09 Feb 2024

Cerebrospinal fluid washes waste from the brain, eventually draining into the lymph nodes and bloodstream—but exactly how this flow bypasses the brain’s barriers to reach the periphery has remained somewhat mysterious. In the February 7 Nature, researchers led by Jonathan Kipnis, at Washington University in St. Louis, report that CSF exits along the outside of veins that connect the parenchyma to the dura mater lining the skull. These so-called bridging vessels (image below) require small openings in the arachnoid mater layer, which otherwise forms an impermeable sheath around the brain.

To visualize these gaps, the authors generated transgenic mice that have all of their arachnoid barrier cells labeled with a green fluorescent protein. Lo and behold, these cells formed “cuffs” around bridging veins. With live imaging, the authors traced molecules injected into the CSF as they flowed through these arachnoid cuff exit (ACE) points to reach the dura. What’s more, traffic went the other way, too. Under neuroinflammatory conditions, immune cells traveled from the dura along these highways into the brain.

“The central nervous system space is physically connected to the dura mater through these gaps in the arachnoid barrier,” Kipnis and first author Leon Smyth wrote to Alzforum.

Other researchers were enthusiastic. “This elegant study significantly contributes to the resolution of a long-debated question regarding the communication between the subarachnoid spaces and the dura,” wrote Michal Schwartz, Javier Peralta Ramos, and Giulia Castellani at the Weizmann Institute of Science in Rehovot, Israel. Kipnis previously studied with Schwartz. Per Kristian Eide at the University of Oslo, Norway, called the findings a potential breakthrough. “[The paper] introduces fresh insights into critical gaps in our existing knowledge,” he wrote to Alzforum.

Many saw broad applications. “The identification of these ACE points has potential implications not just for better understanding of CSF physiology, but also for monitoring disease progression, immune surveillance of the CNS, and immune reactivity,” wrote Berislav Zlokovic and Kassandra Kisler at the University of Southern California in Los Angeles (comments below).

At the same time, researchers noted that it remains to be determined how much of the brain’s CSF outflow goes through ACE points versus other hypothesized routes.

Gaps in the Barrier. Bridging veins (BV) poke through the arachnoid mater (green), allowing dextran tracer (pink) to flow from the subarachnoid space (SAS) to the dura. [Courtesy of Smyth et al., Nature.]

New Route for Brain Access
Tracer studies going back decades have found that CSF drains from inside the brain to cervical lymph nodes, under the ears (McComb, 1983; Eide et al., 2018). More recently, researchers discovered lymphatic vessels in the dura mater of both mice and people, implying these might be the initial collection point for CSF before it proceeds into nearby nodes (Oct 2017 news; Nov 2017 news; Jacob et al., 2022). How CSF reaches the dural lymphatic system was less clear. Theories included that it exits the brain alongside cranial nerves, or that it reaches the periphery via arachnoid granulations, small outpouchings of the arachnoid mater that contact the dura (Upton and Weller, 1985; Gailloud et al., 2001; Spera et al., 2023). The issue remains unsettled.

Direct Connection to Dura? Tracer (purple) injected into mouse cerebrospinal fluid appears first in the dura (left), then the cervical lymph nodes (middle), and finally the bloodstream (right). [Courtesy of Smyth et al., Nature.]

To take a closer look at CSF dynamics, Smyth injected the tracer Evan’s blue into the CSF of wild-type mice, and watched where it went via live imaging. The tracer appeared first in the dura, later in cervical lymph nodes, and last in blood (see video above). Smyth and colleagues confirmed these findings by taking lysates from each tissue and measuring tracer concentration at various time points. Its rapid emergence into the dura suggested to them the existence of a direct route between brain and meninges.

How did Evan’s blue reach the dura? Not by active transport through the arachnoid mater, the scientists think, because a single-nuclei RNA-Seq analysis of barrier cells found few transporters in these cells. Looking closer at the arachnoid, immunostaining of barrier cells revealed them bunched up in cuffs, or ACE points, around bridging veins (see image at right).

To visualize what happens at these points in live mice, the authors generated transgenics with fluorescent arachnoid barrier cells, using the marker Dpp4, which has been shown to be specific for these cells (Sep 2023 news). In live imaging, dextran injected into the CSF moved along bridging veins. Once the tracer passed the ACE point, it spread out into the dura (see video and image below). There, dextran accumulated in “hot spots” near venous sinuses, the channels that collect oxygen-depleted blood. Lymphatic vessels run alongside these sinuses.

Beyond the Gate. Dextran (purple) injected into mouse CSF flows along bridging veins (black) past the arachnoid cuff (green) before spreading into the dura mater (skull is turquoise). [Courtesy of Smyth et al., Nature.]

If molecules can exit the brain through ACE points, do they also enter there? To test this, the authors applied a type of biotin to the surface of the skull in transgenic Dpp4 mice. It diffused through to the dura, where it accumulated around bridging veins. Ten minutes later, it had reached the subarachnoid space.

ACE points permit access to the brain, the authors concluded. This may explain how immune cells in the meninges can communicate with the brain via cytokines (Oct 2019 news). Nonetheless, gatekeepers may still police these entrances. The authors noted that macrophages gather at ACE points and could devour unwanted visitors.

Time Course. Via live imaging of mouse brain (left), researchers watch dextran (purple) injected into mouse CSF flow up a bridging vein past an ACE point (green) and into the dura. Skull is turquoise. [Courtesy of Smyth et al., Nature.]

Most commenters found the data for ACE points convincing, saying the study affords the best look yet at the structure of the arachnoid mater around bridging veins. “This important blood-brain border has been overlooked, mostly because of a lack of tools to dissect its anatomy and cellular and functional components,” Tal Iram at the Weizmann Institute wrote to Alzforum. Sandro Da Mesquita at the Mayo Clinic in Jacksonville, Florida, praised the state-of-the-art imaging techniques. He previously worked with Kipnis. Julie Siegenthaler at the University of Colorado Anschutz Medical Campus, Aurora, called the study the most extensive analysis to date of these regions. “This … provides new tools and an important framework for numerous future studies on how these [ACE points] are altered in aging and disease,” she wrote.

Christer Betsholtz at Uppsala University, Sweden, cautioned that much of the evidence for brain-dura communication remains indirect, and alternate explanations are possible. For example, after tracer injection into the CSF, a small amount may exit the brain along cranial nerves, enter the bloodstream, and reach dura that way. Betsholtz noted that tracer in these experiments accumulated in macrophages that cluster around dural sinuses. These cells may concentrate the signal enough for it to be detectable in the dura before enough tracer has built up in the bloodstream to be visible there. In keeping with this, tracer hot spots in the dura co-localized better with macrophage abundance than with ACE points, he said. “Molecular and cellular passage along bridging veins and ACEs remains hypothetical. More work needs to be done to prove or disprove it,” Betsholtz wrote to Alzforum.

Do ACE Points Control Access for Immune Cells, Molecules?
Several recent papers have reported that immune cells enter the brain in various disease conditions (Jan 2020 news; Jun 2021 news). However, in the healthy brain, the dura harbors a distinct population of immune cells, containing more monocytes, neutrophils, B cells, and T cells than does the subarachnoid space. What keeps them from passing through ACE points into the brain?

Examining their snRNA-Seq dataset, the authors found that dural border cells that reside directly above the arachnoid mater express numerous secreted chemorepellent factors. In the healthy brain, these might well drive immune cells away from the arachnoid layer, the authors speculated. Supporting this idea, they found that two of these repellents, SEMA3A and SEMA3D, kept monocytes at bay in vitro. Knocking down SEMA3A in mouse meninges increased the number of monocytes found in the subarachnoid space.

Does anything change in the diseased brain? To investigate this, the authors used mice with autoimmune encephalomyelitis (EAE), a neuroinflammatory model of multiple sclerosis. Compared with healthy mice, EAE mice expressed less SEMA3A in the meninges, and had more neutrophils and T cells in their subarachnoid space. In addition, live imaging showed myeloid cells moving along bridging veins into the subarachnoid space (see video below).

Immune Migration. In the EAE mouse brain, myeloid cells (green) move down along a bridging vein (purple) toward the subarachnoid space; yellow lines show their trajectories. [Courtesy of Smyth et al., Nature.]

Hints From Human Data
There is some evidence ACE points could be present in human brain, as well. The authors analyzed MRI data from 10 healthy volunteers participating in a multiple sclerosis study. Contrast agent injected into the CSF spread into their dura from bridging veins, just as in mice (see image below). A previous MRI study also showed CSF tracer entering the dura near the sites of bridging veins (Ringstad and Eide, 2020). More recently, a pilot study that used ultrasound to open the blood-brain barrier in Alzheimer’s disease patients found tracer accumulating around dural vessels, as well (Mehta et al., 2023). 

“The concept of ACE points is concordant with recent in vivo human data … This conception of CNS fluid flow updates knowledge on brain-body connections and physiology that has historically been perplexing,” Rupal Mehta at Rush Medical College, Chicago, wrote to Alzforum (comment below).

Commentators wondered about implications for treatment. “Can ACE points be used therapeutically to deliver drugs to the CSF and ultimately the CNS, as an alternative to intrathecal delivery?” Siegenthaler asked. Iram thinks this is worth exploring. “[This] entry point … should catch the attention of pharmaceutical companies that have been focusing mostly on the blood-brain barrier as an entryway for therapeutics,” she wrote.—Madolyn Bowman Rogers

News Citations

1. Lymphatic Vessels Found in Human Brain 13 Oct 2017
2. In Mice, CSF Caught Draining Via Lymphatic Vessels, Not Veins 15 Nov 2017
3. Not So Fast—The Brain Has Three Meningeal Membranes After All 29 Sep 2023
4. Do Immune Cells in the Meninges Help with … Memory? 11 Oct 2019
5. Attack of the Clones? Memory CD8+ T Cells Stalk the AD, PD Brain 14 Jan 2020
6. Private Stock—Brain Taps Skull Bone Marrow for Immune Cells 11 Jun 2021

Paper Citations

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8. Mehta RI, Carpenter JS, Mehta RI, Haut MW, Wang P, Ranjan M, Najib U, D’Haese PF, Rezai AR.
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External Citations

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