Science: Rewrite the textbook! For the first time, scientists have discovered that there may be a fourth layer of meninges in the human brain!

*For medical professionals only

The surface of the brain has 3 layers of meningeal structures from the outside to the inside - dura mater, arachnoid and pia mater, which is the basic knowledge
of brain anatomy that every medical student learns.

However, perhaps it's
time to update this basics.

Recently, the team of Maiken Nedergaard from the University of Rochester Medical Center and the team of Kjeld Møllgård of the University of Copenhagen discovered the fourth layer of meningeal structure in mouse and human brains and named it subarachnoid lymphangioid membrane (SLYM), and the relevant research results were published in the journal Science [1].

Located between the arachnoid and pia mater, SLYM divides the subarachnoid space into superficial and deep layers and envelops the blood vessels in the subarachnoid space, and its morphology and immunophenotype resemble the mesothelium on the surface of peripheral organs
.
Functionally, SLYM not only acts as a lubricating effect, reducing friction between the brain and the skull during exercise, but also separates and controls the flow of cerebrospinal fluid on the surface of the brain and the exchange
of substances.

Anatomical location of SLYM

In addition, SLYM also plays an important role in the brain's immune function, and complete SLYM prevents peripheral immune cells from entering the
brain at will.
At the same time, SLYM has its own population of immune cells that monitor the brain for signs of inflammation or infection through contact with cerebrospinal fluid
.

Overall, the discovery of SLYM is important for further understanding of the clearance of metabolites in the brain and immune function
.

Screenshot of the first page of the paper

The human exploration of the brain began with the understanding
of anatomy.

With the advancement of research technology, many stereotypes of the brain have been shattered, such as the original belief that there is no lymphatic system in the brain, but the discovery of the lymphoid system [2] (also called the glial lymphatic pathway) by Maiken Nedergaard's team in 2012 and the discovery of meningeal lymphatic vessels [3] in 2015 have subverted this understanding
.

The study stemmed from a conjecture by Kjeld Møllgård's team, which is present on the surface of peripheral organs (which encloses and protects organs and contains immune cells), in the brain [4]?

To analyze the membrane structure outside the brain tissue in more depth, Møllgård et al.
used two-photon microscopy to observe the meninges
of the somatosensory cortex of Prox1-EGFP⁺ (Prox1: a transcription factor that determines lymphatic vessel development) mice.
At the same time, the researchers visualized the unlabeled collagen fibers using the second harmonic and labeled blood vessels and astrocytes using Cascade Blue-conjugated dextran and sulforhodamine 101, respectively, to distinguish the layers of structures
outside the brain tissue.

Underneath the dural fiber bundle, they found the presence of a monolayer of continuous flat Prox1-EGFP⁺ cells
mixed with loosely arranged collagen fibers.
This layer of cells, which divides the subarachnoid space into superficial and deep layers and covers the subarachnoid blood vessels, is 14.
2±0.
5 μm thick, thinner than the dura mater (21.
8±1.
3 μm).

The researchers named this membrane SLYM
.

Two-photon imaging in mice shows the presence of a 4th membrane on the surface of the brain

Then the question arises, how permeable is SLYM to matter? This is related to the exchange
of substances on both sides of the membrane.

To test this, Møllgård and his colleagues injected red fluorophore-bound 1 μm diameter microspheres into the shallow layer of the Prox1-EGFP⁺ mouse subarachnoid space, while injecting 1 μm diameter microspheres bound to the blue fluorophore into the deep layers of the subarachnoid space and then observed
using two-photon microscopy.
The results showed that the red beads were confined to the shallow layer while the blue microspheres were confined to the deep layers, indicating that SLYM is not permeable
to spheroids with a diameter of 1 μm.

But many solutes in cerebrospinal fluid, such as cytokines and growth factors, are much smaller than 1 μm
in size.
Therefore, the researchers injected a smaller tracer TMR-glucan (3 kDa) into the deep layers of the subarachnoid space of Prox1-EGFP⁺ mice, and the results showed that TMR-glucan still could not penetrate SLYM
.
However, in mice with dural injury and cerebrospinal fluid leak, injected tracers
were observed on both sides of SLYM.

Based on the above results, Møllgård's team believes that SLYM physically divides the subarachnoid space into superficial and deep layers, and can limit the exchange
of most peptides and proteins between the superficial and deep layers of the subarachnoid space.

SLYM limits the exchange of most peptides and proteins between the superficial and deep layers of the subarachnoid space

Next, Møllgård's team characterized the immunophenotype of SLYM and found that another lymphatic marker in SLYM was positive for PDPN expression and negative for lymphatic endothelial cell receptor 1 (LYVE1).

In addition, cellular tretinoin-binding protein 2 (CRABP2) expression was positive
in SLYM.
Unlike SLYM, lymphatic vessels on the dura mater express all classical lymphatic markers (Prox1-EGFP+, PDPN+, LYVE1+, and VEGFR3⁺, but CRABP2^-).

Notably, analysis of the adult cerebral cortex showed that the subarachnoid space above the pia mater also had a membrane structure
of CRABP2+/PDPN⁺.
Therefore, SLYM is also present
in the human brain.

SLYM is also present in the human brain

To further demonstrate that SLYM is a different membrane structure from the arachnoid membrane, the researchers performed immunostaining for CLDN-11, the main component
that forms the tight junction of the arachnoid barrier cell layer (ABCL).
The staining results showed that CLDN-11 expression was negative in SLYM, while CLDN-11 was densely expressed in both ABCL and choroid plexus stromal cells, indicating that SLYM and arachnoid are two different layers
.
At the same time, the immunolabeling characteristics of SLYM and pia mater were also different
.

From this, the researchers concluded that SLYM is a layer 4 meningeal structure that surrounds the mouse and human brains, differs immunophenotypically from dura mater, arachnoid and pia mater, and exhibits lymphoid features (Prox1-EGFP+, PDPN+, LYVE1^-, CRABP2⁺, VEGFR3).
^-, CLDN-11- and E-Cad^-).

Interestingly, SLYM has the same characteristics
as the mesothelium on the surface of peripheral organs.
Therefore, SLYM may be the mesenchymal layer of the brain, which plays a role
in reducing friction between brain tissue and the skull during exercise.

SLYM differs immunophenotypically from dura, arachnoid and pia mater

So are there other features of SLYM? The answer is yes
.

In brain slices of Prox1-EGFP⁺ mice, the researchers found that Prox1-EGFP⁺SLYM cells often come into direct contact with sinus endothelial cells and form tight junctions, and this tight binding is permeable, allowing small molecules to exchange
between blood and cerebrospinal fluid.

In addition, SLYM also acts as an immune barrier, preventing peripheral immune cells from directly entering the deep cerebrospinal fluid, while immune cells
are resident in SLYM.
After inflammation or brain degenerative changes in the brain, the immune cells in SLYM increased significantly, and LYVE1+, CD206+ and CD68+ macrophages and CD11c⁺ dendritic cells would aggregate on SLYM, indicating that SLYM has the role of
immune surveillance.

SLYM has the function of monitoring the cerebrospinal fluid for signs of infection and inflammation

Overall, the study is the first to discover the presence of a 4th membrane structure on the surface of the brain, which is different from the dura, arachnoid and pia mater, SLYM, which will update the basics
of anatomy in textbooks.

SLYM controls the exchange of cerebrospinal fluid and blood substances, and has immune barrier and immune surveillance functions, which is closely related to various diseases such as central nervous system infection and Alzheimer's disease, and deserves further study
.

References

1.
Mollgard K, Beinlich FRM, Kusk P, Miyakoshi LM, Delle C, Pla V, Hauglund NL, Esmail T, Rasmussen MK, Gomolka RS et al: A mesothelium divides the subarachnoid space into functional compartments.
Science 2023, 379(6627):84-88.

2.
Iliff JJ, Wang M, Liao Y, Plogg BA, Peng W, Gundersen GA, Benveniste H, Vates GE, Deane R, Goldman SA et al: A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid beta.
Sci Transl Med 2012, 4(147):147ra111.

3.
Louveau A, Smirnov I, Keyes TJ, Eccles JD, Rouhani SJ, Peske JD, Derecki NC, Castle D, Mandell JW, Lee KS et al: Structural and functional features of central nervous system lymphatic vessels.
Nature 2015, 523(7560):337-341.

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