Abstract
The arachnoid barrier delineates the border between the central nervous system and dura mater. Although the arachnoid barrier creates a partition, communication between the central nervous system and the dura mater is crucial for waste clearance and immune surveillance1,2. How the arachnoid barrier balances separation and communication is poorly understood. Here, using transcriptomic data, we developed transgenic mice to examine specific anatomical structures that function as routes across the arachnoid barrier. Bridging veins create discontinuities where they cross the arachnoid barrier, forming structures that we termed arachnoid cuff exit (ACE) points. The openings that ACE points create allow the exchange of fluids and molecules between the subarachnoid space and the dura, enabling the drainage of cerebrospinal fluid and limited entry of molecules from the dura to the subarachnoid space. In healthy human volunteers, magnetic resonance imaging tracers transit along bridging veins in a similar manner to access the subarachnoid space. Notably, in neuroinflammatory conditions such as experimental autoimmune encephalomyelitis, ACE points also enable cellular trafficking, representing a route for immune cells to directly enter the subarachnoid space from the dura mater. Collectively, our results indicate that ACE points are a critical part of the anatomy of neuroimmune communication in both mice and humans that link the central nervous system with the dura and its immunological diversity and waste clearance systems.
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Data availability
Raw data of all snRNA-seq are available at the Gene Expression Omnibus under accession number GSE213895. Images of Sema3a and Sema3d expression were accessed from the Allen Brain Atlas ISH browser (https://mouse.brain-map.org/). Source data are provided with this paper.
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Acknowledgements
We thank E. Griffin and A. Apaw for animal care; S. Brophy for laboratory management; all of the members of the Kipnis laboratory for feedback and discussions; G. Strout and J. Wulf for sample preparation; the members of the Flow Cytometry and Fluorescence-Activated Cell Sorting Core for sorting nuclei for sequencing; the staff at the Genome Technology Access Center for performing snRNA-seq; the members of the Washington University in St Louis Pathology Core for paraffin embedding and sectioning; G. Randolph, B. Zinselmeyer and A. Junidi for assistance in intravital imaging of cellular trafficking and for discussions; X. Cui and J. Kouranova for the design and generation of the Dpp4-creERT2 and Slc47a1-creERT2 mice; G. Bhagavatheeshwaran and the staff of the NINDS Neuroimmunology Clinic including J. Duyn, P. van Gelderen, J. de Zwart and J. Liu and the members of the NIMH Functional MRI Facility for assistance with human MRI collection; and all of the volunteers for their contribution to the study. This work was supported by grants from the National Institutes of Health/National Institute on Aging (AG034113 and AG078106) and the Cure Alzheimer’s Fund BEE consortium to J.K. This research was also partially supported by the Intramural Research Program of NINDS to D.S.R. (NS003119). S.V.O. is supported by National MS Society (postdoctoral fellowship grant, FG-2208-40289).
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L.C.D.S. designed and performed experiments, analysed and interpreted data, created the figures and wrote the manuscript. D.X. designed and performed experiments, analysed and interpreted data. S.V.O., M.I.G. and G.N. performed MRI experiments, processed and analysed images, and wrote relevant methods sections. T.D. performed the scRNA-seq data analyses and assisted in writing relevant methods. J.R. designed and performed experiments and provided intellectual contributions. Z.P. assisted with tissue clearing, light-sheet imaging and 3D visualization. M.W.K. performed EAE experiments and scored EAE mice in a blinded manner. A.D. assisted with CSF tracer imaging experiments and co-discovered the perivenous CSF efflux route. T.M. designed experiments and provided intellectual contributions. K.B., S.B. and X.G. assisted in collecting, staining and imaging tissues. S.E.S. provided intellectual contributions. K.B. assisted in collecting, staining and imaging tissues. S.D. performed staining and imaging of human dura-arachnoid granulation specimens. M.A.W. assisted in the design and generation of the transgenic mice. P.B. assisted in two-photon imaging. I.S. performed animal surgeries. K.D. assisted with interpretation and analysis of EM images. D.S.R. designed MRI studies, led and provided resources related to MRI studies, and provided intellectual contributions. J.K. designed the experiments, provided resources and intellectual contribution, oversaw data analysis and interpretation, and wrote the manuscript.
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J.K. is a co-founder of Rho Bio.
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Extended data figures and tables
Extended Data Fig. 1 Direct bulk efflux of CSF to the dura mater.
a. Flat mounted dorsal dura mater 60 min after i.c.m. injection of 70 kDa dextran. Scale = 2 mm, inset = 200 μm. b. Flat mounted dorsal dura mater 60 min after i.v. injection of 70 kDa dextran. Scale = 2 mm, inset = 200 μm. c. Ratio of tracer in the sinus and non-sinus regions of the dura mater 60 min after i.v. or i.c.m. injection. Mean ± SEM. N = 5 (i.v.), 7 (i.c.m.) animals, unpaired, two-tailed Student’s t-test. d. Tracer intensity in the sinus regions of the dura mater 60 min after i.v. or i.c.m. injection. Mean ± SEM. N = 5 (i.v.), 7 (i.c.m.) animals, unpaired, two-tailed Student’s t-test. e. Tracer intensity in the non-sinus regions of the dura mater 60 min after i.v. or i.c.m. injection. Mean ± SEM. N = 5 (i.v.), 7 (i.c.m.) animals, unpaired, two-tailed Student’s t-test. f. Evan’s blue was injected i.c.m. and intravital transcranial imaging performed. Intensity of i.c.m. Evan’s blue tracer in perisinus regions. Mean ± SEM. N = 7 animals. g. Evan’s blue was injected i.c.m. and intravital imaging of the cervical lymph nodes performed. Intensity of i.c.m. Evan’s blue tracer in the cervical lymph nodes. Mean ± SEM. N = 9 animals. h. Evan’s blue was injected i.c.m. and intravital imaging of the femoral vein performed. Intensity of i.c.m. Evan’s blue tracer in the femoral vein. Mean ± SEM. N = 8 animals. i. Transit time (time to cross 5% max threshold) of Evan’s blue signal in the perisinus regions, cervical lymph nodes, and femoral vein. Mean ± SEM. N = 7 (perisinus), 8 (femoral vein),9 (blood) animals, one-way ANOVA with Sidak’s multiple comparisons test. Each point represents an animal. j. Concentration of 70 kDa FITC dextran in the brain following i.c.m. injection. Mean ± SEM. N = 5 (0, 60 mins), 6 (2,5, 10, 15, 30 mins) animals. k. Concentration of 70 kDa FITC dextran in the dura following i.c.m. injection. Mean ± SEM. N = 5 (0, 60 mins), 6 (2,5, 10, 15, 30 mins) animals. l. Concentration of 70 kDa FITC dextran in the dcLN following i.c.m. injection. Mean ± SEM. N = 5 (0, 60 mins), 6 (2,5, 10, 15, 30 mins) animals. m. Concentration of 70 kDa FITC dextran in the scLN following i.c.m. injection. Mean ± SEM. N = 5 (0, 60 mins), 6 (2,5, 10, 15, 30 mins) animals. n. Concentration of 70 kDa FITC dextran in serum following i.c.m. injection. Mean ± SEM. N = 5 (0, 60 mins), 6 (2,5, 10, 15, 30 mins) animals. o. Multiple molecular weight i.c.m. tracers around the transverse sinus 30, 60, and 5 min post injection. Scale = 2 mm, inset = 200 μm. p. Rate of appearance of i.c.m. tracers of different molecular weight and chemical properties in the dura mater. Mean ± SEM. N = 4 (5 min), 5 (15, 30, 60 mins) animals. q. Slope of i.c.m. tracers appearing in the dura. Mean ± SEM. N = 4 (5 min), 5 (15, 30, 60 mins) animals, one-way ANOVA with Sidak’s multiple comparisons test.
Extended Data Fig. 2 Molecular mapping of the leptomeningeal stroma.
a. Representative images of stromal populations in dissected leptomeninges including markers of arachnoid barrier cells (ABCs; E-Cadherin), fibroblasts (CD13, Aldh1a2), macrophages (CD206), and vasculature (αSMA, vWF, PECAM-1). Scale = 50 μm. b. Dotplot of marker genes for cell types, with extended characterization of ABCs, coloured by expression level, and scaled by proportion expressing the gene. c. UMAPs of leptomeninges in different conditions, coloured by cell type. EAE = experimental autoimmune encephalomyelitis. d. Representative images of selected markers for ABCs (Cdh1), DBCs (Slc47a1), pial and perivascular fibroblasts (Cemip). Scale = 100 μm, inset = 20 μm. e. Multiplexed IHC imaging of selected markers for ABCs (Msln, E-Cad, Dpp4, Fn1), relative to the pial basement membrane (pan-laminin). Scale = 100 μm, inset = 20 μm. f. 23-plex IHC imaging of glial markers (Aqp4, GFAP, Iba1), macrophage markers (Lyve1, CD206, Iba1), vascular markers (Cdh5, vWF, αSMA, Cldn5, Podxl), and leptomeningeal stroma (pan: CD13, Pdpn; ABCs: Dpp4; DBCs: Slc38a2; Pial/arachnoid fibroblasts: Collagen I, Slc6a13). Scale = 4 mm, inset = 100 μm. g. Representative images of colocalization of a novel ABC marker (Dpp4) with known ABC marker epithelial membrane antigen (EMA) in human dura-arachnoid granulation sections. Scale = 2 mm, insets = 200 μm. h. UMAP of subclustered pial/arachnoid fibroblasts (11,092 nuclei). i. Marker genes for pial, arachnoid, and interferon (IFN)-responsive fibroblast clusters. j. Ontologies enriched in marker genes for each pial/arachnoid fibroblast subcluster. Enriched gene ontologies were calculated in ENRICHR using the Fisher exact test. k. RNAscope staining of pial/perivascular fibroblasts (Cemip), arachnoid fibroblasts (Rspo2), and ABCs (Cdh1). FB = fibroblasts, PV = perivascular. Scale = 100 μm, inset = 200 μm. l. UMAP of subclustered ABCs (1,871 nuclei). m. Ontologies enriched in marker genes for each ABC subcluster. Enriched gene ontologies were calculated in ENRICHR using the Fisher exact test. n. Enrichment of cell type-specific gene sets from the Descartes cell type atlas in ABCs, coloured by the cell class. Enriched gene ontologies were calculated in ENRICHR using the Fisher exact test. o. Leptomeningeal cell populations coloured by expression score for core mesothelial genes. p. Leptomeningeal cell populations coloured by expression score for core epithelial genes. q. Leptomeningeal cell populations coloured by expression score for core fibroblast genes.
Extended Data Fig. 3 Additional characterization of leptomeningeal stroma.
a. Electron micrographs of leptomeninges following i.c.m. injection of the electron microscopy (EM)-detectable tracer HRP. Representative images of the vesicle uptake by the major stromal cell types. ABC = arachnoid barrier cell. A-FB = arachnoid fibroblast. P-FB = pial fibroblast. Astro. = astrocyte. Arrowheads indicate HRP-positive vesicles, arrows indicate collagen bundles. Scale = 1 μm, inset = 200 nm. b. Density of HRP-positive vesicles in ABCs and leptomeningeal endothelial cells (ECs, including both arterial (aECs) and venous ECs (vECs)). Mean ± SEM. N = 5 animals, unpaired, two-tailed Student’s t-test. Arrowheads indicate HRP-positive vesicles. Arrows indicate collagen bundles. c. Negative stain EM of vesicles in ABCs, leptomeningeal endothelial cells, and capillary endothelial cells (cECs). Arrowheads indicate vesicles. Scale = 250 nm. d. Quantification of the density of vesicles in ABCs, leptomeningeal endothelial cells (aECs and vECs pooled), and capillary endothelial cells (cECs). Mean ± SEM. N = 5 animals, one-way ANOVA with Sidak’s multiple comparisons test. e. Intravital two-photon imaging of uptake of i.c.m. tracers in Cx3cr1-EGFP mice. Arrowhead indicates i.v. tracer-positive macrophage. Scale = 100 μm, inset = 20 μm. f. Experimental design and confocal images of vesicular uptake of i.c.m. 70 kDa dextran by macrophages (Cx3cr1, CD206) but not ABCs (E-Cad) in the leptomeninges of wild type and Cx3cr1-EGFP mice. Arrowheads indicate macrophages taking up tracer. Scale = 20 μm. g. Dotplot of unique and shared junction components in ABCs, vECs, and aECs, coloured by expression level, and scaled by proportion expressing the gene. h. Representative images of ABC tight junctions in negative stain EM, and i.c.m. HRP injected animals. Arrowheads indicate tight junctions. Scale = 100 nm, inset = 50 nm. i. Flat mounted leptomeninges from approximately one dorsal hemisphere, showing ubiquitous continuous E-Cad-positive junctions between ABCs. Arrowheads indicate continuous adherens junctions. Scale = 2 mm, inset = 20 μm. j. Tight junctions formed between ABCs, including the ABC-specific junctional marker Cldn11. Colocalization with the pan-tight junction marker Ocln and ABC adherens junction marker E-Cad are shown. Scale = 25 μm, inset = 2 μm. The diagrams in a and f were created using BioRender.
Extended Data Fig. 4 Additional characterization of leptomeninges targeting mice.
a. Confocal imaging of Prox1-EGFP and Cdh5-CreERT2::tdTom brains co-labelled with markers for ABCs (E-Cad, Dpp4), Fibroblasts (CD13, Aldh1a2), and the pial basement membrane (pan-laminin). Brain = 2 mm, scale = 50 μm, inset = 10 μm. b. Dotplot of Prox1 and Cdh5 expression in leptomeningeal cell types, coloured by expression level, and scaled by percentage expression. c. UMAP of leptomeningeal cells, coloured by expression level of Prox1 and Cdh5. d. Gating strategy and representative histograms of reporter expression in CD45−/CD31−/Pdpn+ fibroblasts. e. Efficiency of Cdh5-CreERT2::tdTom and Prox1-EGFP for targeting fibroblasts and endothelial cells in the leptomeninges and dura. Mean ± SEM. N = 5 animals, unpaired, two-tailed Student’s t-test. f. Representative images of the brain, lung, small intestine, heart, and liver of Dpp4-CreERT2::zsGreen mice. Scale = 2 mm, inset = 200 μm. g. zsGreen coverage in the leptomeninges of Dpp4-CreERT2::zsGreen. Mean ± SEM. N = 10 animals. h. qPCR of Dpp4 gene expression in the leptomeninges of Dpp4-CreERT2 negative and heterozygous mice. Mean ± SEM. N = 6 animals, unpaired, two-tailed student’s t-test. i. Representative images of the brain, lung, small intestine, heart, and liver of Slc47a1-CreERT2::tdTom mice. Scale = 2 mm, inset = 200 μm. j. tdTomato coverage in the leptomeninges of Slc47a1-CreERT2::tdTom. Mean ± SEM. N = 7 animals. k. qPCR of Slc47a1 gene expression in the leptomeninges of Slc47a1-CreERT2 negative and heterozygous mice. Mean ± SEM. N = 4 (Cre negative) 6 (Cre positive) animals, unpaired, two-tailed student’s t-test. l. Intravital two-photon imaging of i.c.m. tracers in Dpp4-CreERT2::zsGreen mice. Arrowheads indicate i.v. tracer-positive cells, arrows indicate arachnoid barrier. Scale = 100 μm, inset = 20 μm.
Extended Data Fig. 5 Mapping the anatomy and stroma of ACE points.
a. RNAscope for the pial/perivascular fibroblast (FB) marker Cemip at an arachnoid cuff exit (ACE) point in a flat mounted dura mater. Scale = 200 μm. b. RNAscope for the dural border cell (DBC) marker Slc47a1 at an ACE point in a flat mounted dura mater. Scale = 200 μm. c. Immunostaining for the arachnoid fibroblast marker Crabp2 at an ACE point in a flat mounted dura mater. Scale = 200 μm. d. Immunostaining for the vascular smooth muscle marker αSMA at an ACE point in a flat mounted dura mater. Scale = 200 μm. e. Prox1-positive fibroblasts (FBs) and lymphatic vessels around an ACE point in a Prox1-EGFP mouse. Scale = 200 μm. f. Matched stereomicroscopy and two-photon intravital imaging of an ACE point around a bridging vein in a Prox1-EGFP mouse. Arrowheads indicate bridging veins. Scale = 2 mm, inset = 200 μm. g. Matched stereomicroscopy and two-photon intravital imaging of an ACE point around a bridging vein in a Dpp4-CreERT2::tdTomato mouse. Arrowheads indicate bridging veins. Scale = 2 mm, inset = 200 μm. h. Volume rendering and diagram of an ACE point in a Cdh5-CreERT2::tdTom mouse. Scale = 200 μm. i. Volume rendering and diagram of an ACE point in a Prox1-EGFP mouse. Scale = 200 μm. j. Volume rendering and diagram of an ACE point in Dpp4-CreERT2::zsGreen mice. Scale = 200 μm. k. Volume rendering and diagram of an ACE point in Slc47a1-CreERT2::tdTom mice. Scale = 200 μm. l. Transition of endothelial phenotype at ACE points. Staining for BBB endothelial marker GLUT-1 to peripheral endothelial marker PLVAP at ACE points. Arrowheads indicate bridging veins. Scale = 200 μm. m. Transition of endothelial phenotype at ACE points. Staining for BBB endothelial tight junction Cldn5, and pan-endothelial junction marker PECAM-1 at an ACE point. Scale = 200 μm, inset = 100 μm, high-magnification = 10 μm. n. Line profile of normalized GLUT1 and E-Cad intensity on bridging veins through the ACE point. N = 27 bridging veins across 5 animals, mean ± SEM of bridging veins.
Extended Data Fig. 6 Additional characterization of CSF efflux at ACE points.
a. Experimental design for examination of CSF tracer accumulation around bridging veins in the dura mater. b. Progressive OVA accumulation around the bridging veins and in the dura mater following i.c.m. injection. Scale 200 μm. c. Progressive dextran (3 kDa) accumulation around the bridging veins and in the dura mater following i.c.m. injection. Scale 200 μm. d. Schematic and map of ACE points across healthy wild-type mice. N = 14 animals, each colour represents the complement of ACE points from a different animal. e. Experimental design for correlative transcranial and two-photon imaging of CSF efflux at ACE points. f. Correlative stereomicroscopy and two-photon imaging of CSF tracer efflux at ACE points in Cdh5-CreERT2::tdTom mice. Scale = 100 μm. g. Correlative stereomicroscopy and two-photon imaging of CSF tracer efflux at ACE points in Dpp4-CreERT2::zsGreen mice. Scale = 100 μm. h. Two-photon intravital imaging of i.c.m. dextran flow to the dura mater through an ACE point in Prox1-EGFP mice. Scale = 200 µm. i. CSF tracer accumulation around bridging veins near the rostral rhinal vein (above the olfactory bulb) in cleared heads. Scale = 1 mm. j. CSF tracer accumulation around bridging veins in tentorial folds of the dura mater. Arrowheads indicate i.c.m.-tracer around tentorial bridging veins. Scale = 1 mm. k. Flat mounted dura mater highlighting ACE points near the rostral rhinal vein and transverse sinus hot-spots (yellow arrowheads), as well as ACE points elsewhere in the dura mater (white arrowheads). Scale = 2 mm, inset = 200 μm. l. Matched intravital transcranial imaging of i.c.m.-injected AlexaFluor-647-labelled bovine serum albumin (BSA) and ex vivo fixed dura mater. Note that the same bridging veins which show CSF flow (yellow arrowheads) in the intravital imaging experiment show tracer labelling while those that did not do not (white arrowheads). Scale = 2 mm, inset = 200 μm. m. Volume renderings of cleared tentorial regions underlying the transverse sinus hot-spots following i.c.m. injection of 70 kDa dextran. Yellow arrowheads depict ACE points with tracer enhancement while white arrowheads depict those without. Scale = 2 mm, inset = 200 μm. The diagrams in a, e, h, i and j were created using BioRender.
Extended Data Fig. 7 Additional characterization of influx of intravenous and dural tracers at ACE points.
a. Experimental paradigm for examining the distribution of i.v. tracers on the brain surface. b. i.v. dextran on the surface of the brain. Scale = 2 mm, inset = 200 μm. c. i.v. dextran in the subarachnoid space and around leptomeningeal vasculature on the surface of the brain. Scale = 200 μm. d. Images of i.v. dextran on the surface of the brain, in the cortex, choroid plexus, hippocampus, and cerebellum 60 min following i.v. injection through either the tail vein or retroorbital route. Scale = 2 mm, inset = 200 μm. e. Intensity of i.v. dextran on the surface of the brain 60 min following i.v. injection through either the tail vein or retroorbital route. Mean ± SEM. N = 5 animals, two-tailed, unpaired Student’s t-test. f. Area of i.v. dextran on the surface of the brain 60 min following i.v. injection through either the tail vein or retroorbital route. Mean ± SEM. N = 5 animals, two-tailed, unpaired Student’s t-test. g. Area of i.v. dextran in the brain 60 min following i.v. injection through either the tail vein or retroorbital route. Mean ± SEM. N = 5 animals, two-tailed, unpaired Student’s t-test. h. Intensity of i.v. dextran in the brain 60 min following i.v. injection through either the tail vein or retroorbital route. Mean ± SEM. N = 5 animals, two-tailed, unpaired Student’s t-test. i. Representative images of biotin staining in flat mounted dura mater from negative control and transcranial biotin tracer duras. Arrowheads represent tracer-positive bridging veins. Scale = 4 mm. j. Quantification of the intensity of biotin tracer in the dura mater and on the dorsal brain surface. Mean ± SEM. N = 3 animals, unpaired two-tailed Student’s t-test. k. Representative images of biotin tracer signal in the leptomeninges of sagittal brain sections. Scale = 1 mm, inset = 100 μm. l. High-resolution image of transcranially applied biotin tracer around a bridging vein in the dorsal dura mater. Scale = 100 μm. Light sheet imaging of transcranially applied biotin and around a bridging vein in a cleared brain. Scale = 100 μm. m. Confocal images of transcranial biotin labelling associated with surface and deep veins, surface arteries, and absent in the capillary bed ten minutes after tracer administration. Scale = 200 μm. n. Stereomicroscopy and sagittal sections of the brain following transcranial application of sulfo-NHS-biotin. Arrowheads indicate enhancement around bridging veins. Scale = 2 mm. o. Intensity of biotin signal in the brain surface in stereomicroscopy images. Mean ± SEM. N = 4 animals, one-way ANOVA with Dunnett’s post-hoc test. 0 v 10 min, p = 0.010, 0 vs 60 min, p < 0.0001. p. Intensity of biotin signal in the leptomeninges, cortex, and choroid plexus in sagittal sections. Mean ± SEM. N = 4 animals, two-way ANOVA with Tukey’s post-hoc test. 0 v 5 min, p = 0.0048, 0 v 10 min, p < 0.0001, 0 vs 60 min, p < 0.0001. The diagram in a was created using BioRender.
Extended Data Fig. 8 Stromal Sema3a restrains myeloid infiltration to the CNS.
a. Volcano plot of differences in gene expression in arachnoid barrier cells (ABCs) in aged mice (20 month), compared to control ABCs (2 month). Benjamini-Hochberg’s adjustment was used to calculate multiplicity-adjusted p-values from from unpaired two-tailed t-tests. b. Selected significantly enriched gene ontologies in differentially expressed genes (DEGs) in control and aged ABCs. The number of DEGs as a fraction of the total gene ontology is given. Enriched gene ontologies were calculated in ENRICHR using the Fisher exact test. c. Images of the BBB marker GLUT-1 in bridging veins in young and aged mice. Scale = 200 μm. d. Expression of GLUT-1 within ACE points in young and aged mice. Mean ± SEM. N = 4 (aged), 5 (young), unpaired two-tailed Student’s t-test. e. Representative gating strategy for leptomeningeal and dural immune populations. Frequency of immune populations in the leptomeninges (lepto) and dura mater of healthy control mice. Mean ± SEM. N = 13 (lepto and dura B cells, cDCs, CD4, CD8), N = 15 (lepto monocytes), N = 18 (macrophages, lepto neutrophils, dura monocytes), N = 21 (dura neutrophils) animals, unpaired two-tailed Student’s t-test. f. ELISA of leptomeningeal Sema3a and Sema3d in healthy wild type mice. Mean ± SEM. N = 4 (Sema3a), 5 animals. g. ELISA of CSF Sema3a and Sema3d in healthy wild type mice. Mean ± SEM. N = 5 animals. h. Representative images of Sema3a and Sema3d ISH in the healthy brain taken from the Allen Brain Atlas. Scale = 1 mm, inset = 100 μm. i. Experimental design to test the effect of semaphorins on monocyte migration in vitro. j. Relative CCL2-induced monocyte transmigration in the presence of Sema3a or Sema3d. Mean ± SEM. N = 5, one-way ANOVA with Sidak’s multiple comparison test. *** - p < 0.001 vs untreated control, # - p < 0.05 vs CCL2-treated, ## - p < 0.01 vs CCL2-treated. Each point represents cells harvested from one animal. k. Representative images of GFP+ perivascular, leptomeningeal, and dural fibroblasts 21 days following i.c.m. injection of AAV8-GFP (2 ×1012 GC) in Col1a2-CreERT2::tdTom mice. Brain, dura = 2 mm, scale = 100 μm. l. Representative images of GFP+ dural border cells on the brain and dura 21 days following i.c.m. injection of AAV8-GFP (2 ×1012 GC) in Slc47a1-CreERT2::tdTom mice. Brain, dura = 2 mm, scale = 100 μm. The diagram in i was created using BioRender.
Extended Data Fig. 9 Changes to stromal and immune cells in the leptomeninges during EAE and aging.
a. Volcano plot of differences in gene expression in arachnoid barrier cells (ABCs) in experimental autoimmune encephalomyelitis (EAE), compared to control ABCs. Benjamini-Hochberg’s adjustment was used to calculate multiplicity-adjusted p-values from unpaired two-tailed t-tests. b. Representative images of GR-1-positive myeloid cells within ACE points in a flat mount of the spinal cord dura mater and leptomeninges of a mouse 17 days following EAE induction. Arrowheads indicate GR-1 positive myeloid cells within the perivascular region of the ACE point. Scale = 100 μm. c. Volcano plot of differences in gene expression in pial/arachnoid fibroblasts in EAE, compared to control pial/arachnoid fibroblasts. Benjamini-Hochberg’s adjustment was used to calculate multiplicity-adjusted p-values from unpaired two-tailed t-tests. d. Selected significantly enriched gene ontologies in differentially expressed genes (DEGs) in control and EAE pial/arachnoid fibroblasts. Enriched gene ontologies were calculated in ENRICHR using the Fisher exact test. e. Volcano plot of differences in gene expression in dural border cells (DBCs) in EAE, compared to control DBCs. Benjamini-Hochberg’s adjustment was used to calculate multiplicity-adjusted p-values from unpaired two-tailed t-tests. f. Selected significantly enriched gene ontologies in differentially expressed genes (DEGs) in control and EAE DBCs. Enriched gene ontologies were calculated in ENRICHR using the Fisher exact test. g. Fold changes in cell abundances in snRNA-seq from control and EAE leptomeninges, with significant differences highlighted. Mean ± 95 % CI. Data were analysed with a permutation test of 1000 iterations to obtain the p-value and bootstrapping to obtain the confidence interval. h. Gating strategy to examine changes to immune cell populations in EAE. i. Frequency of CD45+ immune cells, as a percentage of live cells, in control and EAE leptomeninges. Mean ± SEM. N = 5 animals, unpaired two-tailed Student’s t-test. j. Frequency of CD4+ T cells, as a percentage of CD45+ cells, in control and EAE leptomeninges. Mean ± SEM. N = 5 animals, unpaired two-tailed Student’s t-test. k. Frequency of Ror-γt+ T helper 17 (Th17), as a percentage of CD4+ T cells, in control and EAE leptomeninges. Mean ± SEM. N = 5 animals, unpaired two-tailed Student’s t-test. l. Density of CD3+ T cells around ACE points, sinus regions, and non-sinus regions in the dura of control and EAE mice. Mean ± SEM. N = 3 (sham control), 8 (EAE) animals, two-way ANOVA with Sidak’s multiple comparison adjustment. m. Frequency of MHC-II+/CD38− macrophages, as a percentage of CD206+ leptomeningeal macrophages, in control and EAE leptomeninges. Mean ± SEM. N = 5 animals, unpaired two-tailed Student’s t-test. n. Frequency of MHC-II−/CD38+ macrophages, as a percentage of CD206+ leptomeningeal macrophages (Macs), in control and EAE leptomeninges. Mean ± SEM. N = 5 animals, unpaired two-tailed Student’s t-test. o. Frequency of neutrophils in control and EAE leptomeninges. Mean ± SEM. N = 5 animals, unpaired two-tailed Student’s t-test. p. Frequency of Ly-6Chi monocytes in control and EAE leptomeninges. Mean ± SEM. N = 5 animals, unpaired two-tailed Student’s t-test. q. Representative images of GR-1-positive myeloid cells within ACE points in a flat mount of the spinal cord dura mater and leptomeninges of a mouse 17 days following EAE induction. Arrowheads indicate GR-1 positive myeloid cells within the perivascular region of the ACE point. Scale = 100 μm. r. Representative images of CD3+ T cells within ACE points in a flat mount of the cranial dura mater of a mouse 17 days following EAE induction. Arrowheads indicate T cells within the perivascular region of the ACE point. Scale = 200 μm, inset = 20 μm.
Extended Data Fig. 10 Itga6-laminin interactions govern the entry of immune cells to the CNS around ACE points.
a. Laminin immunolabeling around ACE points and in non-sinus regions of the dura mater. Scale = 200 μm. b. Quantification of laminin staining intensity in non-sinus dura, sinus, and ACE points. Mean ± SEM. N = 5 animals, one-way ANOVA with with Sidak’s multiple comparison adjustment. c. Dotplot of laminin genes in stromal populations of the leptomeninges from single nuclear RNA-sequencing, scaled by percentage of cells expressing a given gene, and its expression level. d. Experimental paradigm for treatment of mice with anti-Itga6 antibodies in EAE and clinical scores of mice treated with isotype control, or anti-Itga6 antibody during EAE induction. Mean ± SEM. N = 6 (anti-Itga6), 9 (isotype) animals, two-way repeated measures ANOVA with Sidak’s multiple comparison adjustment. e. Representative images of CD45 and S100A8 staining in spinal cord sections in isotype control and anti-Itga6 treated mice at the peak of EAE (day 17). Scale = 1 mm, inset = 100 μm. f. Density of CD45+ area in the spinal cord of isotype control and anti-Itga6 treated mice at the peak of EAE (day 17). Mean ± SEM. N = 10 animals, two-tailed, unpaired Student’s t-test. g. Density of S100A8+ neutrophils in the spinal cord of isotype control and anti-Itga6 treated mice at the peak of EAE (day 17). Mean ± SEM. N = 10 animals, two-tailed, unpaired Student’s t-test. h. Representative gating strategy for immune cells in the blood, spinal cord, and dura of EAE mice. i. Ly-6Chi monocyte frequency in the blood, spinal cord, and dura of EAE mice treated with either isotype control or anti-Itga6 antibody at the peak of EAE (day 17). Mean ± SEM. N = 10 (blood, spinal dura), 15 (spinal cord) animals, two-tailed, unpaired Student’s t-test. j. Neutrophil frequency in the blood, spinal cord, and dura of EAE mice treated with either isotype control or anti-Itga6 antibody at the peak of EAE (day 17). Mean ± SEM. N = 10 (blood, spinal dura), 15 (spinal cord) animals, two-tailed, unpaired Student’s t-test. k. Staining for S100A8-positive neutrophils around bridging veins in isotype control and anti-Itga6 treated mice at the peak of EAE (day 17). Scale = 200 μm. l. Density of neutrophils around bridging veins in isotype control and anti-Itga6 treated mice at the peak of EAE (day 17). Mean ± SEM. N = 10 animals, two-tailed, unpaired Student’s t-test. m. T cell frequency in the blood, spinal cord, and dura of EAE mice treated with either isotype control or anti-Itga6 antibody at the peak of EAE (day 17). Mean ± SEM. N = 10 (blood, spinal dura), 15 (spinal cord) animals, two-tailed, unpaired Student’s t-test. n. CD4 T cell frequency in the blood, spinal cord, and dura of EAE mice treated with either isotype control or anti-Itga6 antibody at the peak of EAE (day 17). Mean ± SEM. N = 10 (blood, spinal dura), 15 (spinal cord) animals, two-tailed, unpaired Student’s t-test. o. Staining for CD3-positive T cells around bridging veins in isotype control and anti-Itga6 treated mice at the peak of EAE (day 17). Scale = 200 μm. p. Density of CD3-positive T cells around bridging veins in isotype control and anti-Itga6 treated mice at the peak of EAE (day 17). Mean ± SEM. N = 10 animals, two-tailed, unpaired Student’s t-test. The diagram in d was created using BioRender.
Supplementary information
Supplementary Figure 1
Full western blot images and legend relating to Fig. 3e.
Supplementary Video 1
Intravital imaging of the appearance of i.c.m. injected Evan’s blue on the dorsal surface of the brain, cervical lymph nodes and femoral vein. Related to Extended data Fig. 1f–i.
Supplementary Video 2
Uptake of i.c.m. tracers in Cx3cr1-eGFP mice. Related to Extended data Fig. 3e.
Supplementary Video 3
No uptake of i.c.m. tracers by ABCs in Dpp4-creERT2::zsGreen mice. Related to Extended data Fig. 4l.
Supplementary Video 4
Volume rendering of a bridging vein exiting the subarachnoid space into the dura in a Cdh5-creERT2::tdTom mouse. Related to Extended data Fig. 5h.
Supplementary Video 5
Volume rendering of a bridging vein exiting the subarachnoid space into the dura in a Prox1-eGFP mouse. Related to Extended data Fig. 5i.
Supplementary Video 6
Volume rendering of a bridging vein exiting the subarachnoid space into the dura in a Dpp4-creERT2::tdTom mouse. Related to Extended data Fig. 5j.
Supplementary Video 7
Volume rendering of a dural border cells in proximity to an ACE point in a Slc47a1-creERT2::tdTom mouse. Related to Extended data Fig. 5k.
Supplementary Video 8
Transcranial imaging of perivenous flow of i.c.m. injected tracers. Related to Fig. 2d.
Supplementary Video 9
Two-photon imaging of perivenous efflux of i.c.m. injected tracers into the dura in a Dpp4-creERT2::zsGreen mouse. Related to Fig. 2e.
Supplementary Video 10
Two-photon imaging of perivenous efflux of i.c.m. injected tracers into the dura in a Prox1-eGFP mouse. Related to Extended data Fig. 6h.
Supplementary Video 11
Volume rendering of i.c.m. ovalbumin signal in around bridging veins draining into the rostral rhinal vein near the superior sagittal sinus in the dura mater. Related to Extended data Fig. 6i.
Supplementary Video 12
Two-photon imaging of myeloid cell migration in bridging vein and non-bridging vein-associated regions at the peak of EAE in an Ms4a3-cre::zsGreen mouse. Related to Fig. 5j.
Supplementary Video 13
Two-photon imaging of myeloid cell migration in bridging vein and non-bridging vein-associated regions at the peak of EAE in an Ccr2-RFP::Prox1-eGFP mouse. Related to Fig. 5n.
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Smyth, L.C.D., Xu, D., Okar, S.V. et al. Identification of direct connections between the dura and the brain. Nature 627, 165–173 (2024). https://doi.org/10.1038/s41586-023-06993-7
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DOI: https://doi.org/10.1038/s41586-023-06993-7
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