Time to rewrite the brain anatomy textbook? In addition to the outermost dura, middle arachnoid, and innermost pia membranes surrounding the brain, researchers led by Kjeld Møllgård, University of Copenhagen, and Maiken Nedergaard, University of Rochester Medical Center, New York, identified another layer, called the subarachnoid lymphatic-like membrane. In the January 6 Science, they reported that SLYM lies between the arachnoid and pia membranes, splitting the subarachnoid space. It wraps around blood vessels traversing the pia, restricting solute movement between the blood and cerebrospinal fluid. The membrane also harbors immune cells that likely aid in immune surveillance. Their numbers swell in aged mice, and a physically damaged SLYM compromises glymphatic flow, the scientists report. Both findings might have implications for neurodegenerative disease.
“This is another exciting and thought-provoking work from the group of Maiken Nedergaard that calls for revision to our understanding of meningeal function and anatomy,” wrote Jonathan Kipnis and Leon Smyth, Washington University in St. Louis. Per Kristian Eide, University of Oslo, Norway, called this paper groundbreaking. “The transport of cerebrospinal fluid in the subarachnoid compartment seems to be far more organized than we previously thought,” he wrote (comments below).
A Fourth Brain Layer. Beneath the dura mater (gray), the newly identified SLYM (green) splits the subarachnoid space into an outer compartment and a vessel-rich inner compartment (red) lining brain tissue (purple). [Courtesy of Møllgård et al., Science, 2023.]
Cerebrospinal fluid bathes the brain, bringing in nutrients and clearing out cellular junk. But how the fluid moves through the relatively wide space between the arachnoid and pia mater is unclear. Co-first authors Møllgård, Felix Beinlich, Peter Kusk, and Leo Miyakoshi, all at U Copenhagen, aimed to find out.
The researchers used in vivo two-photon microscopy to peer into the cortices of 3- to 4-month-old mice expressing green fluorescent protein in lymphatic endothelial cells. Lo and behold, under the arachnoid, Møllgård spied a continuous layer of lymphatic endothelial cells intertwined with collagen fibers—the SLYM. It split the subarachnoid space into an upper layer and a vessel-rich lower layer lining the brain (see image above).
Is the SLYM permeable? The scientists injected 1 μm wide red fluorescent particles into the subdural outer layer of the subarachnoid space and blue fluorescent particles into the cisterna magna to fill the inner layer. The particles stayed where they were (see image below). So did the smaller, 3 kDa, dextran, suggesting that the SLYM prevents the exchange of most proteins, peptides, and larger molecules between the CSF and blood.
“It remains to be seen what role the SLYM has in the selective permeability of small molecular weight solutes and their glymphatic circulation,” wrote Roxana Carare, University of Southampton, U.K. (comment below).
How did the researchers know this membrane was distinct? They immunostained brain slices for various lymphatic and membrane-specific markers (see image below). The arachnoid expressed the cell adhesion proteins claudin-11 and E-cadherin. The pia was positive for the meningeal marker CRABP2 with patches of the lymphatic endothelial marker podoplanin and the lymphatic vessel marker VEGRFR3. In contrast, the SLYM widely expressed podoplanin and CRABP2 but no VEGRFR3. It also did not express the lymphatic marker LYVE, suggesting that the SLYM is indeed a meningeal layer, not a lymphatic one.
While all this sleuthing was done in mice, it turns out that people have this fourth membrane, too. Immunostaining human cerebral cortex slices revealed a PDPN- and CRABP2-positive membrane above the pia that ran throughout the entire subarachnoid space.
Podoplanin positivity caught the researchers’ attention because it is expressed by mesothelial membranes that encase and lubricate the movement of peripheral organs against other organs and bones. The authors think that the SLYM may act similarly in the brain, easing friction between the brain and skull and, perhaps, coating blood vessels in the subarachnoid space.
Mesothelial membranes act as an immune barrier, and so might SLYM (Mutsaers et al., 2020). In vivo imaging and immunostaining showed many white blood cells and macrophages embedded within this membrane (see image below). The leukocytes quadrupled in 12-month-old mice compared to healthy 3- to 4-month-old mice. The latter, when injected with lipopolysaccharide, an inflammatory stimulus, similarly ramped up these SLYM immune cells.
Speckled with Immune Cells. Like the dura (gray), the SLYM (green) was filled with white blood cells (red). [Courtesy of Møllgård et al., Science, 2023.]
Such an immune response has direct implications for neurodegenerative disease. A dysfunctional SLYM might blunt immune cell recruitment, the authors contend. Similarly, in mice with a damaged dura, the fluorescent dextran molecule leaked into both sides of the SLYM, suggesting that damage to the meningeal layers may also compromise the blood-brain barrier and alter glymphatic flow.
All told, the authors concluded that the SLYM is a new meningeal membrane that divides the subarachnoid space to organize CSF flow and facilitate immune surveillance.—Chelsea Weidman Burke
- Mutsaers SE, Pixley FJ, Prêle CM, Hoyne GF. Mesothelial cells regulate immune responses in health and disease: role for immunotherapy in malignant mesothelioma. Curr Opin Immunol. 2020 Jun;64:88-109. Epub 2020 May 30 PubMed.
- Møllgård K, Beinlich FR, Kusk P, Miyakoshi LM, Delle C, Plá V, Hauglund NL, Esmail T, Rasmussen MK, Gomolka RS, Mori Y, Nedergaard M. A mesothelium divides the subarachnoid space into functional compartments. Science. 2023 Jan 6;379(6627):84-88. Epub 2023 Jan 5 PubMed.