Conserved meningeal lymphatic drainage circuits in mice and humans

Abstract

Meningeal lymphatic vessels (MLVs) were identified in the dorsal and caudobasal regions of the dura mater, where they ensure waste product elimination and immune surveillance of brain tissues. Whether MLVs exist in the anterior part of the murine and human skull and how they connect with the glymphatic system and extracranial lymphatics remained unclear. Here, we used light-sheet fluorescence microscopy (LSFM) imaging of mouse whole-head preparations after OVA-A555 tracer injection into the cerebrospinal fluid (CSF) and performed real-time vessel-wall (VW) magnetic resonance imaging (VW-MRI) after systemic injection of gadobutrol in patients with neurological pathologies. We observed a conserved three-dimensional anatomy of MLVs in mice and humans that aligned with dural venous sinuses but not with nasal CSF outflow, and we discovered an extended anterior MLV network around the cavernous sinus, with exit routes through the foramina of emissary veins. VW-MRI may provide a diagnostic tool for patients with CSF drainage defects and neurological diseases.

Conflict of interest statement

Disclosures: F. Benbelkacem is an employee of Siemens Healthineers GmbH. B. Stankoff reported personal fees from Sanofi, Merck, and Novartis; and grants from Roche, Merck, and Sanofi outside the submitted work. No other disclosures were reported.

© 2022 Jacob et al.

Figures

Graphical abstract

Figure 1.

iDISCO-LSFM imaging of CSF tracer drainage. (A) Schematic of the experimental workflow. Arrows indicate sites of tracer injection, ICM, Th-Lb, or Lb-Sa. (B) LSFM imaging of OVA-A555 (white) in the pia mater of the spinal cord (SC) 90 min after Th-Lb injection. Asterisk, vertebral body. (C) Coronal section of cervical SC 45 min after Th-Lb injection shows OVA-A555 in the SC and in the dcLNs (green arrowheads). (D) LSFM view of a coronal section of the forebrain shows OVA-A555 in phagocytic cells (inset) along the glymphatic perivascular spaces. Note that brain size is reduced by the iDISCO+ protocol, but meningeal layers are preserved. The dura and superior sagittal sinus (SSS) are in contact with the skull (Sk), and tracer drainage can be followed through the intact calvaria. OVA-A555+ cells are also found along the SSS and the rostral confluence of sinuses (RCS). Cx, cortex. (E) 3D schematic of the lateral view of the head. Cb, cerebellum; CP, cribriform plate; ica, internal carotid artery; ijgv, internal jugular vein; NP, nasopharynx; OB, olfactory bulb; OC, oral cavity; OE, olfactory epithelium; ON, optic nerve. Green arrowheads, scLN. (F–H) Lateral views of OVA-A555 in mice sacrificed 15 min (F) or 45 min (G and H) after Lb-Sa injection. OVA-A555–labeled perivascular glymphatic spaces (F and G). OVA-A555 labeled the dcLN but not the scLN at 15 min (F, green arrowheads), while both dcLN and scLN are labeled at 45 min (G). Lymphatic afferent vessels extend from the NP toward the LNs (G). (H) The same pattern in another mouse with a zoom on the anterior part of the CP and OE. Note OVA-A555+ vessels in the OE and around the NP (G and H). (I) Summary of tracer injections and number of tracer-positive cervical LNs per mouse. (J and K) Quantification of OVA-A555+ DC1s (CD45+/CD11b+/CD11c+), DC2s (CD45+/CD11b−/CD11c+), and MΦ (CD45+/CD11b+/CD11c−) among total CD45+ cells FACS-sorted from the dcLN, mandibular LN (mLN), accessory mandibular LN (amLN), and superficial parotid LN (spLN) of non-injected mice or mice injected into the Th-Lb spine with OVA-A488. n = 6 mice/group. Data show mean + SEM; Mann–Whitney U test (J) and one-way ANOVA with Dunn’s multiple-comparisons test (K); *, P < 0.05; **, P < 0.01. A, anterior; D, dorsal; L, lateral; P, posterior; V, ventral. Scale bar: 500 μm (B–D and H); 800 μm (F and G).

Figure S1.

OVA-A555tracer distribution after ICM and intraspinal injection. (A) Coronal section of the cervical spine after ICM injection of OVA-A555 (white). Note that OVA-A555 spillover labeled paravertebral tissues (PVT, arrowhead) and the brainstem (BS). Asterisk, vertebral body. (B) FITC-dextran tracer distribution 45 min after Th-Lb or Lb-Sa injection. Macroscopic imaging of tracer labeling (white arrowheads) along the caudorostral axis of the spine. SC, spinal cord. (C–F) Coronal sections of the sacral (C and D) and cervical (E and F) spine showing ink deposits (black) in the meninges along the caudorostral axis. Macroscopic imaging of dissected spine segments (C and E) and bright-field microscopic imaging of paraffin-embedded spine sections (D and F). (G) Coronal section of the brainstem (BS) showing ink deposits in the meninges and inside the dcLNs (green arrowheads). Asterisk, vertebral body. (H) Sagittal section of the forehead showing ink deposits in the meninges of the olfactory bulb (OB), in the cribriform plate (CP), the olfactory epithelium (OE), and the nasopharynx (NP). Cx, cortex. (I) LSFM coronal view of the CSF tracer pattern (OVA-A555, white) in the perivascular spaces of the cortex and mesencephalon, and in the scLNs (green arrowheads). (J) Table of additional tracer injection experiments showing the number of mice per group with tracer-labeled cervical LNs 45 and 90 min after injection. (K) Representative flow dot plots used to separate different populations of OVA488+ myeloid cells in cervical LNs in Fig. 1, J and K. A, anterior; D, dorsal; L, lateral; P, posterior; V, ventral. Scale bar: 500 μm (B–F); 2 mm (A and G–I).

Figure 2.

LSFM imaging of known dorsal and laterobasal MLV drainage pathways. (A) Lateral view of OVA-A555 (magenta) in the posterior head 45 min after Lb-Sa injection labeled with anti-LYVE1 (green). OVA-A555 is present in perivascular spaces along the spinal cord (SC), cerebellum (Cb), and cortex (Cx) and in the dcLN (green arrowhead). Roman numbers indicate known MLV circuits: I (SSS), II (transverse sinus TS), III (sigmoid sinus SS and jugular foramen), IV (petrosquamous sinus PSS). NP, nasopharynx (solid line); dashed line, ventral skull border; dashed rectangles, regions magnified in panels I–IV. Inset: Venous sinuses and emissary veins imaged in B–K (light blue) and other venous circuits (dark blue). ijv, internal jugular vein; iptgv, interpterygoid emissary vein; pfv, posterior facial vein; rgv, retroglenoid vein. (B–E) Horizontal (B and C) and sagittal (D and E) views labeled with anti-vWF (B and C) and anti-LYVE1 (D and E) antibodies. The SSS (B, blue arrowheads) connects the rostral confluence of sinuses (RCS) with the caudal confluence of sinuses (COS) and the TS (C and E, blue arrowheads). Note discontinuous OVA-A555 labeling in the perisinusal spaces (B and C). LYVE1+ MLVs along the SSS (D) contain OVA-A555 at the TS (white in E). (F–H) Coronal views at the level of the jugular foramen (jf) labeled with the indicated antibodies and OVA-A555. vWF stained the SS and the jugular vein (ijv, blue arrowhead in F). OVA-A555 labels the jf and the dcLN (green arrowheads in F–H). (G) TUJ1+ cranial nerves exiting the skull through the jf were not colabeled with OVA-A555. (H) LYVE1+ MLVs follow the SS (blue arrowhead) and exit the skull through the jf toward the dcLN. IX, cranial nerve 9 (glossopharyngeal); X, cranial nerve 10 (vagus); X, cranial nerve 11 (spinal accessory); BS, brainstem; dotted line, skull border. (I–K) Coronal views at the PSS exit through the skull. (I) vWF stains the PSS passing through the petrosquamous fissure (interrupted dashed line) to join the pfv via the rgv. (J and K) LYVE1+ MLVs follow the PSS, rgv, and pfv. OVA-A555 accumulated at the petrosquamous fissure level (J). (K) Magnification of the dashed frame in J showing a blind-ended MLV and other OVA-A555+/LYVE1+ lymphatics (white, green arrowhead). Br, brain. Scale bars: 1,000 μm (A–D); 500 μm (E–J); 250 μm (K).

Figure S2.

Perisinusal distribution of CSF tracer and lymphatic uptake hotspots. (A and B) Coronal (A) and sagittal (B) views of the perisinusal distribution of tracer (OVA-A555, magenta, blue arrowheads) along the transversal sinus (TS, vWF+, blue). (C) LYVE1+ MLVs (green) and tracer deposits (magenta) in the rostral convergence of sinuses (RCS). Note tracer accumulation around RCS MLVs (blue arrowhead). OB, olfactory bulb. (D) Schematic lateral view of dural venous sinuses and localization of lymphatic uptake hotspots (1–3) in the caudal portion of CAV. bp, basilar plexus; Cx, cortex; ica, internal carotid artery; IPETS, inferior petrosal sinus; iptgv, interpterygoid emissary vein; pbv, posterior basal vein; SPETS, superior petrosal sinus. (E) Sagittal view of CD31+/PDLX+ blood vessels (red) and OVA-A555 deposits (magenta) around the pituitary gland (PG) and the CAV (blue dashed line). (F–I) Localization of lymphatic uptake hotspots (1–3) in the caudal portion of CAV: horizontal (F) or coronal (G–I) views of tracer deposits (magenta) and vWF+ dural sinuses and veins (blue). (I) Blood vessels (CD31+/PDLX+, orange) at the carotid canal (cc). Dotted line, skull border; ICAV, inter-CAV sinus. Scale bar: 200 μm (A–C); 500 μm (E–I).

Figure 3.

MLV drainage from the caudal CAV. (A) Schematic of venous sinuses, veins, and internal carotid artery at the base of the brain. (B–I) Dashed lines indicate the level of coronal sections in B–I (inset in A), and lymphatics are numbered 1–3. BS, brainstem; bv, basilar vein; ica, internal carotid artery; ICAV, inter-CAV; ijv, internal jugular vein; IPETS, inferior petrosal sinus; iptgv, interpterygoid emissary vein; OB, olfactory bulb; pbv, posterior basal vein; pfv, posterior facial vein; SPETS, superior petrosal sinus. (B) Coronal view of the right CAV. OVA-A555 was injected into the Th-Lb spine 45 min before sacrifice, and the sample was labeled with antibodies recognizing blood endothelium markers CD31 and PDLX that label pituitary gland (PG) vessels as well as dura mater veins and sinuses (blue arrowheads). Blue dashed line, limits of CAV and iCAV. Note tracer deposits (magenta) around the CAV and pbv as well as in the leptomeninges. Br, brain. (C–E) Cavernous MLVs 1 and 2. Lymphatic vasculature (LYVE1+, green in C and D) and TUJ1+ cranial nerves (yellow in E) at coronal levels 1 and 2 (green arrowheads in C and D). Note MLVs close to the OVA-A555+ cavernous perisinusal space (C) and surrounding the foramen of iptgv (D). Cranial nerves were devoid of tracer deposits (E). VI, cranial nerve 6 (abducens); dotted line, skull border. (F–I) Cavernous MLVs 3. Dural veins (vWF, blue in F), lymphatic vasculature (LYVE1+, green in G and H), and cranial nerves (TUJ1+, yellow in I) at coronal (F, G, and I) and sagittal (H) level 3 (green arrowheads in F–H). MLVs contact the cavernous perisinusal space (G and H) and uptake tracer at the intersection of CAV with internal carotid arteries (white in G, magnified in inset). Cranial nerves were devoid of tracer deposits and MLVs (I). III, cranial nerve 3 (oculomotor); IV, cranial nerve 4 (trochlear); V, cranial nerve 5 (trigeminal); A, anterior; cc, carotid canal; D, dorsal; L, lateral; NP, nasopharyngeal cavity; P, posterior; V, ventral. Scale bar: 500 μm (B–I), 100 μm (inset in G).

Figure 4.

MLV drainage from the rostral CAV. (A) Schematic of veins of the ventral forebrain and olfactory bulbs (OB). (B–O) Dashed lines indicate the level of coronal sections shown in B–O, and lymphatics are numbered 4–6. Black circles, optic nerve foramen (of); anterior lacerated foramen (alf). abv, anterior basal vein; iophv, internal ophthalmic vein; IOS, inferior olfactory sinus; olfev, olfactory emissary vein; ON, optic nerve (yellow); ophev, ophthalmic emissary vein; RCS, rostral confluence of sinuses; rrhv, rostro-rhinal vein; SOS, superior olfactory sinus. (B) vWF+ veins (blue) and tracer deposits (OVA-A555, magenta) in the iophv perivenous area and around the ophev at lymphatic uptake site 4. Dotted line, skull border. (C–G) LYVE1+ MLVs (green) and OVA-A555 (magenta) around the CAV at level 4 (green arrowheads). Th-Lb (B–F) or ICM (G) injections of OVA-A555 were performed 45 min before sacrifice. MLVs contact perisinusal OVA-A555 deposits (C). (D) Magnification of dotted frame in C. OVA-A555 is present within MLVs (white arrowheads). (E and F) Coronal (E) and sagittal (F) views of MLVs following the ophev toward the orbital cavity (orange arrows) and connecting ventrally to lymphatics of the nasopharynx (NP, white arrows). (G) Note similar labeling pattern to C; MLVs exit the skull through the alf and prolong along the ophev toward the orbital cavity. (H–K) Meningeal veins (vWF+, blue in H), lymphatic vasculature (LYVE1+, green in I–K), and tracer deposits (OVA-A555, magenta) on coronal (H–J) and sagittal (K) views at section level 5. (H) Tracer accumulated around the confluence of the olfev with the CAV (blue arrowheads). Stippled area, alf. (I–K) LYVE1+ MLVs follow the olfev to exit the skull toward the orbital cavity (orange arrows) and extend ventrally toward the NP (white arrows). OVA-A555 deposits were found in LYVE1+ MLVs (insets in I). Coronal (J) and sagittal (K) view of the orbital cavity. MLVs exit the skull toward the orbital cavity (orange arrows) and the NP (white arrows). (L–O) Rostral end of CAV at section level 6. (L) Tracer accumulated around the confluence of the IOS with the CAV (blue arrowheads). Green arrowheads, MLVs at the rostral end of CAV take up tracer (white in M). MLVs extend rostrally from the CAV along the IOS on the lateral side of each olfactory bulb (N and O, blue arrowhead). OE, olfactory epithelium. (P–S) Horizontal (P and Q) and sagittal (R and S) views of the anterior part of the head showing the IOS, SOS, and RCS. Dural veins (vWF+, blue in P), lymphatic vasculature (LYVE1+, green in Q–S), and tracer deposits (OVA-A555, magenta). (Q) MLVs connect the CAV with the RCS via the IOS and SOS. (R and S) OVA-A555 is accumulated around MLVs at the SOS (Q and R) and RCS (R and S) Scale bar: 300 μm (C–S).

Figure 5.

CSF drainage from the cribriform plate into the nasal cavity. (A) Sagittal view of the nasal cavity and forebrain 45 min after Th-Lb injection of OVA-A555 (magenta) and labeled with LYVE1 (green). Tracer deposits were detected in meningeal perivascular spaces, the cribriform plate (CP), and throughout the olfactory epithelium (OE) and the respiratory epithelium (RE) of the nasal cavity. Dotted line, limit between intra and extracranial regions; dashed lines, nasopharynx (NP); blue arrowheads, CAV; OB, olfactory bulb. (B–E) Lymphatics of the cribriform plate. Lymphatic vasculature (LYVE1+, green in B and C; PROX1+, green in E), meningeal veins (vWF, blue in D), and tracer deposits (magenta) on sagittal (B and E) and coronal (C and D) views of the CP. MLVs were located in the dura of the ethmoid bone (lymphatic bed 7, green arrowheads) and around CP foramina (orange arrowheads in B and C). No LYVE1+ vessels were found to cross the CP toward the olfactory epithelia (B and E), while the outflow of tracer filled the OE and RE (A and D). Inset in B: Magnification of dotted frame showing tracer-labeled phagocytic cells (yellow arrowheads) concentrated close to the ethmoid MLVs. Ethmoid MLVs (C) are not associated with vWF+ blood vessels (D). (E) The ethmoid MLVs (white arrowheads) converge dorsally with the lymphatic vessels prolonging the SOS and exit the nasal cavity via the posterior ethmoid foramen (pef) and the foramen cecum (fc). SP, septum. (F and G) Lymphatic vessels of the nasal cavity. Sagittal views showing the tracer-labeled LYVE1− vasculature of the OE (F, magenta), the LYVE1+ lymphatics of the basal RE and the NP (F, green), and the Vegfr3-expressing vessels of the OE (GFP reporter, green in G). (H) Sagittal schematic of lymphatic circuits of the nasal cavity. Ethmoid MLVs (LYVE1+, light green, 7) do not cross the CP along with olfactory nerves but exit rostrally into the nasal cavity via the pef and fc. OE lymphatics (PROX1+/VEGFR3+/LYVE1−, bluish green) transport CSF, likely collected from perineural drainage along olfactory nerves, then drain into LYVE1+ vessels (dark green) of the basal RE and NP that collect into cervical LNs. (I–K) Lymphatic drainage from the orbital cavity. Tracer deposits (OVA-A555, magenta) and lymphatic vasculature (green in I and J). (I) Ventrolateral view of the nasal lacrimal sac region (NLS, dashed line) shows tracer deposits between the orbital cavity under the eye (E) and the olfactory epithelium (OE). Orbital LYVE1+ lymphatics connect with facial lymphatics (white arrowheads). (J) Lymphatic tracer uptake by orbital PROX1+ vessels (white arrowhead). (K) Macroscopic imaging of a mouse head 10 min after ICM injection with OVA-A555 (magenta) and intra-ocular delivery of OVA-A488 (green). Both OVA-A488 and OVA-A555 collected into the associated mandibular LNs (white); only OVA-A555 drained into the mandibular LNs (magenta). (L–N) Lymphatic drainage from the nose. (L) Coronal view of the nose showing two drainage circuits: dorsal (white arrowheads) and ventral (yellow arrowheads). Dashed circles show the distal part of NLD with LYVE1+ LVs (green) costained with OVA-A555 (magenta). (M) Macroscopic imaging of mandibular LNs (mLN, green arrowhead) labeled with OVA-A488 5 min after nostril OVA488 injection. (N) Sagittal schematic of lymphatic drainage circuits from the orbital cavity and the nose toward cervical LNs. A, anterior; D, dorsal; L, lateral; P, posterior; PEF, posterior ethmoid foramen; V, ventral. Scale bar: 180 μm (insert in B); 500 μm (A–C, E–J, and L); 80 μm (D); 2 mm (K and M).

Figure S3.

CSF drainage through the cribriform plate and inside the nasal cavity. (A–F) Pattern of tracer 45 min after ICM (A) or Th-Lb (B–F) injections of OVA-A555 (magenta) or ink. (A and B) Sagittal (A) and coronal (B) views of the ethmoid MLVs (7). Note tracer deposits at the cribriform plate (CP) border and in LYVE1+ (A, green arrowhead) and PROX1+ (B, green arrowhead) MLVs around olfactory nerve foramina (orange arrowheads). Dotted line, limit between intra- and extracranial regions; OB, olfactory bulb; OE, olfactory epithelium. (C–F) Sagittal (C–E) and coronal (F) views of nasal cavity lymphatics. Tracer deposits (magenta in C, D, and F; ink in E) are colocalized with lymphatic markers: PROX1 (C, green), VEGFR3-YFP (D, green), and LYVE1 (E and F). (C) PROX1+ lymphatics in the turbinate, on the outer aspect of the CP (white arrows in C). (D) VEGFR3+ lymphatics transporting OVA-A555 tracer in the OE (white arrowheads). (E) Two contiguous paraffin sections of the nasal cavity showing ink tracer (black arrowheads) inside narrow LYVE1− vessels and collecting LYVE1+ lymphatics in the respiratory epithelium (RE). (F) LYVE1+ lymphatics, including tracer-labeled vessels around the nasopharynx (NP). SP, septum. (G) Lymphatic drainage from the orbital cavity. Coronal view showing tracer deposits (magenta) and pattern of LYVE1+ lymphatics (green). Lymphatic-tracer uptake is detected around the nasal lacrimal sac (NLS, white labeling, arrowheads). E, eye. (H and I) Lymphatic drainage from the nose 90 min after intraspinal OVA-A555 injection (magenta). Macroscopic (H) or LSFM (I) imaging showing tracer accumulation at the level of the nostril (white arrowheads in H and I) and tracer drainage along dorsal and ventral nasal veins (white arrows in H). Note LYVE1+ lymphatics in the nostril (arrowheads in the magnified frame in I) and along nasal veins (arrows in I). Scale bar: 300 μm (A, H, and I, insert in I); 150 μm (B–F).

Figure S4.

Sacral spinal cord outflow. (A and B) Macroscope imaging of the sacrococcygeal region 90 min after ICM injection of OVA-A555 tracer. (A) Tracer deposits were detected at intervertebral spaces (white arrows, S3–S4, S4-Co1, Co1–Co2) and in the sciatic LNs (siLN, green arrowheads). (B) In the peritoneal cavity, the tracer (white) was detected in collecting lumbar LNs (lbLN) and renal LNs (reLN) (green arrowheads). Co, coccygeal vertebrae; S, sacral vertebrae. (C–E) LSFM sagittal (C) and coronal (D and E) views of clarified sacral vertebrae, spinal cord, and dura mater. (C) Outflow of tracer (red) in the epidural space (white arrows) between S3–S4 and S4-Co1. Asterisks, vertebral bodies. (D and E) Pattern of LYVE1+ lymphatics (green) and tracer (red) in the S3–S4 region. Tracer deposits (red) were detected in the vertebral canal and in siLNs (green arrowheads in D). (E) Magnification of dotted frame in D. Tracer accumulated in the epidural space (ES), including in LYVE1+ lymphatics (yellow, white arrowheads in E). A, anterior; D, dorsal; L, lateral; P, posterior; V, ventral. (F) Quantifications of OVA-A555+ MΦ (CD45+/CD11b+/CD11c−) among total CD45+ cells FACS-sorted from the siLNs and lbLNs of noninjected mice or mice injected into the Th-Lb spine with OVA-A488. n = 6 mice/group. Data show mean + SEM; one-way ANOVA with Dunn’s multiple-comparisons test; *, P < 0.05. (G–K) Light-field microscope images of coronal paraffin sections of S3–S4 vertebrae from mice with intraspinal injection of ink tracer. (G) Low magnification of highly magnified areas shown in H–K. Tracer was detected on the ependymal layer and the pial surface of the spine (H), as well as inside phagocytic cells of the subarachnoid (I and J) and epidural (K) spaces. Note the thin monolayer of dura mater (black arrow in I) and the accumulation of tracer-containing mononuclear cells in subdural arachnoid sacs (black arrow in J). Asterisk, vertebral body. Scale bar: 2 mm (A and B); 500 μm (C–E and G); 10 μm (H–K).

Figure 6.

Meningeal vascular MRI in humans. (A) Schematic of the workflow for meningeal vascular 3D mapping by MRI in humans. Native sequences were acquired from a 3T MRI before and after i.v. gadobutrol injection. 3D-Slicer platform was used for semiautomated signal intensity–based thresholding and segmentation of native sequences. (B–G) Native VW-MRI (B and C) and meningeal vascular 3D mapping (D–G) after gadobutrol injection of a patient with MS (number 7 in Table 1). (B) Native 3D T1 SPACE DANTE in an oblique axial plane crossing the transverse axis of both the superior longitudinal (SSS) and the straight sinuses (StS). The black-blood contrast allowed to darken the lumen of venous sinuses, while dural lymphatics (white) were strongly enhanced by gadobutrol and precisely segregated from the unenhanced perisinusal dura mater (blue arrowheads). (C) Native 3D T1 SPACE DANTE sequence in a sagittal plane along the longitudinal axis of the left internal jugular vein (ijv) covering both the head and neck. cLNs (arrows) surrounding the ijv were enhanced by the contrast agent and distinct from adjacent soft tissues. (D) Oblique posterior view of the dorsolateral group of dural venous sinuses (blue), including the SSS, the StS, the transverse (TS), and the sigmoid sinuses (SS). The ijv represents the major venous outflow of the dorsolateral group of sinuses. Dorsolateral perisinusal fluids (yellow) concentrate in perisinusal areas and include vessel-like compartments (arrows) associated with flattened vesicles (arrowheads). Perisinusal fluids were detected along the internal carotid arteries (ica) and until the dcLNs. (E) Posterior view of the meningeal vascularization in the anterior part of the skull. Left (L) and right (R) CAV are connected at the midline by the superior (S) and inferior (I) coronary sinuses. The superior petrosal sinus (SPS) connects the CAV with the SS, while the inferior petrosal sinus (IPS) connects the CAV with the IJV. The IPS is also connected with the marginal sinus (MS), which drains caudally in the perivertebral venous plexuses. In the intracavernous segments, the ica crosses the CAV before intradural bifurcation in middle (MCA) and anterior (ACA) cerebral arteries. Perisinusal fluids were detected in the perisinusal areas of the CAV, the MS surrounding the foramen magnum, and along the IPS. Exit routes of perisinusal fluids from the skull followed the pericarotid route in the carotid canal, anteriorly, and the perivertebral canal along the vertebral arteries, posteriorly. (F and G) Parasagittal (F) and posterior (G) views of fluid exit routes from the CAV perisinusal area, showing the pericarotid route inside the carotid canal as well as several transforaminal routes along the branches of trigeminal nerve, including the superior orbital fissure (sof) along the ophthalmic branch, the foramen rotundum (fr) along the maxillary branch, and the foramen ovale (fo) along the mandibular branch. Besides their specific trigeminal nerve branches, these foramina contain corresponding emissary veins that collect extracranially in the extracranial veins, including the pterygoid plexuses (PP). No perisinusal flow was observed through the optical canal (oc). Scale bar: 1 cm (B–G).

Figure S5.

Meningeal and skull vascular MRI in humans. (A and B) VW-MRI (A) and meningeal vascular 3D mapping by MRI (B) of a patient with IIH and CSF leak (patient 1 in Table 1). (A) Transcalvarial connections at the level of the superior longitudinal sinus (SSS). A few transcalvarial veins (TcV) connecting the SSS with subcutaneous veins (SuV) and the systemic circulation were associated with tiny nonvenous channels filled with perisinusal fluids (red asterisk). (B) Native 3D T1 SPACE DANTE sequence. Coronal imaging plane across the cribriform plate (CP) and the maxillary sinuses (MS). Intracranially, enhanced gadobutrol signal was detected along the SSS (white arrow) and the perivenous space of several cortical veins (white arrowhead). Extracranially, the olfactory epithelium (OE) was strongly enhanced after gadobutrol injection, but no gadobutrol signal was observed from the dura mater across the CP (blue arrow). Scale bar: 1 cm (A and B). (C–E) Quantitative MRI of human MLVs. Data show mean + SEM; one-way ANOVA with Dunn’s multiple-comparisons test; *, P < 0.05. (C and D) MLV volume measurements between females and males (C) and between neurological disorders (D). For each patient, MLV volume was normalized against the TIV. MLV volume was greater in males (n = 4) than in females (n = 7). No significant difference was observed between groups of patients, except for the male patient with GSD, who showed the greatest MLV volume. (E) MLV/vein volume ratio in the superior sagittal (SSS), the straight (StS), and the lateral (LS) and cavernous (CAV) sinuses. MLVs of CAV have a higher MLV/vein volume ratio compared with other MLV beds and show higher interindividual variation. Data show mean + SEM; one-way ANOVA with Dunn’s multiple-comparisons test; *, P < 0.05.

Figure 7.

Altered MLV imaging overlaps with skull bone erosion in a patient with GSD. (A) 3D reconstruction of a computed tomography scan in superior view showing the large erosion of the right parietal bone (white circle). (B–F) Meningeal vascular 3D mapping by MRI. (B) Superior view showing a large area of gadobutrol slow-flow (yellow) in the region of the vanished bone (white circle) and extending to the perisinusal area of the SSS. (C) Posterior view showing the extension of the perisinusal gadobutrol enhancement (yellow) all along the SSS. (D) Lateral view showing that MLV hypertrophy involved MLVs of the SSS, StS, LS, SS, and CAV. This MLV hypertrophy was associated with a malformation of the StS that was replaced by a large venous plexus (asterisk) encompassing the StS and an abnormally persistent embryonic falcine sinus (FaS). (E) Lateral view reconstruction highlighting the close relationships between the MMA (red arrow) and its related MLVs (black arrows). (F) Anterior view allowing the visualization of the CAV, ica, and vertebral arteries (VA). MLV hypertrophy allowed to visualize the continuity of MMA-related MLVs toward the skull base. Note that, instead of lining the MMA toward the spinosum foramen, MLVs followed the middle meningeal vein toward the sphenoparietal sinus (SPPS) toward the CAV. Scale bar: 1 cm (A–F).

Figure 8.

Schematic representation of the dural venolymphatic complex in mice and humans. (A) Summary schematic of lymphatic CSF drainage circuits in the mouse head. Cerebral veins and dural sinuses (blue) drain blood from the brain. 1–6, novel MLV uptake sites where perivenous glymphatic efflux from these regions communicates with perisinusal areas of the dura mater. Fluorescent green, MLVs; bluish green, olfactory lymphatic vessels; dark green, extracranial lymphatic system; red, internal carotid artery; beige, nasopharynx; amLN, accessory mandibular LN; cc, carotid canal; cpf, cribriform plate foramina; fc, foramen caecum; ica, internal carotid artery; ijv, internal jugular vein; IOS, inferior olfactory sinus; IPETS, inferior petrosal sinus; ipf, interpterygoid foramen; jf, jugular foramen; mLN, mandibular LN; NP, nasopharynx; OE, olfactory epithelium; pef, posterior ethmoid foramen; pfv, posterior facial vein; psf, petrosquamous fissure; PSS, petrosquamous sinus; RCS, rostral confluence of sinuses; RE, respiratory epithelium; SOS, superior olfactory sinus; SS, sigmoid sinus SSS, superior sagittal sinus. (B) Schematic representation of the dural venolymphatic complex and lymphatic outflows in humans. Signal enhancement of gadobutrol (yellow) was depicted around most dural sinuses, including the SSS, the straight sinus (StS), the transverse sinus (TS), the sigmoid sinuses (SS), and the CAV. Gadobutrol flow was also detected in the carotid canal along the ica or in transforaminal routes along the trigeminal nerve branches (gray) through the superior orbital fissure (sof), the foramen rotundum (fr), and the foramen ovale (fo). cLN, cervical LNs; ijv, internal jugular vein; MS, marginal sinus.