Meningeal lymphatic vessels at the skull base drain cerebrospinal fluid

Abstract

Recent work has shown that meningeal lymphatic vessels (mLVs), mainly in the dorsal part of the skull, are involved in the clearance of cerebrospinal fluid (CSF), but the precise route of CSF drainage is still unknown. Here we reveal the importance of mLVs in the basal part of the skull for this process by visualizing their distinct anatomical location and characterizing their specialized morphological features, which facilitate the uptake and drainage of CSF. Unlike dorsal mLVs, basal mLVs have lymphatic valves and capillaries located adjacent to the subarachnoid space in mice. We also show that basal mLVs are hotspots for the clearance of CSF macromolecules and that both mLV integrity and CSF drainage are impaired with ageing. Our findings should increase the understanding of how mLVs contribute to the neuropathophysiological processes that are associated with ageing.

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Fig. 1: Basal mLVs have distinct morphologic features and an anatomic location that is more suitable for CSF uptake and drainage than dorsal mLVs.
Fig. 2: Dynamic contrast-enhanced MRI reveals rapid clearance of CSF mainly through the basal outflow.
Fig. 3: Basal mLVs are hotspots for CSF macromolecular clearance.
Fig. 4: Basal mLVs become lymphoedematous and CSF clearance is delayed with ageing.

Data availability

Source data for quantifications shown in all graphs plotted in figures and extended data figures are available in the online version of the paper. The data sets generated in this study are also available from the corresponding authors upon reasonable request.

Code availability

Custom code used during the current study are also available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank all members of IBS Center for Vascular Research, especially J. Bae and H.-T. Kim for technical support and animal care; K. Alitalo (University of Helsinki) for Vegfr3flox/flox mice; and T. Mäkinen (Uppsala University) for Prox1–CreERT2 mice. This study was supported by the Institute of Basic Science and funded by the Ministry of Science and ICT, South Korea (IBS-R025-D1-2015 to G.Y.K.).

Author information

J.H.A. designed the study, performed the experiments, interpreted the data, and wrote the manuscript; H.C. designed the study, analysed the data, generated the figures, and wrote the manuscript; J.-H.K. and J.-S.H. carried out MRI experiments and analysed the data; S.H.K. performed physiologic monitoring and lateral ventricle infusion for MRI; I.P. revised the manuscript; S.H.S. and S.P.H. helped with the experiments; J.-H.S. designed the study; Y.-K.H. provided materials and interpreted the data; Y.J. and S.-H.P. supervised MRI experiments and interpreted the data; G.Y.K. designed the study, interpreted the data, wrote the manuscript, and directed the project.

Correspondence to Sung-Hong Park or Gou Young Koh.

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The authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Basal mLVs contain valves and have more intraluminal T cells than dorsal mLVs.

a, Schematic images depicting the characteristic structure of the dorsal mLVs around the SSS, transverse sinus, and COS (left) and their discontinuous lymphatic vascular structure (right). b, Representative images of dorsal mLVs around the SSS and COS; they are relatively small in diameter with disconnected vessel structure (white arrows). White dashed boxes are magnified below. Similar findings were observed in n = 6 mice from three independent experiments. Scale bars, 100 µm. c, d, Representative images of dorsal mLVs around the transverse sinus and COS. Discontinuous dorsal mLVs (white arrows) running along the transverse sinus have few protruding mLV branches (c), which are devoid of distinct PROX1high lymphatic valves (d). Similar findings were observed in n = 5 mice from two independent experiments. Scale bars, 500 µm (c); 200 µm (d). e, Schematic images depicting features of basal mLVs around the PSS, sigmoid sinus, PSF, SMF, jugular foramen (JF), and middle meningeal artery (MMA) (left). Note the abundant blunt-ended protrusions of basal mLVs (right). fh, Representative images and magnified views of basal mLVs around the sigmoid sinus, which consist of distinct PROX1high (g) or integrin α9+ (h) lymphatic valves, magnified from the indicated regions. Note the oak leaf-shaped LECs with blunt ends (white arrowheads) in basal mLVs. Similar findings were observed in n = 5 mice from two independent experiments. Scale bars, 500 µm (f); 50 µm (g, h). i, Representative images of basal mLVs, which are larger in diameter than dorsal mLVs with more lymphatic branches extending outwards from venous sinuses. Below, magnified images of white dashed boxes show lymphatic valves (yellow arrowheads) of basal mLVs around the PSF, SMF, and jugular foramen (white arrows). White asterisk indicates the endolymphatic sac. Similar findings were observed in n = 5 mice from two independent experiments. Scale bars, 500 µm. j, Comparisons of the indicated parameters between dorsal and basal mLVs. Each dot indicates a mean value obtained from one mouse and n = 5–6 mice per group from three independent experiments. Mean ± s.d.; two-tailed Mann–Whitney U test. km, Representative images and comparisons of intraluminal T cells (white arrows) in dorsal and basal mLVs, and T cells entering the blunt ends of dorsal and basal mLVs (yellow arrows). Each dot indicates a mean value obtained from one mouse and n = 5 mice per group from two independent experiments. Mean ± s.d.; two-tailed Mann–Whitney U test. Scale bars, 50 µm. Source data

Extended Data Fig. 2 Basal mLVs exhibit typical features of pre-collectors.

a, Representative images of ear skin dermal LVs that consists of capillary LVs (A) with mainly discontinuously sealed button-like LEC junctions (blue arrowheads), pre-collecting LVs (B) with a mix of button- and zipper-like junctions (blue and red arrowheads) and lymphatic valves (white asterisks), and collecting LVs (C) with mostly zipper-like LEC junctions (red arrowheads), SMC coverage (yellow arrowheads) and lymphatic valves (white asterisk). Similar findings were observed in n = 4 mice from two independent experiments. Scale bars, 50 µm. b, Representative images of PROX1high LEC clusters in lymphatic valves of basal mLVs around the PSF with features of pre-collecting LVs, and a magnified view of the dashed box showing integrin α9+ lymphatic valves (white arrowheads) between lymphangion segments (yellow asterisks). Similar findings were observed in n = 4 mice from two independent experiments. Scale bar, 50 µm. ce, Representative images of basal mLVs exhibiting pre-collector characteristics around the PSS and MMA, which are devoid of SMC coverage but possess lymphatic valves (white asterisks) and are heterogeneous in both LYVE-1 expression (low and high; green and brown arrowheads) and LEC junctional pattern (button- and zipper-type; blue and red arrowheads). Black and white images in e are single-channel LYVE-1+ or VE-cadherin+ images magnified from the white dashed box in d. Similar findings were observed in n = 4 mice from two independent experiments. Scale bars, 50 µm. f, Comparison of indicated parameters between basal mLVs and ear skin LVs. Each dot indicates a mean value obtained from one mouse and n = 4 mice per group from two independent experiments. Mean ± s.d.; *P < 0.05 versus basal mLVs, skin capillary LVs, or skin pre-collecting LVs by two-tailed Mann–Whitney U test. n.s., not significant. gi, Representative images and comparisons of capillary and pre-collecting LVs from ear skin and basal mLVs around the sigmoid sinus. White dashed boxes are magnified as indicated. Red arrowheads, button-like junctions; blue arrowheads, zipper-like junctions of LECs. White asterisks, lymphatic valves. Yellow asterisk, a typical oak leaf-shaped LEC in a blunt end tip of initial capillary basal mLVs with button-type junctions. Each dot indicates a mean value obtained from one mouse and n = 4 mice per group from two independent experiments. Mean ± s.d.; P values versus skin capillary LVs or skin pre-collecting LVs, or skin capillary LVs and basal capillary mLVs, calculated by two-tailed Mann–Whitney U test. n.s., not significant. Scale bars, 50 µm. Source data

Extended Data Fig. 3 Postnatal mLVs grow into the cranial cavity through the foramina of the skull base, but do not comprise perineural LVs.

a, Representative gross images of PSF (yellow arrowhead), SMF (two yellow arrowheads) and jugular foramen (three yellow arrowheads) at the skull base. Magnified views of yellow boxes in panels at top right. Similar findings were observed in n = 4 mice from two independent experiments. Scale bars, 1 mm. b, c, Schematic illustration and representative sectional images of adult mouse skull base with basal mLVs (white arrowheads) around the sigmoid sinus and SMF. Note that basal mLVs inside the skull are in close proximity to the subarachnoid space (A, B) and they are connected to extracranial LVs through the SMF (C, D). Green asterisks, artificial spaces formed during sample preparation. Similar findings were observed in n = 4 mice from two independent experiments. Scale bar, 100 µm. d, Representative gross image of the skull base of a neonatal Prox1–GFP mouse at postnatal day 4 (P4). Insets show fluorescent images of extracranial LVs entering the skull through the major skull foramina. Similar findings were observed in n = 4 mice from two independent experiments. Scale bar, 1 mm. e, Representative image of basal mLVs entering the cranial cavity through the skull foramina (yellow arrowheads) at P4. White asterisk, endolymphatic sac. Similar findings were observed in n = 4 mice from two independent experiments. Scale bar, 1 mm. f, Representative images of developing mLVs and PROX1high putative lymphatic valves (white arrowheads) around the PSF (yellow arrowheads). Note that mLVs that grow into the skull through the PSF turn downward and combine with mLVs (P4 and P8, white arrows) growing from the PPA and MMA, and then grow upward (P12, white arrow). Similar findings were observed in n = 4 mice from two independent experiments. Scale bars, 500 µm. g, h, Schematic illustration and representative whole-mount images of postnatal basal mLVs around the SMF (two yellow arrowheads) and jugular foramen (three yellow arrowheads) after removal of cranial nerves. White dashed boxes are magnified as indicated. White asterisk, endolymphatic sac. Similar findings were observed in n = 4 mice from two independent experiments. Scale bar, 500 µm. i, j, Schematic illustration and representative whole-mount images of adult basal mLVs around the SMF (two yellow arrowheads) and jugular foramen (three yellow arrowheads). White asterisk, endolymphatic sac; yellow asterisks, lymphangion segments between the lymphatic valves that are distinguished and far from neurofilament+ nerve fibres (white arrowheads). Similar findings were observed in n = 4 mice from two independent experiments. Scale bar, 500 µm.

Extended Data Fig. 4 Contrast delivery through the lateral ventricle shows a CSF clearance pattern that is similar to that of delivery through the cisterna magna.

a, Phantom MRI experiment among the indicated range of contrast concentrations (1–50%), showing highest enhancement at 10% (peak intensity). The signal intensities of GadoSpin P and artificial CSF (control) are plotted as absolute or normalized values (normalized signal intensity), which is GadoSpin P intensity divided by control intensity. AU, arbitrary unit. b, Representative images of rat mLVs around the jugular foramen (white arrow) demonstrating abundant blunt-end lymphatic capillary branches (yellow arrowheads) and PROX1high LEC clusters depicting lymphatic valves (white arrowheads), magnified from the region within the white dashed box. Similar findings were observed in three independent experiments using n = 3 rats. Scale bar, 100 µm. c, Schematic images for T2-FLAIR MRI with initiation of GadoSpin P infusion into the right lateral ventricle (Rt LV) at a rate of 2 μl min–1 for 15 min under mechanical ventilation and monitoring of vital signs including HR, RR, ETCO2, BT, and EKG. d, Monitoring of indicated parameters during MRI. Grey background indicates a single baseline scan and blue background indicates MRI scans taken during the GadoSpin P infusion. eh, Representative images of 2nd (e), 6th (f), and 12th (g) scans of T2-FLAIR MRI and comparisons of average GadoSpin P intensities within ROIs at the indicated time points (h). The location of each axial scan is displayed in the sagittal view at the top right corner. The ROIs are outlined in green (Rt LV), orange (basal outflow), red (deep cLNs), blue (dorsal area), and purple (SSS, magnified). Similar findings were observed in four independent experiments using n = 4 rats. Scale bars, 5 mm; except SSS, 1 mm. h, Grey background indicates single baseline scan and blue background indicates period of GadoSpin P infusion through the lateral ventricle. Vertical lines indicate time points at which the representative images were taken. Mean ± s.e.m. from four independent experiments using n = 4 rats. AU, arbitrary unit. i, Schematic images illustrating the monitoring of ICP, ABG, and vital signs during and after GadoSpin P infusion into the cisterna magna of rat at a rate of 2 μl min–1 for 15 min. jl, ICP, pH, partial pressure of oxygen (\({p}_{{{\rm{O}}}_{2}}\)), partial pressure of carbon dioxide (\({p}_{{{\rm{CO}}}_{2}}\)), RR, MAP, and HR before, during (blue background) and after contrast infusion. Source data

Extended Data Fig. 5 Dorsal mLVs have a minor role in clearance of macromolecules from the CSF.

a, Schematic diagram depicting QD705 infusion into the cisterna magna (CM) of 8-week-old Prox1GFP mice and subsequent fluorescence imaging at the indicated time points after the QD705 infusion. bd, Schematic and representative images of basal mLVs around jugular foramen and dorsal mLVs around SSS and transverse sinus within the region of interest (black box in schematic) and their profile analysis along the white line at the indicated time points after QD705 infusion. White dotted boxes are magnified at top left of images. Blue background in plots indicates the width of an LV; note that the QD705 peak signal is located within the basal mLV signal (LV) around the jugular foramen, whereas those around the SSS and transverse sinus are located outside the dorsal mLVs. Similar findings were observed in four independent experiments. Scale bars, 200 µm. Source data

Extended Data Fig. 6 Brain ISF macromolecules are cleared preferentially through the basal mLVs.

a, Schematic diagrams depicting stereotactic intraparenchymal infusion of QD705 into the right (Rt) side of the brain of a Prox1–GFP mouse at a rate of 0.2 μl min–1 for 5 min and subsequent fluorescence imaging of whole brain surface, dorsal mLVs, and Rt or left (Lt) basal mLVs and deep cLNs at 30, 60, and 120 min after tracer infusion. Similar findings were observed in n = 4 mice from two independent experiments. b, Representative fluorescence imaging of whole brain surface after infusion of QD705 into the Rt side of brain. White arrowheads, infusion site. Similar findings were observed in n = 4 mice from two independent experiments. Scale bars, 2 mm. ci, Representative images and profile analysis of fluorescence intensity along the white lines in dorsal mLVs around the SSS and right transverse sinus at the indicated time points after tracer infusion. Blue background, width of dorsal mLV (LV); note that QD705 peak signals are not within the LV. Similar findings were observed in n = 4 mice from two independent experiments. Scale bars, 200 µm. jq, Representative images and profile analysis of fluorescence intensity along the white lines at the indicated time points after tracer infusion. White dashed boxes are magnified below; blue background indicates the width of an LV. Similar findings were observed in n = 4 mice from two independent experiments. Scale bars, 500 µm. r, s, Representative images of right or left deep cLNs at the indicated time points after tracer infusion. White dashed box is magnified in the upper right corner as a QD705 single-channel image, indicating drainage of tracer into right deep cLNs (yellow arrowheads). Similar findings were observed in n = 4 mice from two independent experiments. Scale bars, 1 mm. Source data

Extended Data Fig. 7 Differential sensitivity and functionality of dorsal and basal mLVs to LEC-specific depletion of Vegfr3.

a, Diagram depicting LEC-specific depletion of Vegfr3 in 8-week-old Vegfr3iΔLEC mice and analyses of mLVs and blood vessels (BVs), 1 week (Vegfr3iΔLEC (+1 wk)) or 2 weeks (Vegfr3iΔLEC (+2 wk)) after tamoxifen administration for comparison with wild type littermates (WT (+2 wk), 2 weeks after tamoxifen administration). be, Representative images and comparisons of the relative densities of dorsal and basal mLVs and BVs around the SSS, transverse sinus, PSS, sigmoid sinus, PSF, and jugular foramen in wild-type and Vegfr3iΔLEC mice. Each dot indicates a mean value obtained from one mouse and n = 4 mice per group from two independent experiments. Mean ± s.d.; two-tailed Mann–Whitney U test. Note the prominent differences in vessel densities of dorsal mLVs around the SSS and transverse sinus in Vegfr3iΔLEC mice (+1 wk), but the differences in basal mLVs around the sigmoid sinus, PSS, PSF, and jugular foramen become evident in Vegfr3iΔLEC mice (+2 wk), as shown in the volcano plot (e) plotting the relative differences in vessel density on the x-axis and the negative log of the Q value on the y-axis. AU, arbitrary unit; n.s., not significant. Scale bars, 1 mm. f, Schematic diagram of QD705 infusion (1 μl min–1 for 3 min) into the cisterna magna in wild-type or Vegfr3iΔLEC mice, 1 or 2 weeks after tamoxifen administration for fluorescent imaging of draining mandibular LNs and deep cervical LNs (cLNs) 30 min after infusion of QD705. g, h, Representative images and comparison of QD705 intensity in mandibular LNs and deep cLNs (white dashed circle) in wild-type (+2 wk), Vegfr3iΔLEC (+1 wk), or Vegfr3iΔLEC (+2 wk) mice. Each dot indicates a mean value obtained from one mouse, n = 6 mice per group from three independent experiments. Mean ± s.d.; two-tailed Mann–Whitney U test. n.s., not significant. Scale bars, 1 mm. Source data

Extended Data Fig. 8 Ageing is accompanied by dysmorphic changes in basal mLVs with delayed CSF drainage.

a, Schematic images depicting the regions of dorsal mLVs around the transverse sinus. b, c, Representative images and comparisons of the LYVE-1+ area and the diameter of dorsal mLVs around the transverse sinus in young and aged mice. Each dot indicates a value from both sides of the transverse sinus, n = 7 mice per group (LYVE-1+ area around transverse sinus) or a mean value obtained from one mouse, n = 6 mice per group (vessel diameter) from two independent experiments. Mean ± s.d.; P versus young calculated by two-tailed Mann–Whitney U test. n.s., not significant. Scale bars, 500 µm. d, Schematic illustrations depicting the region of basal mLVs around the jugular foramen. e, f, Representative images and comparison of PROX1 fluorescence intensity ratio of valvular LECs to non-valvular LECs of basal mLVs in young (3 months), middle-aged (8–10 months), and aged (24–27 months) mice. White dashed lines demarcate basal mLVs. PROX1 expression is attenuated in lymphatic valve LECs (white arrows) in aged mice. Each dot indicates a value obtained from one mouse and n = 6 mice per group from three independent experiments. Mean ± s.d.; P versus young or middle-aged calculated by two-tailed Mann–Whitney U test. AU, arbitrary unit. Scale bars, 100 µm. g, Schematic diagram of QD705 infusion (1 μl min–1 for 3 min) into the cisterna magna in young, middle-aged, and aged mice and subsequent fluorescence imaging of indicated draining LNs 30 min after the infusion of QD705. h, i, Representative images and comparison of QD705 intensity in mandibular LNs and deep cLNs (outlined with white dashed line) in young, middle-aged, and aged mice. Each dot indicates a value obtained from one mouse and n = 5 mice per group from three independent experiments. Mean ± s.d.; P values versus young or middle-aged calculated by two-tailed Mann–Whitney U test. AU, arbitrary unit. Scale bars, 1 mm. j, k, Representative images and comparisons of the LYVE-1+ LV area in ear skin, tracheal mucosa, and peritoneal side of the diaphragm in young and aged mice. Each dot indicates a mean value obtained from both ears of one mouse, n = 5 mice per group (ear skin), or a mean value obtained from one mouse, n = 4 mice per group (trachea and diaphragm), from two independent experiments. Mean ± s.d.; P values versus young calculated using two-tailed Mann–Whitney U test. n.s., not significant. Scale bars, 250 µm. ln, Representative images and comparisons of collagen IV, PROX1, or FOXC2 expression in skin dermal LECs of lymphatic valves (white arrowheads) in young and aged mice. Each dot indicates a mean value obtained from one mouse and n = 4 (collagen IV and FOXC2) or n = 5 (PROX1) mice per group from two independent experiments. Mean ± s.d.; P values versus young calculated using two-tailed Mann–Whitney U test. AU, arbitrary unit; n.s., not significant. Scale bars, 200 μm. Source data

Extended Data Fig. 9 Distinct changes in dorsal and basal mLVs associated with age.

ad, Representative images of dorsal mLVs around the SSS and COS and comparison of dorsal mLV coverage around the SSS (a, b), and representative images of basal mLVs around the PSS and sigmoid sinus and comparison of LYVE-1+ basal mLV area and number of Prox1+ LECs (c, d) in young (3 months), middle-aged (8–10 months), and aged (24–27 months) mice of indicated sex. Each dot in b and d indicates a mean value from one mouse, n = 6 mice per group from two independent experiments. Mean ± s.d.; and P values versus male, young, or middle-aged calculated by two-tailed Mann–Whitney U test. n.s., not significant. Scale bars, 500 µm. Source data

Extended Data Fig. 10 Characteristic features of dorsal and basal mLVs and their distinct changes associated with ageing.

a, Schematic images depicting the differences between dorsal and basal mLVs and their connection with extracranial LVs. In young adult mice, the dorsal mLVs are disconnected and mostly exhibit zipper-like junctions between LECs. By contrast, basal mLVs, which are located close to the CSF, are characterized by protruding blunt-end capillary lymphatic vessel branches and lymphatic valves, and their LECs are connected with button-like junctions. b, In aged mice, dorsal mLVs are regressed, whereas basal mLVs exhibit hyperplastic changes with impaired lymphatic valves and disrupted junctional structures.

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