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A mesothelium divides the subarachnoid space into functional compartments

Article in Science · January 2023

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RES EARCH

BRAIN ANATOMY itself was 14.2 ± 0.5 mm, hence thinner than
dura (21.8 ± 1.3 mm, n = 6 mice). The dura vas-
A mesothelium divides the subarachnoid space into culature is surrounded by collagen fibers,
whereas SLYM covers the subarachnoid ves-
functional compartments sels. The organization and calibers of the two
sets of vasculature also exhibit distinct differ-
Kjeld Møllgård1*†, Felix R. M. Beinlich2†, Peter Kusk2†, Leo M. Miyakoshi2†, Christine Delle2, ences (Fig. 1, B and C).
Virginia Plá2, Natalie L. Hauglund2, Tina Esmail2, Martin K. Rasmussen2, Ryszard S. Gomolka2, A key question is whether SLYM constitutes
Yuki Mori2, Maiken Nedergaard3* an impermeable membrane that functionally
compartmentalizes the subarachnoid space.
The central nervous system is lined by meninges, classically known as dura, arachnoid, and pia mater. To test this, Prox1-EGFP+ mice were first in-
We show the existence of a fourth meningeal layer that compartmentalizes the subarachnoid space jected with 1-mm microspheres conjugated
in the mouse and human brain, designated the subarachnoid lymphatic-like membrane (SLYM). SLYM is to a red fluorophore into the subdural outer
morpho- and immunophenotypically similar to the mesothelial membrane lining of peripheral organs and superficial compartment of the subarachnoid
body cavities, and it encases blood vessels and harbors immune cells. Functionally, the close apposition space along with an injection of 1-mm micro-
of SLYM with the endothelial lining of the meningeal venous sinus permits direct exchange of small spheres conjugated to a blue fluorophore dis-
solutes between cerebrospinal fluid and venous blood, thus representing the mouse equivalent of the tributed within the inner deep subarachnoid
arachnoid granulations. The functional characterization of SLYM provides fundamental insights into space compartment by cisterna magna injec-
brain immune barriers and fluid transport. tion (Fig. 2A). In vivo two-photon microscopy
showed that the red microspheres were con-

E
fined to the outer superficial compartment,
merging evidence supports the concept lagen fibers, while the vascular volume was whereas the blue microspheres remained
that cerebrospinal fluid (CSF) acts as a labeled by a Cascade Blue conjugated dextran, trapped in the inner deep subarachnoid space

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quasi-lymphatic system in the central and astrocytes were labeled by sulforhod- compartment. Quantitative analysis showed
nervous system (1). Cardiovascular pul- amine 101 (SR101, intraperitoneally) (15, 16). that the 1-mm microspheres did not cross
satility drives CSF inflow along periar- Below the parallel-oriented collagen bundles SLYM from either side. Yet, many solutes in
terial spaces into deep brain regions (2, 3), in dura, we noted a continuous monolayer of CSF, such as cytokines and growth factors, are
where CSF exchange with interstitial fluid, flattened Prox1-EGFP+ cells intermixed with considerably smaller than 1 mm in diameter (17).
facilitated by glial aquaporin 4 (AQP4) water loosely organized collagen fibers. This sub- Therefore, we sought to determine whether a
channels (4), takes place. Fluid and solutes arachnoid lymphatic-like membrane (SLYM) small tracer could pass through SLYM. In these
from the neuropil are cleared along multiple divides the subarachnoid space into an outer experiments, tetramethylrhodamine (TMR)–
routes, including perivenous spaces and cra- superficial compartment and an inner deep dextran (3 kDa) was administered into the
nial nerves, for ultimate export to the venous compartment lining the brain (Fig. 1A). Quan- deep inner subarachnoid space via the cister-
circulation via meningeal and cervical lym- titative in vivo analysis of the somatosensory na magna in Prox1-EGFP+ mice. In six mice, the
phatic vessels (5, 6). CSF reabsorption may cortex revealed that the thickness of SLYM small tracer did not cross the EGFP-expressing
also occur at the sinuses via the arachnoid
granulations—although this has not been
described in rodents (7–10). Despite the ef-
forts dedicated to studying CSF flow along the
glymphatic-lymphatic path, it remains to be
determined how CSF is transported within
the large cavity of the subarachnoid space
(11, 12). In this study, we explored how CSF
and immune cell trafficking are organized
within the subarachnoid space surrounding
the brains of mice and humans.
The meningeal membranes were first ana-
lyzed by in vivo two-photon microscopy in the
somatosensory cortex of Prox1-EGFP+ reporter
mice (Prox1, prospero homeobox protein 1;
EGFP, enhanced green fluorescent protein). Fig. 1. In vivo imaging depicts a fourth meningeal layer. (A) In vivo two-photon imaging of Prox1-EGFP+
Prox1 is a transcription factor that determines reporter mice viewed through a closed cranial window placed over the somatosensory cortex. Maximum
lymphatic fate (13, 14). Second harmonic gen- projection and three-dimensional (3D) views depict the spatial distribution of dura mater collagen fibers
eration was used to visualize unlabeled col- (gray) detected by second harmonic generation. Prox1-EGFP+ cells (green) intermixed with the irregular
sparse collagen fibers (purple) localized below dura. This subarachnoid lymphatic-like membrane is
1
Department of Cellular and Molecular Medicine, Faculty of
abbreviated SLYM. Blood vessels outlined by Cascade Blue conjugated dextran (red, 10 kDa, iv) are located at
Health and Medical Sciences, University of Copenhagen, 2200 the cortical surface. (Inset) A lateral view of the 3D reconstruction with all the layers displayed individually
Copenhagen, Denmark. 2Division of Glial Disease and along the z axis to facilitate spatial comprehension. (B) Two-photon imaging over the sensorimotor cortex
Therapeutics, Center for Translational Neuromedicine, Faculty of
in a Prox1-EGFP reporter mouse. The vasculature was outlined by intravenous injection of TMR-dextran
Health and Medical Sciences, University of Copenhagen, 2200
Copenhagen, Denmark. 3Division of Glial Disease and (2000 kDa), and z-stacks were collected. Representative 3D reconstruction of the z-stacks. The vasculature
Therapeutics, Center for Translational Neuromedicine, University in dura (magenta) is embedded in collagen fibers (white). In contrast, the vasculature in the subarachnoid
of Rochester Medical Center, Rochester, NY 14642, USA. space (red) is overlaid by SLYM (green). (C) Orthogonal sections through the z-stack show that the
*Corresponding author. Email: [email protected]
(M.N.); [email protected] (K.M.) vasculature in dura is surrounded by collagen fibers. SLYM is located beneath dura, in close apposition
†These authors contributed equally to this work. with the large-caliber subarachnoid vessels.

Møllgård et al., Science 379, 84–88 (2023) 6 January 2023 1 of 5

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SLYM (Fig. 2B and fig. S1). Yet, in mice with acterize the meningeal membranes. To preserve binding protein 2 (CRABP2) (Fig. 3, A and D),
dural damage and leakage of CSF, the tracer the integrity of the meningeal membranes, which is restrictively expressed in dural and
was observed on both sides of the EGFP+ mem- sections were next obtained from whole heads arachnoid cells during early development (21).
brane (fig. S1). Thus, SLYM divides the sub- of Prox1-EGFP+ mice. Immunohistochemistry In contrast to SLYM, lymphatic vessels in dura
arachnoid space into an upper superficial and revealed that Prox1-EGFP+ cells lined the ven- were positive for all the classical lymphatic
a lower deep compartment for solutes ≥3 kDa. tral parts of the entire brain surface (Fig. 3A). antigens, Prox1-EGFP+, PDPN+, LYVE1+, and
SLYM is therefore a barrier that limits the ex- Immunolabeling showed that the Prox1-EGFP+ VEGFR3+, but was CRABP2− (fig. S2). Nota-
change of most peptides and proteins, such as SLYM cells were positive for another lymphatic bly, analysis of adult human cerebral cortex
amyloid-b and tau, between the upper and marker, podoplanin (PDPN) (19), but not for the depicted that above the pia mater, a CRABP2+/
lower subarachnoid space compartments. lymphatic vessel endothelial receptor 1 (LYVE1) PDPN+ membrane was present in the entire
Live brain imaging avoids fixation artifacts (20) (Fig. 3, A, lower right panels, and D). SLYM subarachnoid space (Fig. 3, B and C). Thus,
(18) but cannot immunophenotypically char- also labeled for the cellular retinoic acid– SLYM also surrounds the human brain. We

Fig. 2. SLYM represents a barrier that subdivides

the subarachnoid space into two compartments.
(A) Representative image of a 3D view of maximum
projection collected after dual injections of red
microspheres (red, 1 mm) into the outer superficial
subarachnoid space (subdural) and blue micro-
spheres delivered into the inner deep subarachnoid
space by cisterna magna injection (blue, 1 mm) in a

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Prox1-EGFP+ mouse. Graphs show a comparison
of red microspheres versus blue microspheres
detected in both the outer and inner subarachnoid
space (SAS). Two-tailed unpaired t test; outer
SAS, P < 0.01; inner SAS, P < 0.01; n = 4 mice.
(B) Representative in vivo z-stack of a Prox1-EGFP
mouse injected with a 3-kDa TMR-conjugated dextran CSF tracer delivered via the cisterna magna. Upper panels depict SLYM (green) and the perivascular distribution
of the dextran (red) as well as the two channels merged. Lower panel displays the merge of the two channels and orthogonal optical sections showing that tracer
is confined to below the membrane. Graph shows the mean tracer intensity detected below and above the membrane. Two-tailed unpaired t test with Welch’s correction,
P < 0.01, n = 6 mice. Significance shown as **P < 0.01. au, arbitrary units; CM, cisterna magna; d, dorsal; v, ventral.

Fig. 3. Immunophenotypic characterization

of SLYM in the mouse and human brain.
(A) Sections of Prox1-EGFP+ mouse brain after
decalcification of the whole head counterstained
with Mayer’s hematoxylin (M-HE, purple) show
that SLYM (arrowheads) is positively immunolabeled
for CRABP2 (brown) and Prox1-EGFP+/PDPN+/
LYVE1−/VEGFR3− and encases the entire brain,
covering its dorsal and ventral portions (purple
and blue insets, respectively). (B) Adult human
brain sections immunolabeled for CRABP2 and PDPN
reveal the presence of SLYM (arrowheads) that
enwraps the subarachnoid space blood vessels
(arrow). Ependymal and pia mater cells are also
PDPN+ (asterisks). (C) Serial sections of the same
adult human material immunolabeled for Prox1,
PDPN, CLDN11, E-CAD, and LYVE1. SLYM is indicated
by arrowheads. (D) Confocal images of SLYM
immunolabeling showing positive labeling for
PDPN and CRABP2 (both in red). No signal was
detected for LYVE1 or VEGFR3. (E) Schematic
representations of the immunophenotypical
characterization of the meningeal layers, meningeal
lymphatic vessels, and arachnoid trabeculations.
For arachnoid trabeculae, CRABP2* signifies that
the trabeculae are CRABP2− in the outer SAS
but CRABP2+ in the inner SAS. For pia, PDPN*
indicates that pia is PDPN+ in many regions of pia, but not all. VEGFR3* signifies that pia was VEGFR3+ only in a few regions. Aq, aqueduct; BA, basilar artery;
BS, brain stem; BV, blood vessel; Cb, cerebellum; Ctx, cerebral cortex; Ep, ependyma; LV, lateral ventricle.

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infer that the SLYM monolayer of Prox1-EGFP+ Pial cells covering the cortical surface also ex- phatic vessels were also observed in the lungs
cells organizes into a membrane rather than hibited an immune-labeling profile that dif- and intestinal tract (fig. S5, B and C). Thus,
vessel structures and exhibits a distinctive fered from that of SLYM (figs. S3 and S4). We SLYM may represent the brain mesothelium
set of lymphatic markers (Fig. 3E). To dis- conclude that SLYM constitutes a fourth men- and, as such, covers blood vessels in the sub-
tinguish SLYM from the structures forming ingeal layer surrounding the mouse and hu- arachnoid space (Fig. 1) (26). The mesothelium
the arachnoid mater, we used immunolabeling man brain displaying lymphatic-like features is present where tissues slide against each
against claudin-11 (CLDN-11), a main constit- (Prox1-EGFP+, PDPN+, LYVE1−, CRABP2+, other and is believed to act as a boundary
uent of the tight junctions that create the VEGFR3−, CLDN-11−, and E-Cad−) and that lubricant to ease movement (27). Physiological
arachnoid barrier cell layer (ABCL) (22). CLDN- SLYM is phenotypically distinct from dura, the pulsations induced by the cardiovascular sys-
11 was densely expressed in ABCL as well as in arachnoid, and pia mater (Fig. 3E). Interest- tem, respiration, and positional changes of the
the stromal cells of the choroid plexus, but ingly, SLYM expressed PDPN, sharing a trait head are constantly shifting the brain within
SLYM was CLDN-11− (fig. S3, A and B). Ad- with the mesothelium lining the body cavities the cranial cavity. SLYM may, like other meso-
ditionally, ABCL was distinctively positive for (26). Accordingly, we observed PDPN+ cells thelial membranes, reduce friction between
E-cadherin (E-Cad) (Fig. 3C), as previously re- lining the kidney, as well as PDPN+ podocytes the brain and skull during such movements.
ported (23, 24). We also compared SLYM to the in the kidneys of adult C57BL/6J mice (fig. Does SLYM have additional functions? The
arachnoid trabeculae (25), collagen-enriched S5A). In a human fetus, a PDPN+ membrane arachnoid villi and granulations are defined
structures that span the subarachnoid space, corresponding to pericardium, pleura, and peri- as protrusions of the arachnoid membrane
finding that cells surrounding the arachnoid toneum encases the developing heart, lungs, into the lateral walls of the sinus veins and
trabeculae are Prox1-EGFP−/LYVE1− (fig. S3C). and intestinal tract, respectively. PDPN+ lym- are believed to act as passive filters that drain

Fig. 4. SLYM forms subarachnoid villus–like

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structures at the venous sinus walls in mice.
(A and B) Schematic diagrams illustrating the
region of interest. (C and D) Parasagittal
consecutive sections from a decalcified mouse
whole head stained for (C) the SLYM marker
Prox1-EGFP and (D) the arachnoid barrier cell
marker E-Cad. Rectangular insets on the left in
(B), (C), and (D) are shown in higher magnification
on the right. A Prox1-positive arachnoid villus–
like structure (AV) and a vein from the dorsal
venous system are in direct contact with the
transverse sinus wall (SW), which is lacking an
intervening ABCL [inset in (C) and (D)]. The ABCL
[arrows in inset in (C)] are not stained for Prox1,
in contrast to the strongly stained SLYM layer,
whereas the opposite pattern of reactivity is
depicted in the adjacent section [inset in (D)], where
ABCL is positive for E-Cad and SLYM is negative.
Arrowheads point to pia. In (C), the narrow dura
layer, indicated by small arrows, is facing the
venous endothelial layer (VEC), indicated by
slender darker arrows. (C) and (D) are the same
magnification, as are their insets. (E) Confocal
imaging of Prox1-EGFP and E-Cad shows that the
signals do not colocalize.

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CSF from the subarachnoid space into the was detected from the exposed cortex, includ- ent in the vascular compartment (fig. S7, A
venous sinus system (7–10). The arachnoid ing from the sinus venous wall (fig. S7D). In to C), we propose that the apposition of the
villi and granulations are present in the brains another set of control experiments, GeNL was venous endothelia and SLYM represents ro-
of humans, primates, and larger animals such injected into the soft ear tissue, while FFz dent arachnoid villus–like structures, com-
as dogs, but not in the brains of rodents was delivered intravenously. Consistent with parable to those in human brain.
(28, 29). We critically reexamined this issue to the notion that peripheral blood vessels are The mesothelium surrounding peripheral
evaluate the distribution of SLYM in relation leaky (11), light emission was clearly observed organs acts as an immune barrier (26). Does
to the superior sagittal and transverse sinus. in the region of the ear injected with Nano- SLYM also impede the entry of exogenous
Sections obtained from decalcified heads of Luc but not in surrounding noninjected re- particles into CSF? In vivo two-photon imag-
Prox1-EGFP+ mice showed that Prox1-EGFP+ gions of the same ear. No signal was observed ing of Prox1-EGFP+ mice injected intraven-
SLYM cells often were in direct contact with in the venous compartment, likely reflecting ously with rhodamine 6G (Rhod6G) to label
the venous sinus endothelial cells (Fig. 4A). that blood flow rapidly diluted the biolumines- leukocytes (35) showed that a large number
Thus, the arachnoid barrier cell layer (CLDN- cence signal (fig. S7E). Together, this analysis of Rhod6G+ myeloid cells are embedded in
11+/E-Cad+), which normally separates dura shows that a small molecule, FFz, can enter SLYM (Fig. 5A). The number of Rhod6G+
from the subarachnoid space, was lacking the central nervous system (CNS) from the leukocytes in dura and SLYM was directly
in discrete areas allowing SLYM to directly blood and activate an enzyme, NanoLuc, pres- comparable, suggesting a prominent role of
contact the venous sinus wall (Fig. 4B). Prox1- ent in CSF, resulting in the generation of SLYM in CNS immune responses, which sup-
EGFP+ SLYM cells were not positive for CLDN- photons along the wall of the venous sinus. ports the finding that leptomeninges are den-
11 or E-Cad, which distinguish the arachnoid On the basis of the juxtaposition of SLYM sely populated with immune cells (36) (Fig.
barrier cell layer (fig. S6). and the venous endothelium in histological 5A). How do systemic inflammation and aging
Are the close appositions of SLYM and the examination (Fig. 4A), the selective generation affect the immune cell populations residing in
venous endothelial cells permeable, allowing of photons when luciferase was injected into SLYM? Ex vivo analysis of brain sections
the exchange of small molecules between CSF, and the fact that the substrate was pres- obtained from Prox1-EGFP+ mice showed that,

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blood and CSF? To test this, we used the prin-
ciples of bioluminescence, wherein the con-
vergence, in the same compartment, of an
enzyme with its substrate is needed to trigger
light emission. First, we delivered the lucifer-
ase enzyme from Oplophorus gracilirostris
(NanoLuc) fused to the fluorescence tag
mNeongreen (GeNL, 44 kDa) (30) into CSF
via the cisterna magna of wild-type (C57bl/6)
mice, and allowed it to circulate for 30 min
to ensure thorough distribution by the glym-
phatic system. The distribution of GeNL was
verified by mNeongreen fluorescence. Then,
the blood-brain barrier (BBB)–impermeable
substrate fluorofurimazine (FFz, 433 Da) (31)
was administered intravenously (fig. S7, A
to C) (32). After intravenous injection of FFz,
a bright bioluminescence signal catalyzed by
GeNL was detected specifically near the large
venous sinus wall (fig. S7, A and B). The bio-
luminescence signal was particularly strong
around the confluence of sinuses (fig. S7B).
The distribution of the bioluminescence sig-
nal was quantified by plotting the mean sig-
nal intensity profiles perpendicular to the
venous wall of the transverse sinus and supe-
rior sagittal sinus. The mean bioluminescence
signal profiles intersected with the fluores-
cence signal profiles of the intravascular tracer Fig. 5. SLYM hosts a large number of myeloid cells. (A) (Left) In vivo two-photon microscopy of Prox1-
(TMR-dextran, 70 kDa) or with shadow im- EGFP+ mice injected with Rhod6G (red) shows that SLYM (EGFP+, green) is permeated by myeloid cells
aging of the inverted GeNL signal outlining similar to dura (collagen fibers, gray). Middle panels show orthogonal sections depicting Rhod6G + cells in
the vascular wall (fig. S7C). Thus, the biolu- dura and SLYM, respectively. (Right) In vivo quantification of the number of Rhod6G+ cells present in dura
minescence signal was restricted to the venous and SLYM. Values are expressed as mean ± SEM, two-tailed unpaired t test with Welch’s correction, P =
wall of the two major sinuses lacking a BBB 0.5748, n = 7 mice. (B) Representative image showing the accumulation of CD45+ cells along the pial
(33, 34), consistent with the notion that FFz vessels. (C) The percentage of area covered by CD45+ cells was significantly increased both in aged (12- to
is BBB-impermeable and requires the cata- 13-month-old) mice and in response to inflammation (LPS 4 mg/kg, ip, 24 hours). Values are expressed as
lyzation enzyme NanoLuc to generate photons mean ± SEM, two-tailed unpaired t test with Welch’s correction, P < 0.005, n = 3 mice. (D) LYVE1 macrophages
(fig. S7, A to C). In control experiments, FFz were also found in the SLYM layer, being more prominent in aged and LPS-treated animals than in healthy
was delivered intravenously, while the GeNL young Prox1-EGFP mice. (E) The mannose receptor CD206 was detected at similar levels in the young,
injection into CSF was omitted. In these con- aged, and LPS-treated groups, suggesting that SLYM may act as a niche for border-associated mouse
trol experiments, no bioluminescence signal macrophages. Significance shown as *P < 0.05. Ctr, control.

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in the control group, CD45+ cells were abun- association with the brain surfaces, is likely to 30. K. Suzuki et al., Nat. Commun. 7, 13718 (2016).
dant, located mostly along pial vessels in the play a prominent role in this surveillance. 31. Y. Su et al., Nat. Methods 17, 852–860 (2020).
32. M. P. Hall et al., ACS Chem. Biol. 7, 1848–1857
surface of the brain (Fig. 5B). This observa- Herein, we showed a large increase in the num- (2012).
tion, together with the significant increase ber and diversity of immune cells residing in 33. J. Rustenhoven et al., Cell 184, 1000–1016.e27
in CD45+ in inflammation-prone conditions SLYM in response to acute inflammation and (2021).
34. P. Mastorakos, D. McGavern, Sci. Immunol. 4, eaav0492
[aging and lipopolysaccharide (LPS)–treated natural aging. Physical rupture of SLYM could, (2019).
mice, 4 mg/kg of body weight, intraperito- by altering CSF flow patterns, explain the pro- 35. H. Baatz, M. Steinbauer, A. G. Harris, F. Krombach, Int. J.
neally (ip), 24 hours] (Fig. 5C), suggests that longed suppression of glymphatic flow after Microcirc. Clin. Exp. 15, 85–91 (1995).
36. A. Merlini et al., Nat. Neurosci. 25, 887–899
SLYM can act as a CD45+ recruiting and/or traumatic brain injury as well as the height- (2022).
proliferating site in pathological conditions. ened posttraumatic risk of developing Alz- 37. A. Louveau et al., J. Clin. Invest. 127, 3210–3219
Of note, the dose of LPS used (4 mg/kg) did heimer’s disease (41, 42). Rupture of SLYM (2017).
38. S. Da Mesquita, Z. Fu, J. Kipnis, Neuron 100, 375–388
not affect the BBB (fig. S8). Additional im- will also permit the direct passage of immune
(2018).
mune markers showed that LYVE1+ (Fig. 5D), cells from the skull bone marrow (33, 43) into 39. Z. Xu et al., Mol. Neurodegener. 10, 58 (2015).
CD206+ (Fig. 5E), and CD68+ (fig. S9) macro- the inner subarachnoid space, with direct ac- 40. L. Wang et al., Brain Pathol. 29, 176–192 (2019).
phages can be found in SLYM, together with cess to the brain surfaces, possibly explaining 41. A. Z. Mohamed, P. Cumming, J. Götz,
F. Nasrallah; Department of Defense Alzheimer’s Disease
dendritic cells (CD11c+) (fig. S9). Despite the the prolonged neuroinflammation after trau- Neuroimaging Initiative, Eur. J. Nucl. Med. Mol. Imaging 46,
absence of CD3+ and CD19+ lymphocytes (fig. matic brain injury (44). SLYM may also be 1139–1151 (2019).
S9), our results indicate that SLYM functions directly involved in CNS immunity, in addi- 42. J. D. Flatt, P. Gilsanz, C. P. Quesenberry Jr., K. B. Albers,
R. A. Whitmer, Alzheimers Dement. 14, 28–34 (2018).
as a niche for immunological surveillance. Thus, tion to being host to many immune cells. 43. S. Brioschi et al., Science 373, eabf9277 (2021).
in young, healthy mice, SLYM hosts CD45+ Lymphatic-like tissues can transform quickly 44. J. J. Iliff et al., J. Neurosci. 34, 16180–16193
cells, but the number and diversity of innate in the setting of inflammation, which in the (2014).
45. N. B. Pikor, A. Prat, A. Bar-Or, J. L. Gommerman,
immune cells rapidly expands in LPS-induced brain may be of notable relevance for dis- Front. Immunol. 6, 657 (2016).
inflammation and was also significantly al- eases such as multiple sclerosis (45).

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tered in aged mice. We conclude that SLYM AC KNOWLED GME NTS

fulfills the characteristics of a mesothelium RE FERENCES AND NOTES We acknowledge P. S. Froh and H. Nguyen (Department of
Cellular and Molecular Medicine, Faculty of Health and Medical
by acting as an immune barrier that prevents 1. J. J. Iliff et al., Sci. Transl. Med. 4, 147ra111 (2012).
Sciences, University of Copenhagen, Denmark) for their excellent
exchange of small solutes between the outer 2. H. Mestre et al., Nat. Commun. 9, 4878 (2018). technical assistance for the histology and immunohistochemistry
3. N. E. Fultz et al., Science 366, 628–631 (2019). of the decalcified samples. We also thank D. Xue for expert
and inner subarachnoid space compartments
4. H. Mestre et al., eLife 7, e40070 (2018). graphical support, B. Sigurdsson for analysis, and H. Hirase,
and by covering blood vessels in the sub- 5. A. Louveau et al., Nature 523, 337–341 (2015). N. Cankar, and N. C. Petersen for critical reading of the manuscript.
arachnoid space. 6. A. Aspelund et al., J. Exp. Med. 212, 991–999 Funding: Funding was provided by Lundbeck Foundation grant
(2015). R386-2021-165 (M.N.), Novo Nordisk Foundation grant
Discussion 7. L. H. Weed, J. Anat. 72, 181–215 (1938). NNF20OC0066419 (M.N.), the Vera & Carl Johan Michaelsen’s
8. A. Key, G. Retzius, Studien in der Anatomie des Legat Foundation (K.M.), National Institutes of Health grant
The critical roles of the meningeal membranes R01AT011439 (M.N.), National Institutes of Health grant
Nervensystems und des Bindegewebes (Samson & Wallin,
lining the brain have only recently been 1876). U19NS128613 (M.N.), US Army Research Office grant MURI
acknowledged (5, 37). It is now known that 9. W. E. le Gros Clark, J. Anat. 55, 40–48 (1920).
W911NF1910280 (M.N.), Human Frontier Science Program grant
RGP0036 (M.N.), the Dr. Miriam and Sheldon G. Adelson Medical
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Research Foundation (M.N.), and Simons Foundation grant 811237
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sels in the meninges and that suppression of (M.N.). The views and conclusions contained in this article are
11. M. K. Rasmussen, H. Mestre, M. Nedergaard, solely those of the authors and should not be interpreted as
this drainage accelerates protein aggregation Physiol. Rev. 102, 1025–1151 (2022). representing the official policies, either expressed or implied, of
and cognitive decline in animal models of 12. A. Drieu et al., Nature 611, 585–593 (2022). the National Institutes of Health, the Army Research Office, or the US
neurodegeneration (38–40). SLYM is Prox1+/ 13. I. Choi et al., Blood 117, 362–365 (2011). Government. The US Government is authorized to reproduce and
PDPN+/LYVE1−/CRABP2+/VEGFR3−/CLDN-11−/ 14. J. T. Wigle et al., EMBO J. 21, 1505–1513 distribute reprints for Government purposes notwithstanding any
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E-Cad− and thereby distinct from the tradi-
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tional meningeal membranes, including dura, (2012). writing of the manuscript. Author contributions: K.M. and M.N.
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into two compartments, suggesting that CSF 18. H. Mestre, Y. Mori, M. Nedergaard, Trends Neurosci. 43, Competing interests: The authors declare that they have no
competing interests. Data and materials availability: All data are
transport is more organized than currently 458–466 (2020).
available in the main text or the supplementary materials.
acknowledged. For example, SLYM covering 19. M. Tomooka, C. Kaji, H. Kojima, Y. Sawa, Acta Histochem.
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Møllgård et al., Science 379, 84–88 (2023) 6 January 2023 5 of 5

 A mesothelium divides the subarachnoid space into functional compartments
Kjeld MøllgårdFelix R. M. BeinlichPeter KuskLeo M. MiyakoshiChristine DelleVirginia PláNatalie L. HauglundTina
EsmailMartin K. RasmussenRyszard S. GomolkaYuki MoriMaiken Nedergaard

Science, 379 (6627), • DOI: 10.1126/science.adc8810

An extra layer lines the brain

The traditional view is that the brain is surrounded by three layers, the dura, arachnoid, and pia mater. Møllgård
et al. found a fourth meningeal layer called the subarachnoid lymphatic-like membrane (SLYM). SLYM is
immunophenotypically distinct from the other meningeal layers in the human and mouse brain and represents a
tight barrier for solutes of more than 3 kilodaltons, effectively subdividing the subarachnoid space into two different
compartments. SLYM is the host for a large population of myeloid cells, the number of which increases in response to
inflammation and aging, so this layer represents an innate immune niche ideally positioned to surveil the cerebrospinal

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fluid. —SMH

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