Innate immune cells recruited to inflammatory sites have short life spans and originate from the marrow, which is distributed throughout the long and flat bones. While bone marrow production and release of leukocyte increases after stroke, it is currently unknown whether its activity rises homogeneously throughout the entire hematopoietic system. To address this question, we employed spectrally resolved in vivo cell labeling in the murine skull and tibia. We show that in murine models of stroke and aseptic meningitis, skull bone marrow-derived neutrophils are more likely to migrate to the adjacent brain tissue than cells that reside in the tibia. Confocal microscopy of the skull–dura interface revealed myeloid cell migration through microscopic vascular channels crossing the inner skull cortex. These observations point to a direct local interaction between the brain and the skull bone marrow through the meninges.
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The authors thank M. Ericsson (HMS Electron Microscopy Facility) for skull sample preparation, sectioning, and assistance with EM imaging. We acknowledge D. Capen (Center for Systems Biology and Program in Membrane Biology/Division of Nephrology, MGH) for help with interpretation of electron microscopy data. The authors thank the MGH mouse imaging program and the Center for Skeletal Research Core (NIH P30 AR066261) for assistance with imaging. This work was funded in part by grants from the National Institutes of Health (NS084863 and HL139598), the American Heart Association (16SDG30190009), the Cure Alzheimer’s Fund, the Global Research Lab (GRL) program (NRF-2015K1A1A2028228) of the National Research Foundation by the Korean government, and by fellowships from the Netherlands Organisation for Scientific Research (NWO, Rubicon Grant: 835.15.014), the Deutsche Forschungsgemeinschaft (RO5071/1-1), and the MGH Research Scholar program.
The authors declare no competing interests.
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Integrated supplementary information
a, Neutrophil recruitment to the brain after intravenous or marrow injection of cell tracker. Two-tailed Mann-Whitney test, naive (n = 7 IV, n = 6 skull tag, 4 experiments), P = 0.52; stroke 6hrs (n = 3 IV, n = 8 skull tag, 3 experiments), P = 0.28; stroke 24hrs (n = 3 IV, n = 6 skull tag, 3 experiments), P = 0.71; b, Viability (upper panel) and cellularity (lower panel) of bone marrow after intravenous or marrow injection of the red (skull) and green (tibia) dyes in naive mice (n = 7 IV, n = 8 Tag, 6 experiments). Two-tailed Mann Whitney test, viability skull, P = 0.01; viability tibia, P = 0.02; cellularity skull P > 0.99; cellularity tibia, P = 0.61. c, Gating for microglia and uptake of red cell tracker 4 hrs after cisternal carrageenan injection (single experiment), after subdural injection of 10 µl (upper left), no tracker (bottom left), 10 µl in the calvarium (upper right), IV injection of 10 µl (bottom right). Data are mean ± s.e.m.. Mann-Whitney test, ns indicates not significant. d, Brain sections of a naive Cx3cr1GFP mouse, after 10 µl red cell tracker injected in the sub-dural area (upper panel, arrow shows injection site) or locally in the skull marrow (lower panel), single experiment.
Bottom right panel shows gates for cells originating from tibia (green tracker, FITC channel) and skull (red tracker, APC channel) based on the signal obtained in the circulation after IV injection (bottom left panel).
a-c, Frequency of cells tracked from skull and tibia marrow in (a) stroke, 6hrs, n = 11, 5 experiments; 1 day, n = 13 for brain and spleen and n = 12 for blood, 5 experiments; 2 days, n = 7, 2 experiments; skull ***P = 0.001 and **P = 0.002 at 6hrs, *P = 0.022, *P = 0.03 at day 1; *P = 0.03, ns P = 0.22 at 2 days; tibia ns P = 0.46 at 6hrs, ns P = 0.69 at day 1, (b) carrageenan, n = 6, 4 experiments, Kruskall Wallis test, skull P = 0.51, tibia ns P = 0.65, (c) myocardial infarction, n = 5, 1 experiment, Kruskall Wallis test skull ns P = 0.11, tibia ns P = 0.32. d-f, Frequency of tracked cells in respective organs relative to the circulation after (d) stroke, at 6 hrs, n = 11, 5 experiments, ***P = 0.002, 1 day, n = 12, 5 experiments, P = 0.064 and 2 days, n = 5, 2 experiments, ns P = 0.13, (e) carrageenan injection, n = 6, 4 experiments and (f) after myocardial infarction, n = 5, 1 experiment. Data are mean ± s.e.m., ns indicates not significant, two-tailed Wilcoxon test unless otherwise specified.
Cells originating from skull (red, arrow) and tibia (green, arrow head) in brain one day after induction of aseptic meningitis (n = 2 mice). Cells are outside (a,b,d) or inside (c) the vasculature. Speckles in c are present in all channels and present autofluorescence. Collapsed Z stacks show cells at a depth of 20-100 µm below the brain tissue surface.
N = 12 per group, 3 experiments. Data are mean ± s.e.m. Two-tailed Mann-Whitney test, neutrophils, P = 0.29 and Ly6Chi monocytes, P = 0.052.
Supplementary Figure 6 Competitive in vitro transmigration assay through activated endothelium comparing skull and tibial neutrophils.
Cells from 6 mice (6 skull-tibia pairs, red or green fluorescence staining for location-specific tracking) were subjected to migration through a TNFα-activated brain endothelium towards fMLP. Results are migrated cells as % of initial population. Data are mean ± s.e.m.. P = 0.69, two-tailed Wilcoxon test.
Dural vasculature one day after stroke induced by permanent occlusion (representative of two experiments). Arrows show cells inside vasculature, arrow heads indicate cells outside of vasculature.
Supplementary Figures 1–7
Supplementary Video 1 - Neutrophils exiting a channel in vitro after intracisternal carrageenan injection.
Representative of 4 experiments. Channel exits are indicated with arrows. Dashed arrows indicate meningeal blood vessels linked to the channel. The bone marrow cavity is indicated by an asterisk. Scale bar indicates 50µm.
In vivo imaging of a channel crossing the interior skull cortex in a 4 week old mouse one day after stroke (single experiment). Arrow indicates the cell’s direction towards the brain. Scale bar indicates 50µm.
As in supplementary movie 2, but 6hrs after permanent middle cerebral artery occlusion (single experiment). Scale bar indicates 50µm.
This movie was obtained in vitro from a sham control and shows no cell exit. Representative of 4 experiments. Scale bar indicates 50µm. Arrow indicates the exit of the channel at the dural. The asterisk indicates skull bone marrow.
As Supplementary movie 4, recorded in vitro after stroke. Scale bar indicates 50µm.
Animation of a high resolution data set shows mouse skull surface rendering (single experiment). Toward the end of the movie, arrows indicate microscopic channels.
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Herisson, F., Frodermann, V., Courties, G. et al. Direct vascular channels connect skull bone marrow and the brain surface enabling myeloid cell migration. Nat Neurosci 21, 1209–1217 (2018). https://doi.org/10.1038/s41593-018-0213-2
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