Smyth LC, Xu D, Okar SV, Dykstra T, Rustenhoven J, Papadopoulos Z, Bhasiin K, Kim MW, Drieu A, Mamuladze T, Blackburn S, Gu X, Gaitán MI, Nair G, Storck SE, Du S, White MA, Bayguinov P, Smirnov I, Dikranian K, Reich DS, Kipnis J. Identification of direct connections between the dura and the brain. Nature. 2024 Mar;627(8002):165-173. Epub 2024 Feb 7 PubMed.
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Weizmann Institute of Science
Weizmann Institute of Science
Weizmann Institute of Science
Over the last 20 years, it became evident that adaptive and innate immune cells play a key role in lifelong brain plasticity and protection in health, with far-reaching implications for brain aging and diseases. This transformative understanding raised questions regarding brain anatomy, specifically, how these cells attain access to the brain in health, and how interactions with immune cell populations are modified under disease conditions. This elegant study from the Kipnis lab significantly contributes to the resolution of a long-debated question regarding the communication between the subarachnoid spaces and the dura.
Kipnis' group reports the identification of previously unknown gateways between the dura and the brain, coined arachnoid cuff exit (ACE) points. These enable the direct efflux of CSF from the subarachnoid space to the dura and orchestrate immune cell trafficking in the reverse direction. The authors further suggest that similar structures are present in humans. The evidence for such a route and its mechanisms of regulation represent a noteworthy advance in the understanding of communication of the brain with the immune system.
This is an exciting discovery for the field of brain immunity, because it helps address one of the biggest mysteries regarding the brain-immune interplay, which has remained enigmatic since we first showed that the brain is dependent on immune cells for its plasticity. Kipnis was a student in my lab at that time, and he subsequently demonstrated that immune cells residing in the meninges can remotely affect the brain. Nevertheless, it was not understood how these cells were regulated.
Overall, the discovery of ACE points provides a mechanism for the brain's protection from an unwanted or excessive immune response, and could also provide additional potential therapeutic targets under various brain pathologies.
View all comments by Giulia CastellaniOslo University Hospital / University of Oslo
This paper is of great importance. It potentially represents a breakthrough in our comprehension of how substances in the CSF of the subarachnoid space communicate with the dura mater, which houses lymphatic vessels. It introduces fresh insights into critical gaps in our existing knowledge, addressing questions such as the anatomic efflux routes of CSF from the subarachnoid space, the entry of immune cells and products from the peripheral circulation to the CSF and brain, and the permeability of the arachnoid membrane.
The paper is also relevant to debated aspects of the glymphatic concept, including the connection between the proposed pial perivenous efflux site and the dural lymphatic structures. These inquiries are crucial for comprehending CSF physiology.
The work by Smyth et al. uncovered discontinuities in the arachnoid barrier, where bridging veins from the brain traverse the arachnoid membrane, entering the dura mater. These discontinuities create perivenous openings denoted as arachnoid cuff exit (ACE) points, facilitating the passage of substances between the subarachnoid space and the dura. Consequently, locations where bridging veins enter the dura serve as focal points for CSF efflux.
The potential implications of ACE sites are broad, enabling the exchange of cells between the dura and CSF in the subarachnoid space. Importantly, ACE points were also identified as entry points for substances from veins to the CSF, shedding light on the discussion of how molecules from the systemic circulation, such as intravenous contrast agents, enter the CSF.
This recent paper raises several questions: What is the relative importance of ACE points for CSF efflux? Is CSF efflux at the ACE sites actively controlled, or do these sites represent passive efflux pathways where CSF bulk flow is primarily regulated by hydrostatic and osmotic pressure gradients?
View all comments by Per Kristian EideUniversity of Southern California
University of Southern California, Keck School of Medicine
Smyth et al. present several new observations about the nature and function of the meningeal layers surrounding the brain. Using transcriptomic data, they generated new mouse models to visualize and study anatomical structures that function as routes across the arachnoid barrier. Although recent work has demonstrated that cells in the leptomeningeal layers surrounding the brain survey and detect molecular cues in the cerebrospinal fluid (CSF), how the fluid, molecules, and immune cells move between layers of the meninges remain open questions. Smyth et al. describe arachnoid cell “cuffs” around venous vessels, which, they show, control transport of solutes and immune cells between the subarachnoid space and the dura, and regulate the drainage of cerebrospinal fluid from brain. At the same time, these cuff points limit transport of molecules from the dura to the subarachnoid space.
The identification of these arachnoid cuff exit (ACE) points has potential implications not just for better understanding of the CSF system physiology, but also for monitoring disease progression, immune surveillance of the CNS, and immune reactivity. Indeed, the authors observed increased immune cell transits via these cuff points in an experimental autoimmune encephalomyelitis (EAE) model. In their model, the authors did not focus on whether the cuff points themselves underwent any changes in structure, function, or composition with EAE. However, it was previously reported that inflammatory conditions can alter the structure of the meningeal layers (Mapunda et al., 2023; Pietilä et al., 2023), making this an important question for future investigation.
How large a role the ACE route plays in the human CNS and in other diseases requires more research. That said, these cuff points may also open new avenues to therapeutic strategies if drug delivery into the CNS can be realized via these transit points, and if this is more effective than crossing the blood-brain barrier. Future studies should also investigate how much of a role these cuff points play in CNS fluid and cell movement and in CNS immune surveillance compared to other possible routes for exchange of solutes and cells in the brain.
View all comments by Kassandra KislerWeizmann Institute of Science
The extent of novel anatomical observations at the leptomeningeal-brain interface coming out of this lab is astounding. This important blood-brain border has been overlooked, mostly because of lack of tools to dissect its anatomy and cellular and functional components. In this paper, the Kipnis lab combined single-cell transcriptomics with very carefully timed in-vivo imaging to identify subsets of leptomeningeal barrier cells and the way CSF flows between them.
I think that as with the discovery of CSF efflux through the brain lymphatics, this newly discovered exit route offers novel ways to manipulate CSF flow and debris clearance in aging and neurodegenerative diseases.
Furthermore, that this is also an entry point for molecules and immune cells from the blood to the brain (through the CSF) should catch the attention of pharmaceutical companies that have been focusing mostly on the blood-brain barrier as an entry way for therapeutics.
View all comments by Tal IramMayo Clinic
This major scientific study coming from the Kipnis lab shines light onto the architecture of the mammalian meninges, and its crucial roles in mediating the molecular and cellular communication between the brain, its border tissues, and the periphery.
Despite the growing amount of data showing that cerebrospinal fluid molecular content can reach the outermost meningeal dura, and ultimately be drained by the dural lymphatics into the cervical lymph nodes, we had a very limited idea, and even less data, on how and where this communication takes place. The experimental evidence supporting the existence of anatomical regions in the mammalian meningeal arachnoid layer that mediate this communication between brain fluids, the meningeal dura and later the peripheral lymphatic and immune systems is finally here.
Using very elegant experimental mouse models and techniques, combined with human biospecimen analyses and state-of-the-art imaging modalities, this study was able to provide strong evidence for the existence of a rapid exchange, by a somewhat direct route, of molecular and cellular content between the subarachnoid space and the dura, via what the authors call arachnoid cuff exit points. Basically, the authors show that these cuffs in the arachnoid layer are due to penetrating bridging veins that cross it on their way to the dural venous sinuses. At these anatomical points the arachnoid layer barrier cells cannot form a tight barrier, and the arachnoid becomes permissive, allowing the passage of both molecules and immune cells into the dura.
The authors point out, and I totally agree, that this discovery opens new avenues of research in the field of neuroimmunology. For example, it will be important to understand the contribution of the arachnoid cuff exit points to CSF outflow into the dura and lymphatic system, when compared to the arachnoid villi-to-venous outflow pathways that are found in human arachnoid granulations.
It will also be interesting to look at how these arachnoid cuffs change, cellularly and structurally, in aging and in different aging-related diseases, where CSF composition and turnover are known to be altered, meningeal lymphatic drainage is poor, and neuroinflammation is a major component. The authors provide evidence suggesting that the arachnoid cuffs serve as “a way into” the subarachnoid and cerebrospinal fluid for dura-derived molecules and immune cells in models of central nervous system autoimmunity. So one would assume that, at some point during disease initiation, there is a shift in the flow directionality of molecules and immune cells via the arachnoid cuffs. It will be interesting to explore the mechanisms behind it, and whether this directional shift in flow through these cuffs around the bridging veins is also observed in aging-related chronic pathological conditions.
View all comments by Sandro Da MesquitaUniversity of Colorado, Anschutz Medical Campus
This new work shows extensive anatomical and functional evidence that spots where bridging veins cross from the leptomeninges to the dura are entry-exit points for small and large molecular weight tracers. This is in contrast to the arachnoid barrier layer, shown in the past, and confirmed in this study, to occlude paracellular transport via presence of tight junctions.
The authors perform extensive fixed tissue and intravital imaging analysis using existing (Cdh5-CreERT and Prox1-GFP) and newly generated Cre lines (Dpp4-CreER and Slc47a1-CreER) to visualize arachnoid barrier (AB) and other leptomeningeal and dural fibroblast populations. This permits an extensive characterization of the cellular arrangements around these exit points, which alternatively appear as gaps in the AB layer or “cuffs” of AB cells as the vein exits the leptomeninges into the dura.
This is the most extensive analysis to date of these regions and provides new tools and an important framework for future studies on how these arachnoid cuff exits (ACE) are altered in aging and disease. Importantly, future studies targeting ACE points to disrupt or block flow of molecules will determine their functional relevance in brain health, permitting these and other questions to be answered:
The authors use their methods to study ACE points as potential entry sites for immune cells into the subarachnoid space in healthy mice and in an animal model of multiple sclerosis (EAE). Their data in Figure 5j,n show convincing evidence with intravital imaging at peak EAE that monocytes are moving from the supra-arachnoid (marked by Prox1) indicative of the dura, to SAS, below the Prox1-GFP layer, using the ACE-bridging vein as an entry point.
However, regarding the extravasation of monocytes from leptomeningeal vasculature that occurs in neuroinflammation, it is difficult to discern if this represents an entry point that substantially contributes to disease pathology. The authors do provide functional data to address this point by zeroing in on integrin-alpha6 (expressed by immune cells and endothelial cells) as a potential receptor-extracellular matrix interaction that is permissive to ACE point crossing, similar to what was described by Dorothy Sipkins and colleagues for cancer cell access to the SAS (Yao et al., 2018).
The authors show, using systemic administration of anti-itga6, that clinical disease score is reduced and neutrophil density at ACE points is lower, but the total immune cell entry into the spinal cord shows some mixed results. While clearly of interest and an important avenue of research, more studies are needed to interrogate how immune cell entry via ACE points contributes to disease progression versus other routes of entry, such as across the vasculature. Here, too, functional studies to block ACE points will be valuable.
While I recognize this is beyond what the authors discuss, and not the focus of this study, I'd like to draw attention to the electron microscopy studies following HRP injection into the cisternal magna to target the SAS. They are particularly interesting, and relevant to recent discussions of meningeal anatomy and function, including reported barrier properties of Prox1+ cells (referred to as SLYM or a fourth meningeal layer (Møllgård et al., 2023; Jan 2023 news).
In Supplemental Figure 3a, HRP (45 kDa) reaches the AB layer (including being taken up in vesicles by AB cells), arguing against size-restrictive barrier properties of adjacent inner arachnoid cells, in particular Prox1+ cells, as had been reported by another group (Møllgård et al., 2023). Notably, HRP passes into the subpial space from the SAS, consistent with reports by Mapunda et al., 2023, that the pial layer, though continuous and containing VE-cadherin junctions, does not form a size restrictive barrier. Notably, Smyth et al.'s EM in Figure 1C also shows arachnoid barrier and arachnoid fibroblasts fused above a single subarachnoid space. This matches recent studies by Pietilä et al., 2023, and decades of EM studies on a variety of species, supporting a single arachnoid mater comprised of fused sublayers (Sep 2023 news).
Ideally, these types of studies could be repeated by combining Dpp4-CreER (AB) and Prox1-GFP (inner arachnoid) both in EM and in 2-photon live imaging to help resolve recent controversies regarding functional meningeal anatomy and CSF flow.
Overall, I find this to be a timely, innovative and high-interest study using numerous advanced techniques that will enable future work on peripheral-CNS communication in health and disease.
References:
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View all comments by Julie SiegenthalerDepartment of Pathology, RUSH Medical College
The authors provide an important cellular and functional description of the arachnoid barrier cell layer that explains how it is simultaneously capable of separating and adjoining compartments (namely, the subarachnoid space and dural interstitium), thereby accomplishing barrier functions while also permitting fluid and cellular communication between meningeal layers in mice.
They refer to arachnoid barrier defects next to veins as arachnoid cuff exits or “ACE” points, that correspond to tracer hot spots. The concept of ACE points is concordant with recent in vivo human data from individuals with Alzheimer’s disease, that suggest convergence of efflux fluid at junction points around meningeal cerebral veins as they enter dura (Mehta et al., 2023). This conception of CNS fluid flow updates knowledge on brain-body connections and physiology that has been perplexing historically. It is an important finding for interpreting and investigating disease pathogenesis in the brain, and will be interesting to study in Alzheimer’s disease.
References:
Mehta RI, Carpenter JS, Mehta RI, Haut MW, Wang P, Ranjan M, Najib U, D'Haese PF, Rezai AR. Ultrasound-mediated blood-brain barrier opening uncovers an intracerebral perivenous fluid network in persons with Alzheimer's disease. Fluids Barriers CNS. 2023 Jun 16;20(1):46. PubMed.
View all comments by Rupal MehtaRockefeller Neuroscience Institute of West Virginia University
This interesting study examined mouse and human perivenous physiology, which has been a relatively overlooked area of investigation. The authors found that dynamic changes occur in both species within the spaces around veins, and further depict immune cellular changes at the sites of dural entry by veins in mice, where there are gaps in the arachnoid barrier cell layer. Further exploration of the imaging changes in perivenous and arachnoid cuff exit (ACE) regions may elucidate neurobiology of diseases. Moreover, understanding ways in which the physiology of ACE regions can be therapeutically targeted and/or modulated in humans will be valuable.
View all comments by Rashi MehtaMake a Comment
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