCE DATA FIG. 5 Source data for graphs SOURCE DATA FIG. 6 Source data for graphs SOURCE DATA EXTENDED DATA FIG. 2


Source data for graphs SOURCE DATA EXTENDED DATA FIG. 3 Source data for graphs SOURCE DATA EXTENDED DATA FIG. 6 Source data for graphs SOURCE DATA EXTENDED DATA FIG. 7 Source data for graphs


SOURCE DATA EXTENDED DATA FIG. 8 Source data for graphs SOURCE DATA EXTENDED DATA FIG. 10 Source data for graphs RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS


ARTICLE 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 Download citation * Received: 02 June 2020 * Accepted: 08 December 2020 * Published: 18 January 2021 * Issue Date: February 2021 * DOI:


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