This is an exciting study—it provides the first view into tracer movement through the human subarachnoid space over just a few hours. They find that tracers move in a specific pathway along the arteries before entering brain tissue. This information is important to understand the pathways of waste clearance in the human brain, which have been very challenging to image due to the tiny, intricate structures involved.
Divide and Conquer: Compartmentalization of the Brain’s Subarachnoid Space Facilitates Rapid Periarterial CSF Flow
Over the past decade, there has been significant interest in comprehending the underlying anatomy and physiological mechanisms that regulate the exchange of cerebrospinal fluid (CSF) and interstitial fluid (ISF) within the brain. Throughout this period, the initial characterization of the glymphatic pathway by Nedergaard and colleagues (Iliff et al., 2012), demonstrating the swift movement of CSF into the brain parenchyma through periarterial spaces and the clearance of ISF within perivenous spaces, has been repeatedly replicated by many groups in multiple model systems. While most of these studies have been conducted in non-human subjects, there is a growing body of research, including contributions from Eide and Ringstad's group, validating the existence and function of the glymphatic pathway in humans using non-invasive MRI-based techniques (Eide and Ringstad, 2015; Ringstad et al., 2017; Eide et al., 2018; Ringstad and Eide, 2022).
In this article by Eide and Ringstad, the authors delve deeper into characterizing the anatomical and functional aspects of leptomeningeal perivascular CSF flow dynamics in humans under both healthy and diseased conditions. They utilize intrathecal gadobutrol-enhanced T1-weighted MRI and report the compartmentalization of the human subarachnoid space into what they propose is an inner perivascular subarachnoid space (PVSAS) and an outer generalized subarachnoid space (SAS). The PVSAS predominantly surrounds major arteries from the circle of Willis, such as the anterior cerebral artery (ACA), middle cerebral artery (MCA), and posterior cerebral artery (PCA). The authors observe direct CSF solute propagation between the basal cisterns and the PVSAS, and confirm antegrade CSF flow within the PVSAS of large surface arteries before reaching the adjacent cerebral cortex. Functionally, they note slowed CSF tracer appearance in periarterial subarachnoid spaces with increased pulsatile intracranial pressure, suggesting potential impairments in diseases like communicating hydrocephalus. Additionally, they find an enlarged PVSAS area in idiopathic normal-pressure hydrocephalus (iNPH), associated with altered CSF and solute flow dynamics and tracer accumulation in specific cortical regions.
The study by Eide and Ringstad deserves commendation for its impact, relevance, and methodological rigor. Nevertheless, it raises crucial questions, particularly regarding the membrane that segregates the inner perivascular space from the broader subarachnoid space. While the authors propose this membrane is part of the arachnoid, they provide limited data supporting an arachnoid rather than pial identity. Counter to this conception of a subarachnoid perivascular space, previous anatomical studies suggest that surface arteries and veins run in a sub-pial plane, with Virchow-Robin spaces formed by pial membrane invagination as surface arteries penetrate the brain parenchyma (Zhang et al., 1990). Further, recent work from Nedergaard's group identifies a potential 4th meningeal membrane, termed the subarachnoid lymphatic-like membrane (SLYM), distinct from the arachnoid, based on its expression of lymphatic endothelial cell markers (LYVE-1, PROX-1, and PDPN) (Plá et al., 2023). Consequently, it is also possible that this SLYM layer demarcates perivascular spaces from the subarachnoid space. Future investigations focusing on labeling for arachnoid barrier cells, pial cells, and lymphatic markers in postmortem human samples may clarify this membrane's identity.
Moreover, Eide and Ringstad's study reveals that tracer initially appears in the PVSAS of major arteries near the circle of Willis, suggesting CSF entry at proximal locations within the basal cisterns. The exact anatomical site of this entry, and the cellular and molecular components forming it, remain unknown, warranting further exploration. Further, this perivascular membrane appears to function akin to myelin on an axon in promoting rapid axial CSF and solute movement down arteries, while restricting radial movement into the broader SAS. The mechanisms underlying this barrier function, however, remain elusive. If this membrane indeed consists of arachnoid barrier cells, it may contain tight junction proteins that prevent perivascular contents from entering the surrounding SAS. Over time (three hours), there appears to be signal equilibration between the PVSAS and the SAS, leading the authors to suggest that the membrane is semipermeable. Supporting this, they demonstrate rapid signal enrichment in the adjacent cortex after tracer appearance in the PVSAS. This suggests similarities to fenestrated pia in Virchow-Robin spaces (Zhang et al., 1990) or SLYM, which allows fluid and solute movement up to 3 kDa in size (Plá et al., 2023). Future research should focus on understanding the factors contributing to this membrane's semipermeability, such as molecular weight or charge.
While most of the tracer is concentrated around arteries, there is also signal, though less pronounced, around surface veins (see supplemental figure 4). This hints at possible communication between the PVSAS of arteries and veins or the direct entry of tracer into the perivenous subarachnoid space from the basal cisterns. The latter scenario seems less probable, given prior research indicating that CSF and tracer predominantly flow toward the brain through periarterial spaces rather than perivenous spaces (Iliff et al., 2012). Furthermore, it is unlikely that tracer is entering the perivenous space directly from the parenchyma (the clearance side of the glymphatic pathway) at such an early stage (less than one hour). Therefore, there is likely direct solute communication from periarterial spaces to adjacent perivenous spaces, similar to what is observed in the adjacent cerebral cortex. This raises similar questions to those previously mentioned regarding factors limiting the movement of CSF and solute across this semipermeable membrane. Is the limited tracer observed in the perivenous space due to characteristics of the tracer molecule itself, such as its size? Would a larger molecule, exceeding the size of gadobutrol (690 Da), face greater restriction entering the perivenous space, or would its size hinder its movement into the brain parenchyma, causing it to accumulate in the perivenous space? These questions necessitate further thorough investigation, as their answers may provide insights into why proteinopathies such as cerebral amyloid angiopathy predominantly affect arteries rather than veins. Additionally, it would be intriguing to expand upon the remarkable temporal resolution showcased in this study to a range of 3-6 hours. Previous research conducted by the authors' group has identified enhancements in tracer activity around bridging veins and within the parasagittal dura (Ringstad and Eide, 2022). Recent findings from our own group indicate that arachnoid barrier discontinuities, referred to as arachnoid cuff exit (ACE) points, coincide with the entry of these bridging veins into the dura. This phenomenon allows CSF to permeate into the dura, playing a crucial role in CSF efflux (Smyth et al., 2024).
In conclusion, Eide and Ringstad's work presents the first human in vivo MRI evidence of periarterial space segregation, facilitating rapid CSF and solute movement from basal cisterns to adjacent cerebral cortex. Yet, key questions persist regarding the identity of the compartmentalizing membrane, the etiology of this membrane’s semipermeability, and the site of CSF entry into perivascular spaces. Future studies probing these aspects may illuminate fundamental mechanisms impacting brain fluid dynamics in health and disease.
References:
Iliff JJ, Wang M, Liao Y, Plogg BA, Peng W, Gundersen GA, Benveniste H, Vates GE, Deane R, Goldman SA, Nagelhus EA, Nedergaard M.
A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid β.
Sci Transl Med. 2012 Aug 15;4(147):147ra111.
PubMed.
Eide PK, Ringstad G.
MRI with intrathecal MRI gadolinium contrast medium administration: a possible method to assess glymphatic function in human brain.
Acta Radiol Open. 2015 Nov;4(11):2058460115609635. Epub 2015 Nov 17
PubMed.
Ringstad G, Vatnehol SA, Eide PK.
Glymphatic MRI in idiopathic normal pressure hydrocephalus.
Brain. 2017 Oct 1;140(10):2691-2705.
PubMed.
Eide PK, Vatnehol SA, Emblem KE, Ringstad G.
Magnetic resonance imaging provides evidence of glymphatic drainage from human brain to cervical lymph nodes.
Sci Rep. 2018 May 8;8(1):7194.
PubMed.
Ringstad G, Eide PK.
Molecular trans-dural efflux to skull bone marrow in humans with CSF disorders.
Brain. 2022 May 24;145(4):1464-1472.
PubMed.
Zhang ET, Inman CB, Weller RO.
Interrelationships of the pia mater and the perivascular (Virchow-Robin) spaces in the human cerebrum.
J Anat. 1990 Jun;170:111-23.
PubMed.
Plá V, Bitsika S, Giannetto MJ, Ladron-de-Guevara A, Gahn-Martinez D, Mori Y, Nedergaard M, Møllgård K.
Structural characterization of SLYM-a 4th meningeal membrane.
Fluids Barriers CNS. 2023 Dec 14;20(1):93.
PubMed.
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.
Research concerning cerebrospinal fluid (CSF) flow and drainage is an emerging subject area, with numerous independent research groups conducting investigations to uncover its underlying mechanisms in animal models. Thus, it is crucial to understand CSF dynamics in humans, which allows for the translation of these novel findings into the clinic. In this report, Dr. Eide and Dr. Ringstad have investigated the features of early tracer dissemination after intrathecal injection of gadobutrol in the lumbar area in humans, and made a fascinating observation that the tracer distributes in the brain from the basal cisterns in an antegrade perivascular fashion around the major arteries before distributing in the subarachnoid space (SAS). This pattern of tracer distribution in the human brain has not been reported before and provides valuable insight. The authors interpret these findings such that the gadobutrol signal outlines a not-yet-described perivascular space for cerebrospinal fluid transport around the major arteries in the human brain. They propose this space to be an extension of the subarachnoid space that is separated by a semipermeable membrane and call it “perivascular subarachnoid space (PVSAS).”
As a Ph.D. student in neuroimmunology, I am fascinated by the anatomy and function of the meninges and the role of CSF in CNS immunity. Therefore, this exciting study performed in humans caught my interest and I critically read through the manuscript and presented it to my peers and supervisors as a journal club. While the data are original and very interesting, I admit, with due respect, that a number of conclusions drawn based on the MRI imaging are difficult to understand and, from my perspective, may benefit from considering other options for data interpretation. I listed these points below in hopes of stimulating discussion regarding this exciting data.
1: What is the evidence for the proposed semipermeable membrane?
Based on the observed distribution of intrathecally injected gadobutrol around the arteries subsequent to its arrival at the basal cisterns exterior to the gyrencephalic brain surface, the authors propose the existence of a novel membrane that compartmentalizes the human subarachnoid space and have named this transiently tracer-enhancing area ensheathed by the proposed membrane as the PVSAS. Why did they not consider the role of the pia mater in their study? To my understanding, electron microscopy studies have shown that the pia mater covers the floor of the SAS and is continuous with the fibroblasts forming the adventitia of the vessels. However, the anatomy of pia where the arteries penetrate the brain parenchyma is a matter of debate, and thus it is not yet clear if these perivascular spaces are continuous with the SAS or rather with the subpial space. Furthermore, the authors mention in the discussion that the resolution of MRI does not allow identification of their proposed membrane, yet still limit the interpretation of their data to a model supposing the existence of a membrane. What would be alternative interpretations for the observed tracer distribution? Could the distribution pattern be explained by the physics of fluid movement within the SAS, with a resultant faster distribution of contrast agent at regions close to arteries?
2: Why are the results presented without specifying the conditions of the patients undergoing the MRI scans?
Natural to a study with humans, the present cohort includes patients with various diseases. Because these diseases affect the CNS and its vasculature, distinguishing the state of healthy brains (in this case non-CSF-affecting diseases, according to the authors) versus diseased brains is important for drawing conclusions. I found the results to lack sufficient clarity regarding the assignment of the MRI scans to the respective disease conditions throughout Figures 1 to 8. While the study encompasses patients with diverse diseases, the paper only addresses the differences in tracer appearance between the reference and idiopathic normal pressure hydrocephalus groups in Figure 9; and appears to show no data of patients with spontaneous intracranial hypotension, arachnoid cyst, pineal cyst, idiopathic intracranial hypertension, and communicating hydrocephalus.
3: What is the variability of CSF tracer distribution between individuals?
Figures 3f and 4e show the first-time tracer appearance in different branches of the ACA and MCA, respectively. According to these figures, gadobutrol appears around the A2 and M2 branches of the arteries at approximately 50±10 minutes after the injection. However, according to Figures 7e and 8c and Supplementary Figure 6, first-time tracer appearance around these branches varies between five to 250 minutes, suggesting a high variability of tracer distribution between individuals. Thus Figures 3 and 4 preclude appreciation of the range observed across the entire cohort. Could this miss potentially important variabilities in CSF flow and tracer distribution between individuals or conditions?
4: What is the precise method for image analysis?
It was really interesting to see the visualization and quantification of the irregular tracer diffusion around the vasculature in the dementia brains. Since the paper lacks details of the analysis method, I am very interested to learn more about the analysis of such images. Specifically, knowing whether the measurement of the gadobutrol-enhanced area included or excluded the arterial lumen, whether sphericity was considered, and the details of the calibration methods for determining the size of the vessels in the MRI images would be informative. Comparing the data reported here with previously reported size measurements of cerebral arteries, the area seems to be too large for the spaces between gyri even in the presence of cortical atrophy in dementia patients (Gutierrez et al., 2014). More detailed methods would help us compare these novel findings with previously described dimensions of vessels and perivascular spaces.
5: Question about a citation.
The discussion mentions that Møllgård et al. reported that damage to the “subarachnoidal lymphatic like membrane—SLYM” proposed by these authors to divide the subarachnoid space impairs periarterial solute transport. This paper does not mention such a phenomenon, nor does it discuss the effects of damage to SLYM and its effect on periarterial solute transport. This paper does mention in the supplementary material that “surgical damage caused tears in dura and SLYM resulting in loss of the barrier properties of SLYM” (Møllgård et al., 2023), but does not correlate this to periarterial solute transport.
6: How did the study measure the barrier threshold of the proposed membrane?
The authors report that, “Our study concludes about the existence of a perivascular subarachnoid space, abbreviated PVSAS, surrounding larger arteries at the surface of the gyrencephalic brain, delineated by a semipermeable membrane that aligns well with the barrier threshold of 3 kDa described for “SLYM,” which also was impermeable to 1 μm wide fluorescent particles.” What is the proof or supporting data for this claim in the present study? As mentioned by the authors, the resolution of T1-weighted scans acquired with 3-Tesla MRI does not allow one to obtain such data.
7: Can we interpret these findings beyond select concepts?
In this paper, the authors seem to relate their observations to the concepts of glymphatic flow and the existence of the “SLYM,” both of which are subject to significant debate (Abbott et al., 2018; Engelhardt et al., 2017; Hladky and Barrand, 2014; Miao et al., 2024; Pietilä et al., 2023; Smith et al., 2017). Consideration of the present findings in the context of other currently discussed concepts of CSF flow would be highly desirable. From the point of view of a junior researcher, this study offers valuable data from numerous patients in need of treatment, through the extensive efforts of a team of esteemed scientists. I would welcome a broader discussion of these novel observations in the context of the known brain anatomy and with consideration of all concepts of CSF flow. I would like to thank Drs. Eide and Ringstad for their novel work, and for considering my comments.
References:
Abbott NJ, Pizzo ME, Preston JE, Janigro D, Thorne RG.
The role of brain barriers in fluid movement in the CNS: is there a 'glymphatic' system?.
Acta Neuropathol. 2018 Mar;135(3):387-407. Epub 2018 Feb 10
PubMed.
Engelhardt B, Vajkoczy P, Weller RO.
The movers and shapers in immune privilege of the CNS.
Nat Immunol. 2017 Feb;18(2):123-131. Epub 2017 Jan 16
PubMed.
Gutierrez J, Rosoklija G, Murray J, Chon C, Elkind MS, Goldman J, Honig LS, Dwork AJ, Morgello S, Marshall RS.
A quantitative perspective to the study of brain arterial remodeling of donors with and without HIV in the Brain Arterial Remodeling Study (BARS).
Front Physiol. 2014;5:56. Epub 2014 Feb 19
PubMed.
Hladky SB, Barrand MA.
Mechanisms of fluid movement into, through and out of the brain: evaluation of the evidence.
Fluids Barriers CNS. 2014;11(1):26. Epub 2014 Dec 2
PubMed.
Miao A, Luo T, Hsieh B, Edge CJ, Gridley M, Wong RT, Constandinou TG, Wisden W, Franks NP.
Brain clearance is reduced during sleep and anesthesia.
Nat Neurosci. 2024 Jun;27(6):1046-1050. Epub 2024 May 13
PubMed.
Correction.
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.
Pietilä R, Del Gaudio F, He L, Vázquez-Liébanas E, Vanlandewijck M, Muhl L, Mocci G, Bjørnholm KD, Lindblad C, Fletcher-Sandersjöö A, Svensson M, Thelin EP, Liu J, van Voorden AJ, Torres M, Antila S, Xin L, Karlström H, Storm-Mathisen J, Bergersen LH, Moggio A, Hansson EM, Ulvmar MH, Nilsson P, Mäkinen T, Andaloussi Mäe M, Alitalo K, Proulx ST, Engelhardt B, McDonald DM, Lendahl U, Andrae J, Betsholtz C.
Molecular anatomy of adult mouse leptomeninges.
Neuron. 2023 Dec 6;111(23):3745-3764.e7. Epub 2023 Sep 29
PubMed.
Smith AJ, Yao X, Dix JA, Jin BJ, Verkman AS.
Test of the 'glymphatic' hypothesis demonstrates diffusive and aquaporin-4-independent solute transport in rodent brain parenchyma.
Elife. 2017 Aug 21;6
PubMed.
We thank Dr. Sarmad Peymaei for his comments on our article. The article includes a number of pictures to illustrate what we observed. We retrieved a few images retrieved from our article and show them below as input to Dr Peymaei’s comments. A few responses to his questions:
What is the evidence for the proposed semipermeable membrane? The evidence relies on the pattern of contrast enrichment around the major artery trunks (anterior, middle, and posterior cerebral arteries) within the subarachnoid space (SAS). As shown in Figures 1a, 9a, and Suppl Fig 11a, b, the contrast enrichment forms a sharp doughnut-shaped demarcation around the artery toward the subarachnoid space (SAS). In Figure 4g it is further shown that when the SAS is being enriched by contrast, there is stronger enrichment within the perivascular subarachnoid space (PVSAS) around the arteries. Enrichment of SAS occurs later than enrichment of PVSAS, indicating that the membrane is semipermeable. The histopathological organization of the membrane cannot be answered from this imaging study, but the study provides strong evidence for compartmentalization of the SAS. Notably, the arteries studied here are not subpial, but within the SAS. The sharp demarcation of contrast around the arteries shown in the images below cannot be explained by physics of fluid movement within the SAS. Why should there be unidirectional transport around vessels residing within an open non-compartmentalized fluid compartment? And how should physics of fluid movement explain such a distinct demarcation of contrast?
Why are the results presented without specifying the conditions of the patients undergoing the MRI scans? The main objective of the present work is to describe the functional organization of a compartmentalized SAS, independent of underlying disease. The reference patients have no identified cerebrospinal fluid (CSF) disorder; information from these patients cannot be attributed to CSF disease. Supplementary material includes description of the various patient groups. We further describe how the dementia subtype iNPH differs from the reference subjects.
What is the variability of CSF tracer distribution between individuals? While there is variation at the individual level regarding first appearance of tracer within the PVSAS, a consistent finding at the individual level was antegrade tracer propagation along the arteries towards cortex cerebri.
What is the precise method for image analysis? We believe this aspect has been addressed in the article. However, with regard to several of the present questions, visual inspection of the images provides answers to the inquiries.
The article provides multiple images, supporting the interpretation that a PVSAS compartmentalizes the SAS. The temporal profile of tracer enrichment within PVSAS and SAS further suggests this compartmentalization is created by a semipermeable membrane. The tracer used has a molecular size of 605 Da, while the previously reported subarachnoid lymphatic like membrane (SLYM) was reported impermeable to solutes 3 kDa in size (Møllgård et al., 2023).
Figure 1a shows the distinct donut shape of contrast enrichment (white color visualizing the perivascular subarachnoid space, PVSAS) around the middle cerebral artery (A) within the subarachnoid space (SAS) that has black color. There is a sharp demarcation between PVSAS and SAS, indicative of a membrane, here deonted perivascular membrane (PVM), even though the submillimeter membrane cannot be visualized directly with magnetic resonance imaging, having a resolution of 1 mm.
Figure 4g shows that some time after contrast injection, the SAS is being enriched by contrast as well, even though the enrichment is still stronger within the PVSAS. However, also at this time, there is a sharp demarcation between the PVSAS and the SAS.
Figure 9a illustrates that the PVSAS (contrast-filled cavity) is altered in patients with the dementing disease idiopathic normal pressure hydrocephalus (iNPH). In these patients, the PVSAS shows altered configuration, and the artery has another location, residing more towards the margin of the PVSAS
Supplementary Figure 11 a-b. A higher magnification from a reference subject (left) and an iNPH patient (right) further visualizes the sharp demarcation between the PVSAS and the surrounding SAS is evident.
References:
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.
Comments
Boston University
This is an exciting study—it provides the first view into tracer movement through the human subarachnoid space over just a few hours. They find that tracers move in a specific pathway along the arteries before entering brain tissue. This information is important to understand the pathways of waste clearance in the human brain, which have been very challenging to image due to the tiny, intricate structures involved.
View all comments by Laura LewisWashington University School of Medicine in St. Louis
Washington University in St. Louis
Washington University in St. Louis, School of Medicine
Divide and Conquer: Compartmentalization of the Brain’s Subarachnoid Space Facilitates Rapid Periarterial CSF Flow
Over the past decade, there has been significant interest in comprehending the underlying anatomy and physiological mechanisms that regulate the exchange of cerebrospinal fluid (CSF) and interstitial fluid (ISF) within the brain. Throughout this period, the initial characterization of the glymphatic pathway by Nedergaard and colleagues (Iliff et al., 2012), demonstrating the swift movement of CSF into the brain parenchyma through periarterial spaces and the clearance of ISF within perivenous spaces, has been repeatedly replicated by many groups in multiple model systems. While most of these studies have been conducted in non-human subjects, there is a growing body of research, including contributions from Eide and Ringstad's group, validating the existence and function of the glymphatic pathway in humans using non-invasive MRI-based techniques (Eide and Ringstad, 2015; Ringstad et al., 2017; Eide et al., 2018; Ringstad and Eide, 2022).
In this article by Eide and Ringstad, the authors delve deeper into characterizing the anatomical and functional aspects of leptomeningeal perivascular CSF flow dynamics in humans under both healthy and diseased conditions. They utilize intrathecal gadobutrol-enhanced T1-weighted MRI and report the compartmentalization of the human subarachnoid space into what they propose is an inner perivascular subarachnoid space (PVSAS) and an outer generalized subarachnoid space (SAS). The PVSAS predominantly surrounds major arteries from the circle of Willis, such as the anterior cerebral artery (ACA), middle cerebral artery (MCA), and posterior cerebral artery (PCA). The authors observe direct CSF solute propagation between the basal cisterns and the PVSAS, and confirm antegrade CSF flow within the PVSAS of large surface arteries before reaching the adjacent cerebral cortex. Functionally, they note slowed CSF tracer appearance in periarterial subarachnoid spaces with increased pulsatile intracranial pressure, suggesting potential impairments in diseases like communicating hydrocephalus. Additionally, they find an enlarged PVSAS area in idiopathic normal-pressure hydrocephalus (iNPH), associated with altered CSF and solute flow dynamics and tracer accumulation in specific cortical regions.
The study by Eide and Ringstad deserves commendation for its impact, relevance, and methodological rigor. Nevertheless, it raises crucial questions, particularly regarding the membrane that segregates the inner perivascular space from the broader subarachnoid space. While the authors propose this membrane is part of the arachnoid, they provide limited data supporting an arachnoid rather than pial identity. Counter to this conception of a subarachnoid perivascular space, previous anatomical studies suggest that surface arteries and veins run in a sub-pial plane, with Virchow-Robin spaces formed by pial membrane invagination as surface arteries penetrate the brain parenchyma (Zhang et al., 1990). Further, recent work from Nedergaard's group identifies a potential 4th meningeal membrane, termed the subarachnoid lymphatic-like membrane (SLYM), distinct from the arachnoid, based on its expression of lymphatic endothelial cell markers (LYVE-1, PROX-1, and PDPN) (Plá et al., 2023). Consequently, it is also possible that this SLYM layer demarcates perivascular spaces from the subarachnoid space. Future investigations focusing on labeling for arachnoid barrier cells, pial cells, and lymphatic markers in postmortem human samples may clarify this membrane's identity.
Moreover, Eide and Ringstad's study reveals that tracer initially appears in the PVSAS of major arteries near the circle of Willis, suggesting CSF entry at proximal locations within the basal cisterns. The exact anatomical site of this entry, and the cellular and molecular components forming it, remain unknown, warranting further exploration. Further, this perivascular membrane appears to function akin to myelin on an axon in promoting rapid axial CSF and solute movement down arteries, while restricting radial movement into the broader SAS. The mechanisms underlying this barrier function, however, remain elusive. If this membrane indeed consists of arachnoid barrier cells, it may contain tight junction proteins that prevent perivascular contents from entering the surrounding SAS. Over time (three hours), there appears to be signal equilibration between the PVSAS and the SAS, leading the authors to suggest that the membrane is semipermeable. Supporting this, they demonstrate rapid signal enrichment in the adjacent cortex after tracer appearance in the PVSAS. This suggests similarities to fenestrated pia in Virchow-Robin spaces (Zhang et al., 1990) or SLYM, which allows fluid and solute movement up to 3 kDa in size (Plá et al., 2023). Future research should focus on understanding the factors contributing to this membrane's semipermeability, such as molecular weight or charge.
While most of the tracer is concentrated around arteries, there is also signal, though less pronounced, around surface veins (see supplemental figure 4). This hints at possible communication between the PVSAS of arteries and veins or the direct entry of tracer into the perivenous subarachnoid space from the basal cisterns. The latter scenario seems less probable, given prior research indicating that CSF and tracer predominantly flow toward the brain through periarterial spaces rather than perivenous spaces (Iliff et al., 2012). Furthermore, it is unlikely that tracer is entering the perivenous space directly from the parenchyma (the clearance side of the glymphatic pathway) at such an early stage (less than one hour). Therefore, there is likely direct solute communication from periarterial spaces to adjacent perivenous spaces, similar to what is observed in the adjacent cerebral cortex. This raises similar questions to those previously mentioned regarding factors limiting the movement of CSF and solute across this semipermeable membrane. Is the limited tracer observed in the perivenous space due to characteristics of the tracer molecule itself, such as its size? Would a larger molecule, exceeding the size of gadobutrol (690 Da), face greater restriction entering the perivenous space, or would its size hinder its movement into the brain parenchyma, causing it to accumulate in the perivenous space? These questions necessitate further thorough investigation, as their answers may provide insights into why proteinopathies such as cerebral amyloid angiopathy predominantly affect arteries rather than veins. Additionally, it would be intriguing to expand upon the remarkable temporal resolution showcased in this study to a range of 3-6 hours. Previous research conducted by the authors' group has identified enhancements in tracer activity around bridging veins and within the parasagittal dura (Ringstad and Eide, 2022). Recent findings from our own group indicate that arachnoid barrier discontinuities, referred to as arachnoid cuff exit (ACE) points, coincide with the entry of these bridging veins into the dura. This phenomenon allows CSF to permeate into the dura, playing a crucial role in CSF efflux (Smyth et al., 2024).
In conclusion, Eide and Ringstad's work presents the first human in vivo MRI evidence of periarterial space segregation, facilitating rapid CSF and solute movement from basal cisterns to adjacent cerebral cortex. Yet, key questions persist regarding the identity of the compartmentalizing membrane, the etiology of this membrane’s semipermeability, and the site of CSF entry into perivascular spaces. Future studies probing these aspects may illuminate fundamental mechanisms impacting brain fluid dynamics in health and disease.
References:
Iliff JJ, Wang M, Liao Y, Plogg BA, Peng W, Gundersen GA, Benveniste H, Vates GE, Deane R, Goldman SA, Nagelhus EA, Nedergaard M. A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid β. Sci Transl Med. 2012 Aug 15;4(147):147ra111. PubMed.
Eide PK, Ringstad G. MRI with intrathecal MRI gadolinium contrast medium administration: a possible method to assess glymphatic function in human brain. Acta Radiol Open. 2015 Nov;4(11):2058460115609635. Epub 2015 Nov 17 PubMed.
Ringstad G, Vatnehol SA, Eide PK. Glymphatic MRI in idiopathic normal pressure hydrocephalus. Brain. 2017 Oct 1;140(10):2691-2705. PubMed.
Eide PK, Vatnehol SA, Emblem KE, Ringstad G. Magnetic resonance imaging provides evidence of glymphatic drainage from human brain to cervical lymph nodes. Sci Rep. 2018 May 8;8(1):7194. PubMed.
Ringstad G, Eide PK. Molecular trans-dural efflux to skull bone marrow in humans with CSF disorders. Brain. 2022 May 24;145(4):1464-1472. PubMed.
Zhang ET, Inman CB, Weller RO. Interrelationships of the pia mater and the perivascular (Virchow-Robin) spaces in the human cerebrum. J Anat. 1990 Jun;170:111-23. PubMed.
Plá V, Bitsika S, Giannetto MJ, Ladron-de-Guevara A, Gahn-Martinez D, Mori Y, Nedergaard M, Møllgård K. Structural characterization of SLYM-a 4th meningeal membrane. Fluids Barriers CNS. 2023 Dec 14;20(1):93. PubMed.
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.
View all comments by Jonathan KipnisUniversity of Bern
Research concerning cerebrospinal fluid (CSF) flow and drainage is an emerging subject area, with numerous independent research groups conducting investigations to uncover its underlying mechanisms in animal models. Thus, it is crucial to understand CSF dynamics in humans, which allows for the translation of these novel findings into the clinic. In this report, Dr. Eide and Dr. Ringstad have investigated the features of early tracer dissemination after intrathecal injection of gadobutrol in the lumbar area in humans, and made a fascinating observation that the tracer distributes in the brain from the basal cisterns in an antegrade perivascular fashion around the major arteries before distributing in the subarachnoid space (SAS). This pattern of tracer distribution in the human brain has not been reported before and provides valuable insight. The authors interpret these findings such that the gadobutrol signal outlines a not-yet-described perivascular space for cerebrospinal fluid transport around the major arteries in the human brain. They propose this space to be an extension of the subarachnoid space that is separated by a semipermeable membrane and call it “perivascular subarachnoid space (PVSAS).”
As a Ph.D. student in neuroimmunology, I am fascinated by the anatomy and function of the meninges and the role of CSF in CNS immunity. Therefore, this exciting study performed in humans caught my interest and I critically read through the manuscript and presented it to my peers and supervisors as a journal club. While the data are original and very interesting, I admit, with due respect, that a number of conclusions drawn based on the MRI imaging are difficult to understand and, from my perspective, may benefit from considering other options for data interpretation. I listed these points below in hopes of stimulating discussion regarding this exciting data.
1: What is the evidence for the proposed semipermeable membrane?
Based on the observed distribution of intrathecally injected gadobutrol around the arteries subsequent to its arrival at the basal cisterns exterior to the gyrencephalic brain surface, the authors propose the existence of a novel membrane that compartmentalizes the human subarachnoid space and have named this transiently tracer-enhancing area ensheathed by the proposed membrane as the PVSAS. Why did they not consider the role of the pia mater in their study? To my understanding, electron microscopy studies have shown that the pia mater covers the floor of the SAS and is continuous with the fibroblasts forming the adventitia of the vessels. However, the anatomy of pia where the arteries penetrate the brain parenchyma is a matter of debate, and thus it is not yet clear if these perivascular spaces are continuous with the SAS or rather with the subpial space. Furthermore, the authors mention in the discussion that the resolution of MRI does not allow identification of their proposed membrane, yet still limit the interpretation of their data to a model supposing the existence of a membrane. What would be alternative interpretations for the observed tracer distribution? Could the distribution pattern be explained by the physics of fluid movement within the SAS, with a resultant faster distribution of contrast agent at regions close to arteries?
2: Why are the results presented without specifying the conditions of the patients undergoing the MRI scans?
Natural to a study with humans, the present cohort includes patients with various diseases. Because these diseases affect the CNS and its vasculature, distinguishing the state of healthy brains (in this case non-CSF-affecting diseases, according to the authors) versus diseased brains is important for drawing conclusions. I found the results to lack sufficient clarity regarding the assignment of the MRI scans to the respective disease conditions throughout Figures 1 to 8. While the study encompasses patients with diverse diseases, the paper only addresses the differences in tracer appearance between the reference and idiopathic normal pressure hydrocephalus groups in Figure 9; and appears to show no data of patients with spontaneous intracranial hypotension, arachnoid cyst, pineal cyst, idiopathic intracranial hypertension, and communicating hydrocephalus.
3: What is the variability of CSF tracer distribution between individuals?
Figures 3f and 4e show the first-time tracer appearance in different branches of the ACA and MCA, respectively. According to these figures, gadobutrol appears around the A2 and M2 branches of the arteries at approximately 50±10 minutes after the injection. However, according to Figures 7e and 8c and Supplementary Figure 6, first-time tracer appearance around these branches varies between five to 250 minutes, suggesting a high variability of tracer distribution between individuals. Thus Figures 3 and 4 preclude appreciation of the range observed across the entire cohort. Could this miss potentially important variabilities in CSF flow and tracer distribution between individuals or conditions?
4: What is the precise method for image analysis?
It was really interesting to see the visualization and quantification of the irregular tracer diffusion around the vasculature in the dementia brains. Since the paper lacks details of the analysis method, I am very interested to learn more about the analysis of such images. Specifically, knowing whether the measurement of the gadobutrol-enhanced area included or excluded the arterial lumen, whether sphericity was considered, and the details of the calibration methods for determining the size of the vessels in the MRI images would be informative. Comparing the data reported here with previously reported size measurements of cerebral arteries, the area seems to be too large for the spaces between gyri even in the presence of cortical atrophy in dementia patients (Gutierrez et al., 2014). More detailed methods would help us compare these novel findings with previously described dimensions of vessels and perivascular spaces.
5: Question about a citation.
The discussion mentions that Møllgård et al. reported that damage to the “subarachnoidal lymphatic like membrane—SLYM” proposed by these authors to divide the subarachnoid space impairs periarterial solute transport. This paper does not mention such a phenomenon, nor does it discuss the effects of damage to SLYM and its effect on periarterial solute transport. This paper does mention in the supplementary material that “surgical damage caused tears in dura and SLYM resulting in loss of the barrier properties of SLYM” (Møllgård et al., 2023), but does not correlate this to periarterial solute transport.
6: How did the study measure the barrier threshold of the proposed membrane?
The authors report that, “Our study concludes about the existence of a perivascular subarachnoid space, abbreviated PVSAS, surrounding larger arteries at the surface of the gyrencephalic brain, delineated by a semipermeable membrane that aligns well with the barrier threshold of 3 kDa described for “SLYM,” which also was impermeable to 1 μm wide fluorescent particles.” What is the proof or supporting data for this claim in the present study? As mentioned by the authors, the resolution of T1-weighted scans acquired with 3-Tesla MRI does not allow one to obtain such data.
7: Can we interpret these findings beyond select concepts?
In this paper, the authors seem to relate their observations to the concepts of glymphatic flow and the existence of the “SLYM,” both of which are subject to significant debate (Abbott et al., 2018; Engelhardt et al., 2017; Hladky and Barrand, 2014; Miao et al., 2024; Pietilä et al., 2023; Smith et al., 2017). Consideration of the present findings in the context of other currently discussed concepts of CSF flow would be highly desirable. From the point of view of a junior researcher, this study offers valuable data from numerous patients in need of treatment, through the extensive efforts of a team of esteemed scientists. I would welcome a broader discussion of these novel observations in the context of the known brain anatomy and with consideration of all concepts of CSF flow. I would like to thank Drs. Eide and Ringstad for their novel work, and for considering my comments.
References:
Abbott NJ, Pizzo ME, Preston JE, Janigro D, Thorne RG. The role of brain barriers in fluid movement in the CNS: is there a 'glymphatic' system?. Acta Neuropathol. 2018 Mar;135(3):387-407. Epub 2018 Feb 10 PubMed.
Engelhardt B, Vajkoczy P, Weller RO. The movers and shapers in immune privilege of the CNS. Nat Immunol. 2017 Feb;18(2):123-131. Epub 2017 Jan 16 PubMed.
Gutierrez J, Rosoklija G, Murray J, Chon C, Elkind MS, Goldman J, Honig LS, Dwork AJ, Morgello S, Marshall RS. A quantitative perspective to the study of brain arterial remodeling of donors with and without HIV in the Brain Arterial Remodeling Study (BARS). Front Physiol. 2014;5:56. Epub 2014 Feb 19 PubMed.
Hladky SB, Barrand MA. Mechanisms of fluid movement into, through and out of the brain: evaluation of the evidence. Fluids Barriers CNS. 2014;11(1):26. Epub 2014 Dec 2 PubMed.
Miao A, Luo T, Hsieh B, Edge CJ, Gridley M, Wong RT, Constandinou TG, Wisden W, Franks NP. Brain clearance is reduced during sleep and anesthesia. Nat Neurosci. 2024 Jun;27(6):1046-1050. Epub 2024 May 13 PubMed. Correction.
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.
Pietilä R, Del Gaudio F, He L, Vázquez-Liébanas E, Vanlandewijck M, Muhl L, Mocci G, Bjørnholm KD, Lindblad C, Fletcher-Sandersjöö A, Svensson M, Thelin EP, Liu J, van Voorden AJ, Torres M, Antila S, Xin L, Karlström H, Storm-Mathisen J, Bergersen LH, Moggio A, Hansson EM, Ulvmar MH, Nilsson P, Mäkinen T, Andaloussi Mäe M, Alitalo K, Proulx ST, Engelhardt B, McDonald DM, Lendahl U, Andrae J, Betsholtz C. Molecular anatomy of adult mouse leptomeninges. Neuron. 2023 Dec 6;111(23):3745-3764.e7. Epub 2023 Sep 29 PubMed.
Smith AJ, Yao X, Dix JA, Jin BJ, Verkman AS. Test of the 'glymphatic' hypothesis demonstrates diffusive and aquaporin-4-independent solute transport in rodent brain parenchyma. Elife. 2017 Aug 21;6 PubMed.
View all comments by Sarmad PeymaeiOslo University Hospital / University of Oslo
We thank Dr. Sarmad Peymaei for his comments on our article. The article includes a number of pictures to illustrate what we observed. We retrieved a few images retrieved from our article and show them below as input to Dr Peymaei’s comments. A few responses to his questions:
The article provides multiple images, supporting the interpretation that a PVSAS compartmentalizes the SAS. The temporal profile of tracer enrichment within PVSAS and SAS further suggests this compartmentalization is created by a semipermeable membrane. The tracer used has a molecular size of 605 Da, while the previously reported subarachnoid lymphatic like membrane (SLYM) was reported impermeable to solutes 3 kDa in size (Møllgård et al., 2023).
Figure 1a shows the distinct donut shape of contrast enrichment (white color visualizing the perivascular subarachnoid space, PVSAS) around the middle cerebral artery (A) within the subarachnoid space (SAS) that has black color. There is a sharp demarcation between PVSAS and SAS, indicative of a membrane, here deonted perivascular membrane (PVM), even though the submillimeter membrane cannot be visualized directly with magnetic resonance imaging, having a resolution of 1 mm.
Figure 4g shows that some time after contrast injection, the SAS is being enriched by contrast as well, even though the enrichment is still stronger within the PVSAS. However, also at this time, there is a sharp demarcation between the PVSAS and the SAS.
Figure 9a illustrates that the PVSAS (contrast-filled cavity) is altered in patients with the dementing disease idiopathic normal pressure hydrocephalus (iNPH). In these patients, the PVSAS shows altered configuration, and the artery has another location, residing more towards the margin of the PVSAS
Supplementary Figure 11 a-b. A higher magnification from a reference subject (left) and an iNPH patient (right) further visualizes the sharp demarcation between the PVSAS and the surrounding SAS is evident.
References:
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.
View all comments by Per Kristian EideMake a Comment
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