Anatomical correlates for the newly discovered meningeal layer in the existing literature: A systematic review

Anatomical correlates for the newly discovered meningeal layer in the existing literature: A systematic review

Abstract

The existence of a previously unrecognized subarachnoid lymphatic-like membrane (SLYM) was reported in a recent study. SLYM is described as an intermediate leptomeningeal layer between the arachnoid and pia mater in mouse and human brains, which divides the subarachnoid space (SAS) into two functional compartments. Being a macroscopic structure, having missed detection in previous studies is surprising. We systematically reviewed the published reports in animals and humans to explore whether prior descriptions of this meningeal layer were reported in some way. A comprehensive search was conducted in PubMed/Medline, EMBASE, Google Scholar, Science Direct, and Web of Science databases using combinations of MeSH terms and keywords with Boolean operators from inception until 31 December 2023. We found at least eight studies that provided structural evidence of an intermediate leptomeningeal layer in the brain or spinal cord. However, unequivocal descriptions for this layer all along the central nervous system were scarce. Obscure names like the epipial, intermediate meningeal, outer pial layers, or intermediate lamella were used to describe it. Its microscopic/ultrastructural details closely resemble the recently reported SLYM. We further examined the counterarguments in current literature that are skeptical of the existence of this layer. The potential physiological and clinical implications of this new meningeal layer are significant, underscoring the urgent need for further exploration of its structural and functional details.

1 INTRODUCTION

The meningeal coverings act as protective barriers enveloping the brain and spinal cord. Their arrangement and architecture are pertinent and imperative in the health and disease of the central nervous system (CNS). The traditional understanding of the CNS describes three meningeal layers—from outer to inner—dura mater, arachnoid mater, and pia mater (Standring, 2021).

Dura mater lines the cranial cavity and is an independent layer. However, the arachnoid and pia create a closed compartment along the brain and spinal cord surfaces—the subarachnoid space (SAS) being filled with cerebrospinal fluid (CSF). The SAS is traversed by the numerous “arachnoid trabeculae” attached to the arachnoid and pia mater. Recently, Møllgård et al. reported the existence of a new leptomeningeal layer in mouse and human brains located between arachnoid and pia, dividing the subarachnoid space into superficial outer and deep inner compartments (Møllgård et al., 2023). They described a “subarachnoid lymphatic-like membrane (SLYM)” as a one to two-cell thick mesothelial membrane not allowing the passage of moieties more than 1 μm in size and three kilodaltons in weight (Møllgård et al., 2023). Thus, it divides the SAS containing CSF into two functional compartments (Figure 1).

An intermediate leptomeningeal layer dividing the SAS into two functional compartments would have significant physiological and clinical implications. It would drastically revise the existing concepts of the brain's protective barriers and those of CSF dynamics. Moreover, the current understanding of the waste clearance mechanism in the CNS is also likely to change with this discovery. The absence of a well-defined lymphatics network in the CNS has always been puzzling. Although recent studies have established the presence of lymphatics in the walls of dural venous sinuses (reviewed in Kumar et al., 2019), it is not sufficient to explain how waste clearance is carried over through SAS. Some authors proposed that a perivascular sheath around the vessels traversing SAS creates an alternative glia-mediated “glymphatic system,” which brings about CNS waste clearance (reviewed by Kumar et al., 2019; Plog & Nedergaard, 2018). However, the formation of this sheath is least explained with the traditional description of meningeal arrangement in SAS. Now, indications for the presence of an intermediate layer in the SAS have raised new expectations, including a possible explanation of the formation of the glymphatic system.

The paradigm shift in established concepts brought in by the discovery of SLYM is intriguing and hence calls for meticulous scrutiny to explore the veracity of the claim of the previously unidentified meningeal layer. This becomes even more relevant since some recent studies have vehemently denied the existence of this new layer (Mapunda et al., 2023; Pietilä et al., 2023). Yet, on the other hand, a study has provided further evidence to strengthen the original claim (Plá et al., 2023). Thus, a controversy either mushroomed or persisted surrounding the existence of this newly identified layer.

Current evidence for the existence of this new layer is based on histological immunophenotypical characterization and in vivo microscopic animal imaging. The macroscopic details along the length of the neural axis and ultrastructural details of this layer are still awaited. Despite being a macroscopic structure, it escaped prior scientific detection even by advanced neuroimaging modalities. This is surprising, and exploring whether published literature provides any previous description of this layer is pertinent.

This article examined the published literature for prior indications of the intermediate leptomeningeal layer and its alignment with existing knowledge. Further, we reviewed the counterarguments in current literature that are skeptical of this layer's existence.

2 METHODS

2.1 Information sources and search strategy

We performed a systematic review of the published reports of meninges in CNS components of mammals, including humans, following the “Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)” guidelines, 2020 (http://www.prisma-statement.org/). The online databases, including PubMed/Medline, EMBASE, Google Scholar, Science Direct, and Web of Science, were searched for the original studies using multiple combinations of controlled vocabulary terms and free keywords with Boolean operators. Advanced search builder was used for PubMed, applying all fields to widen the search. Further, a more specialized systematic search with targeted terms involving physiological and clinical terms was performed to discuss the physiological and clinical significance and relevance of the newly reported meningeal layer.

2.2 Keywords and search strings

MeSH terms: (meninges, dura mater, arachnoid, pia mater, cerebrospinal fluid, blood–brain barrier, anatomy, humans, primates, brain, spinal cord, central nervous system, subarachnoid space, magnetic resonance imaging, microscopy, electron, scanning, transmission, ultrasonography, computed tomography, lymphatic vessels, glymphatic system, growth and development, inflammation).

Free keywords: subarachnoid lymphatic-like membrane (SLYM), epiphysial layer, inner pial layer, outer pial layer, circulation, blood-CSF barrier, dissection, trabeculae, layers, clinical significance, immune cells, mesothelium, ligamentum denticulatum, linea splendens, filum terminale, and so forth. For search strings see Supplementary file 1.

2.3 Eligibility criteria

Original reports of macroscopic, microscopic/ultrastructural, neuroimaging, and clinical studies of brain or spinal meninges in animals (mammals only) and humans were included. The studies published up to 31 December 2023 were eligible without language restrictions. Any case reports, commentary, and newsletters were excluded.

2.4 Data selection, extraction, and synthesis

Full articles reporting original details on the meninges were included in the study. Data collection was performed in two steps. In the first step, the titles and abstracts were screened. The articles in languages other than English were translated using Google Translate. Duplicate studies and those that did not meet the eligibility criteria were excluded. In the second step, full-text articles were assessed for the included studies. Reasons for excluding any article at the second step are given in Figure S1 (Supplementary file 2). No prioritization approach was adopted during the search to avoid a selection bias. Citation details and abstracts of all retrieved studies were downloaded into the Mendeley bibliography manager for the record. Three authors independently completed data collection. Two independent investigators performed the quality assessment of the included articles using the “Anatomical Quality Assessment (AQUA)” tool for the anatomical studies included in meta-analyses and systematic reviews, as given in Table S1 (Supplementary file 3) (Henry et al., 2017).

All co-authors contributed to and reviewed the qualitative analysis. Mutual discussions resolved any investigative disagreement. The final inferences were made based on the qualitative synthesis from the collected data. No quantitative analysis or statistical testing was performed.

4 DISCUSSION

4.1 Macroscopic and microscopic/ultrastructural anatomy

The recent study by Møllgård et al. claimed the identification of a new leptomeningeal layer in mouse and human brains using immunohistology, in vivo two-photon microscopy, and CSF tracer-based methods (Møllgård et al., 2023). The existing literature principally details the architecture of only three meningeal layers, i.e., dura, arachnoid, and pia mater. However, occasional studies in animals and humans provide indications of an intermediate leptomeningeal layer in the SAS of the brain and spinal cord (Table 1) (Angelov & Vasilev, 1989; Key & Retzius, 1875; Krisch et al., 1984; Mestre et al., 2022; Millen & Woollam, 1961; Nicholas & Weller, 1988).

The macroscopic description of meninges in history can be traced back to as early as 1600 BC, in the Edwin Smith Papyrus from ancient Egypt (Adeeb et al., 2013). The given macroscopic descriptions of meninges in Papyrus perhaps indicated the dura mater (Adeeb et al., 2013). In the fourth century BC, the Greek philosopher Aristotle, based on animal dissections, described two layers of meninges: the stronger one near the skull and the delicate one covering the brain, indicative of dura mater and pia mater, respectively (Adeeb et al., 2013). Although the presence of arachnoid mater between dura and pia mater was first described as early as the third century BC by Herophoilus, its detailed descriptions were given much later by Frederick Ruysch, a Dutch anatomist, in 1699 (Adeeb et al., 2013). Thus, a conventional order of referring outer to inner meningeal layers was set in practice as dura, arachnoid, and pia mater. Later studies provided extensive structural details for each layer (Coles et al., 2017).

The discovery of the SLYM between the arachnoid and pia changes the set order of the meningeal layers. The outermost meningeal layer—dura mater (dura- tough, mater-mother) is a thick, dense collagenous membrane called pachymeninx (patchy -thick). In contrast, the intermediate layer, the arachnoid mater, and the innermost layer, the pia mater, are called leptomeninges (lepto-thin) (Standring, 2021). SLYM is a new candidate in the category of leptomeninges. Being positioned between the arachnoid and pia mater in SAS, it has to be considered the intermediate leptomeningeal layer. In the revised order of the meninges, it should be recognized as the third meningeal layer from the outside, displacing the pia mater to the fourth layer.

Prior mention of an intermediate leptomeningeal layer in published literature in human SAS is limited to the spinal cord. As early as 1875, Key and Retzius described the inner leptomeninx as pia intima and epipial tissue. Pia intima directly posed on the nervous tissue and resembled the current description of the pia mater (Key & Retzius, 1875). The epipial tissue layer was described as a meshwork of collagenous fibers with a superficial, irregular, but deeper circular arrangement. Key and Retzius's descriptions (Key & Retzius, 1875) did not receive general support until 1961 when Millen and Woollam (1961) reconfirmed these findings in normal human adult and fetal spinal cords. Millen and Woollam noted that the epipial tissue layer was thick on the sides of the spinal cord and over the anterior median fissure. On the sides, it formed the collagenous core of the ligamentum denticulatum. In the anterior median fissure, thickened epipial tissue formed linea splendens (a glistening band formed by leptomeningeal thickening along the length of the anterior median fissure) that enclosed anterior spinal vessels. The epipial tissue also dipped into the depth of the anterior median fissure (Millen & Woollam, 1961).

Moreover, the filum terminale, the tubular extension from the terminal part of the spinal cord, was also described as formed of the epipial tissue layer. The epipial tissue layer was noted as a continuous sheet and only interrupted at the sites of entry and exit of the spinal nerve rootlets, forming a round margin around the nerves. The subarachnoid vessels lay between the epipial tissue layer strands before entering the spinal cord's substance (Millen & Woollam, 1961). The authors could not find its presence in the brain beyond the medulla oblongata. Later, in 1988, Nicholas and Weller described the presence of an intermediate meningeal layer between the arachnoid and pia in the human spinal cord using scanning electron microscopy (SEM) (Nicholas & Weller, 1988). Their portrayal of the intermediate meningeal layer in the spinal cord resembled the epipial tissue layer described by Key and Retzius (1875) and Millen and Woollam (1961). Nicholas and Weller also described the intermediate meningeal layer contributing to the ligamentum denticulatum and enveloping anterior spinal vessels at the anterior median fissure.

Interestingly, the epipial tissue layer (Millen & Woollam, 1961) or the intermediate meningeal layer (Nicholas & Weller, 1988) was described by the authors as the collagen fiber bundles arranged in lattices forming fenestrated sheets, thus giving free passage to CSF. Also, there was no mention of the leptomeningeal cells lining them. The permeability for CSF and absence of leptomeningeal cell lining in these descriptions disagrees with the portrayal of SLYM as presented by Møllgård et al. However, descriptions by Møllgård et al. were limited to the brain as their paper did not provide any data from the spinal cord.

The historical reports of the epipial tissue layer (Millen & Woollam, 1961) or intermediate meningeal layer (Nicholas & Weller, 1988) in the spinal cord were little followed in later studies. Descriptions of the epipial tissue layer in the formation of ligamentum denticulatum, linea splendens, and filum terminale (Millen & Woollam, 1961) did not attract much attention partly because these structures have been conventionally described as the modifications of the pia mater. We discussed Nicholas and Weller's SEM findings in detail under the sub-section “Hidden indications from SEM data.”

The earliest indication of an intermediate meningeal layer in rat and rabbit brain SAS is observable in two consecutive studies published in 1983–1984 by Krisch et al. (1983, 1984). The authors used IHC and EM to show the presence of a two-cell thick intermediate lamella that compartmentalized the brain SAS into pial and arachnoid spaces (Figure 2). This intermediate lamella comprised a single-cell outer pial layer enjoined with a similar thickness inner arachnoid layer. Both layers of the intermediate lamella were also continued as a “perivascular sheath.” The pial space became extremely narrow at some sites and again reopened at other sites. Replacing the conventional description of two leptomeningeal layers, the authors proposed “inner,” “intermediate,” and “outer leptomeninges,” encompassing SAS's inner and outer leptomeningeal spaces.

When authors injected horse radish peroxidase (HRP) in either the pial or arachnoid space, the dye was limited to that space only, indicating the impermeability of the intermediate lamella bordering these spaces (Krisch et al., 1984). Interestingly, intracerebral injection of HRP labeled the intercellular clefts of the intermediate lamella as well as the outer arachnoid layer but not the pial or arachnoid spaces, indicating unexplained channels of communications between intercellular clefts across the leptomeningeal layers and brain parenchyma (Krisch et al., 1984).

Kurucz et al., in a series of neuro-endoscopic studies in fresh human cadavers, described inner arachnoid membranes in brain SAS that match the positional description given for SLYM (Kurucz et al., 2013a, 2013b). The authors demonstrated that the inner arachnoid membrane was distinct from the outer arachnoid, with delicate trabeculae separating the layers. However, as a significant limitation, they provided only localized and fragmented descriptions, and it was unclear if the inner arachnoid membrane presented as a single and continuous layer along the neural axis. The SAS was more complicated at specific locations, which lodged large vessels and anastomotic channels, such as in the Sylvian fissure, interpeduncular, carotid, and pontine cisterns. Multiple inner arachnoid layers were shown at these places, some sieved and appearing as trabecular networks (Kurucz et al., 2013a, 2013b).

Interestingly, immediately before Møllgård et al. discovered SLYM, another study by Mestre et al. described the epipial tissue layer in mouse brains (Mestre et al., 2022). The authors presented an epipial layer of the pia mater in the cerebral cortex using advanced light and electron microscopy combined with routine and immunohistological stainings and CSF tracers. Mestre et al. 's descriptions closely resembled those of SLYM. However, their study primarily focused on delineating the contribution of the epipial layer in forming a perivascular sheath and lacked detailed exploration along the brain surfaces. Keeping with the earlier descriptions of the epipial tissue layer in the spinal cord, the authors seemed to consider it a component of the pia mater, as they did not examine its immunophenotypic differences from the arachnoid and pia mater (Mestre et al., 2022). The pial and epipial layer cells were identified as the reticular fibroblasts, displaying three generations of cytoplasmic processes. The cytoplasmic processes of the reticular cells were shown adjoined with the help of junctional complexes (i.e., tight junctions), forming a microporous meshwork. They further showed that the epipial layer formed perivascular sheaths around the cerebral arteries that facilitated CSF tracer accumulation, indicating its role in CSF circulation. The authors also showed degenerative changes in the epipial vascular sheath in mice models of aging and Alzheimer's disease (Mestre et al., 2022). As an exception, the description of the epipial layer by Mestre et al. also showed a deviation from that of SLYM by Møllgård et al. as the later demonstrated absence of tight junctions (Møllgård et al., 2023). However, observations from Møllgård et al. were based on the expression of a tight junction marker, claudin-11, that differentiated SLYM from arachnoid mater (Møllgård et al., 2023). An EM-based exploration of the junctional complexes in SLYM is advisable to resolve these differences.

Macroscopically, all three earlier known meningeal layers cover the CNS in its entirety. While the outer two layers are closely apposed, the inner two enclose a fluid-filled trabeculated space—SAS (Coles et al., 2017; Standring, 2021). The dura mater lines the cranial cavity and vertebral canal. The arachnoid mater maintains a loose apposition to the brain surface; however, it bridges over the sulci, cisterns, and between various brain parts. In contrast, the pia mater is tightly apposed to the brain surface and follows it into the sulci and cisterns (Coles et al., 2017; Standring, 2021). The SAS is just a capillary layer thick at the gyri and flattened surface in CNS, but it is deep and contains a substantial amount of CSF in the sulci and cisterns (Coles et al., 2017; Standring, 2021). Histologically, the arachnoid mater is thin and translucent—made of a few layers of flattened cells (Pawlina & Ross, 2020.). Arachnoid trabeculae, spongy connective tissue columns consisting of collagen fibers and fibroblasts, arise from the arachnoid mater (Coles et al., 2017; Pawlina & Ross, 2020). Compared to the two outer layers, which include a substantial amount of fibers, the pia mater is a delicate, single-cell-layer membrane (Coles et al., 2017; Pawlina & Ross, 2020). The basement membrane of the pia mater is fused with that of the glia limitans—the astrocytic sheath covering the brain parenchyma, with a nominal subpial space, thus closely adhering to the brain surface (Adeeb et al., 2013; Dasgupta & Jeong, 2019; Standring, 2021).

Although no macroscopic examinations were conducted, patterns revealed from the histological images in Møllgård et al. 's study suggest that the SLYM is very thin (14.2 ± 0.5 μm in mice brains) but has a macroscopically visible structure (Møllgård et al., 2023). Like the arachnoid mater, it passes over the sulci. Thus, the inner compartment would be more profound at the sulci than the outer CSF compartment. However, its arrangement over the cisterns and fissures is still not documented. Although the SLYM seems present in the entire length of SAS, Møllgård et al. did not provide any data from the spinal cord. The vessels have been described to be present in the inner compartment. The authors histologically showed that SLYM jackets around the vessels. However, whether it gets carried forward as a tubular sheath is unclear from their study (Møllgård et al., 2023). Studies exploring its gross details and neurosurgical/dissection methods to show it macroscopically are awaited. Microscopically, it is only a one to two-cell thick membrane intermixed with loosely organized collagen fibers. Møllgård et al. described it as an impermeable membrane that does not allow passage of moieties larger than one μm and three kilodaltons, which limits the exchange of most of the peptides and proteins, such as amyloid-beta and tau. Thus, the new layer divides SAS into two functional compartments (Møllgård et al., 2023). Studies providing its ultrastructural details are still lacking. Based on the histology and immunophenotypic characterization by Møllgård et al., SLYM closely resembles the lymphatic vessels (PDPN + Prox + Lyve −). It is immunophenotypically distinct from the arachnoid mater, pia mater, and arachnoid trabeculae. The leptomeningeal cells in SLYM lack tight or adherence junctions as they showed negative expressions of claudin-11 and E-cadherin (Møllgård et al., 2023). The trabeculae were shown to anchor SLYM to the arachnoid mater above and the pia mater below. Møllgård et al. did not mention if there were distinct immunophenotypic differences between the trabeculae in the outer and inner compartments; hence, this remains to be explored (Møllgård et al., 2023). The subarachnoid blood vessels were shown primarily in the inner compartment between SLYM and the pia mater (Figure 1) (Møllgård et al., 2023).

Unfortunately, no EM-based descriptions are currently available that can help differentiate SLYM ultrastructurally from other meningeal layers. Based on the histological features and permeability details presented by Møllgård et al., such as negative expressions of tight and adherence junction markers, claudin-11and E-cadherin, respectively, it can be speculated that its architecture is closer to the pia mater than arachnoid mater. In the EM, the arachnoid appears as a thick translucent sheet (about 200 μm in humans) composed of closely packed leptomeningeal cells joined by adherence junctions (i.e., desmosomes) and devoid of basement membranes (Saboori, 2021; Saboori & Sadegh, 2015; Weller et al., 2018). Its outer barrier layer abuts the dura mater, where tight junctions join the cells. The inner layer of the arachnoid facing the CSF –the reticular layer—has a thin underlying layer of collagen that extends into trabeculae. A thin layer of leptomeningeal cells underlines that reticular layer and extends over the subarachnoid trabeculae. In comparison, the pia mater comprises a thin layer of leptomeningeal cells joined mainly by the gap junctions with the occasional presence of desmosomes (Weller, 2005; Weller et al., 2018).

In addition to these structural details, the authors presented robust evidence in mouse brains suggesting that SLYM may have a role in CNS immune responses as it harbored immune cells (CD45 + macrophages) that increased with aging and in the presence of inflammation (Møllgård et al., 2023).

4.2 Embryogenesis

The developmental evidence for an intermediate leptomeningeal layer is scarce in the literature. In a rare study in 1989, Angelov and Vasilev, using light and electron microscopy, described the presence of inner and outer pial layers in the embryonic rat brains enclosing blood vessels in between (Angelov & Vasilev, 1989). This description resembles the inner compartment of the SAS in the study by Møllgård et al., with inner and outer pial layers representing the pia mater and SLYM, respectively.

4.2.1 Contradictions with proposed mesothelial derivation

In mammals, including humans, the primordial source for meninges development is the mesenchymal tissue surrounding the neural tube: the primary meninx (also called primitive meninx or meninx primitiva). The primary meninx is divided into a dense outer layer and an inner reticular layer. The inner layer is considered to be the meningeal mesenchyme. The meningeal primordium is further separated into pachymeninx (dura mater) and leptomeninx (arachnoid and pia mater) by the dural limiting layer. While pachymeninx contains longitudinally arranged fibroblasts, the leptomeninx is a meshwork of loosely organized cells. With further development, the leptomeninx undergoes cavitation, leading to the formation of arachnoid trabeculae and the subarachnoid space bounded by the arachnoid and pia mater at its outer and inner limits, respectively (Batarfi et al., 2017; Dasgupta & Jeong, 2019; Schoenwolf et al., 2021).

This primordial source also gives rise to the skull and the scalp. Further, the primary meninx also gives rise to a perineural vascular plexus, which further develops into meningeal blood vessels. The histological observations suggest that the mesenchymal cells in the primitive meninx originate primarily from the neural crest cells (Batarfi et al., 2017; Dasgupta & Jeong, 2019; Schoenwolf et al., 2021). The neural crest cells are, in turn, derived from the ectoderm at the margin of the neural tube following mesenchymal transformation (Schoenwolf et al., 2021). Certain studies also suggest a contribution from the paraxial mesoderm in deriving primitive meninx (Couly et al., 1992; O'Rahilly & Müller, 1986). Notably, the paraxial mesoderm has an established contribution to the axial skeleton, including the head region (Tani et al., 2020); hence, it is a likely contender for developing leptomeninges. However, evidence is scarce on the role of the lateral plate or intermediate mesoderm in developing the axial skeleton and any meningeal layers.

Møllgård et al. described the SLYM as a mesothelium based on the immunohistochemical expression of podoplanin (PDPN), which is commonly expressed in the mesothelial membranes of the body cavities around the heart, lungs, and abdominal organs (Møllgård et al., 2023). Based on the PDPN expression, the authors compared the embryonic derivation of SLYM to the mesothelial lining of these body cavities, hinting at their shared embryonic development. These body cavities are embryologically derived from the intraembryonic coelom in the lateral plate mesoderm. If accepted, the proposed mesothelial derivation of the SLYM relates it developmentally to lateral plate mesoderm rather than paraxial mesoderm or neural crest cells. It, therefore, strongly contradicts the existing paradigm about the embryogenesis of the meningeal layers (Prummel et al., 2020; Schoenwolf et al., 2021) and may even call for the revision of the fundamental understanding of CNS development. Any such proposition will require a radical revision of human embryology and the evolution of chordates as it necessitates the presence of intraembryonic coelom (thus lateral plate mesoderm) inside the skull or vertebral column (Schoenwolf et al., 2021), which is quite a radical conceptual revision and would need extraordinary evidence and comprehensive work.

Moreover, in stark contrast to the proposed single-layer structure in SLYM, mesothelial derivatives are double-layered, enclosing fluid-filled cavities (Schoenwolf et al., 2021). Also, PDPN, used as a mesothelial marker, is not specific to intra-embryonic coelomic mesothelium. It is also expressed in derivatives of paraxial mesoderm, such as lymphatic vessels (Stone & Stainier, 2019), and intermediate mesoderm, such as podocytes in the glomerulus of the kidney (Tanaka et al., 2022). It is also expressed in many non-mesodermal derivatives, such as type 1 pneumocytes and thymic medullary epithelial cells, which are endodermal in origin (Astarita et al., 2012). Thus, PDPN alone provides a critical lead but does not necessarily justify any mesodermal component from which SLYM may be derived. The immunohistochemical examination of the markers for all mesodermal parts will be indispensable in establishing its embryonic derivation (Dasgupta & Jeong, 2019).

4.3 Physiological and clinical relevance

Functional compartmentalization of the SAS by SLYM necessitates a new conceptualization of the dynamic of CSF circulation. Per the traditional description, the CSF is secreted by the choroid plexus in the cerebral ventricles (chiefly in the lateral but also the third and fourth). From the lateral ventricle, CSF enters the third ventricle through the interventricular foramen (of Monro) and then through the cerebral aqueduct into the fourth ventricle. From the fourth ventricle, it enters SAS around the brain and spinal cord through the openings in the inferior aspect of the roof of the fourth ventricle (foramen of Magendie in the midline and foramen of Luschka bilaterally). From SAS, CSF is drained into the dural venous sinuses through arachnoid granulations (Standring, 2021). The presence of SLYM has added one more layer of complication to the previously described CSF course through SAS. It is currently not known if the outer and inner compartments created by the SLYM have any anatomical communication along the complete length of SAS.

CSF has an established role in clearing brain waste (Plog & Nedergaard, 2018). Compartmentalization may have an impact on the composition of the CSF. It is unclear whether CSF pressure and CSF circulation vary between the compartments. Arguably, the inner compartment bounded between the new membrane and pia mater lies in a close approximation of the brain parenchyma; it will majorly receive the brain waste. On the contrary, the outer compartment will contain a clearer CSF, as there is a limited exchange of solutes between the two compartments.

The compartmentalization of SAS by SLYM complicates the course. One might ask: how does the CSF exiting through the median and lateral openings at the fourth ventricle enter two separate compartments? And how is it absorbed into the dural venous sinuses? As SLYM does not allow passage of molecules greater than one μm and three kilodaltons (Møllgård et al., 2023), it remains unexplained how larger molecules generated as brain waste will be removed from the inner compartment if it has no direct access to the dural venous sinuses.

The presence of SLYM creates an additional layer in the blood-CSF/brain barrier. The blood vessels passing through the inner compartment may carry a sleeve of SLYM; hence, it is likely to contribute to the formation of the glymphatic system (Møllgård et al., 2023), the proposed perivascular route of waste clearance from the CNS (Kumar et al., 2019; Plog & Nedergaard, 2018). This also gives rise to an alternative possibility that the brain waste may directly drain into outer space through the perivascular sheath present along the cerebral vessels.

Contrary to the belief that the brain is an immune-privileged organ, the physiological presence of immune cells, primarily leucocytes, in meningeal spaces and circulation in CSF is now an established phenomenon (Gadani et al., 2017; Nevalainen et al., 2022; Walker et al., 2018; Wolf et al., 2009). The meningeal immune cells are believed to monitor the entry of pathogens to the CNS. Møllgård et al. showed that SLYM not only shares the immunophenotypic characteristics with the lymphatic vessels but also shows the presence of receptors of immune cells (Møllgård et al., 2023), which indicates that it creates an immunogenic checkpoint against the pathogens invading the SAS. A traumatic rupture or pathological degeneration of SLYM will allow the mixing of the dirty and clear CSF and permit the access of pathogens in the inner compartment and, consequently, to the CNS parenchyma. Thus, an intact and healthy SLYM creates an additional protective barrier to the CNS.

Aging and consequent immune cell dysregulation have been implicated in the pathogenesis of many neurodegenerative disorders, such as Alzheimer's and Parkinson's, amyotrophic lateral sclerosis, and multiple sclerosis (Mayne et al., 2020). Possible degenerative changes in the SLYM with aging may lead to their pathogenesis.

The discovery of the new meningeal layer also necessitates revising the drug delivery approaches to the CNS. Inter-individual variations in the clinical and adverse effects of subarachnoid drug administration for spinal anesthesia/analgesia are common (Fettes et al., 2009). Anatomical factors are suggested to play a role (Tuominen et al., 1992); however, the exact mechanisms have not been understood. SLYM may help to explain this phenomenon. Neural absorption of an anesthetic agent injected into the respective CSF compartments of spinal SAS may vary, which is likely to be reflected in their clinical and adverse effects.

4.4 Counterarguments against the claims of a discovery

At odds with Møllgård et al. 's claim of breakthrough discovery, two recent studies presented strong reservations about the very existence of SLYM (Mapunda et al., 2023; Pietilä et al., 2023). Pietilä et al., in a single-cell RNA-Sequencing (Sc-RNA seq) study of mouse meningeal fibroblasts, identified six different expression profiles (type 1–6) of leptomeningeal cells. Transgenic reporter labeling showed that type 3 fibroblasts matched the Prox1 + cells in the arachnoid mater juxtaposed to the barrier cells (type 4 fibroblasts). They noted that Prox1 + cells are discontinuous, possibly intermixing with type 2 fibroblasts in the inner arachnoid layer. The inner arachnoid was identified distal to the barrier cell layer based on the specific ultrastructural features revealed in TEM. They further showed that the inner arachnoid connects with the barrier cell layer with the adherens junctions. Although the authors did not perform any immunostaining for Prox1 + cells in TEM, it was performed for Dpp4—a marker of the barrier cell layer. A localization study with Prox1 and Dpp4 in sequential sections may be needed to give more precise information on the proximity of Prox1 + cells with the barrier cell layer (Pietilä et al., 2023).

Moreover, Møllgård et al. did not describe the characterization of the fibroblasts lining the arachnoid trabeculae. Of note, the fibroblastic layers transversely traverse through the SAS, creating distinct compartments that can be conferred from the schematic representations of TEM images of mammalian brains in classic historical literature (Figures 2 and 3) (Krisch et al., 1983, 1984; Nabeshima et al., 1975; Orlin et al., 1991). Although Møllgård et al. cite these precedences to corroborate their immunohistochemical findings, they performed no TEM imaging. A more detailed TEM imaging of the SAS with immunolabeling of the structures in context may be necessary to conclude the existence of the SLYM.

Another study by Mapunda et al. performed two-photon live imaging of injected fluorescent tracers in VE-cadherin-GFP reporter mice (Mapunda et al., 2023). The authors showed that VE-cadherin—a marker of the adherens junctions in the vascular endothelium—was abundantly expressed by leptomeningeal cells of the arachnoid and pia mater. They further observed that tracers injected into the bloodstream appeared in the subdural space, perhaps due to leaks from the vessels with fenestrated endothelium, but did not enter the subarachnoid space. In contrast, tracers injected into the CSF filled the SAS between the arachnoid and pia mater but did not cross into the subdural space. The authors claimed these tracers showed the existence of a single barrier beneath the dura mater—the arachnoid mater, and a single SAS compartment. Using immunostaining, they further showed that Prox1 + cell layer co-expressed VE-cadherin and clung to E-cadherin-positive ABCs, nullifying the possibility of CSF-filled space between them. Notably, they did not use Prox1-GFP transgenic mice, which could precisely show if the tracers were injected into the SAS beneath the Prox1 + cell layer. Hence, missing the Prox1 + layer in their in vivo optical imaging of the injected tracer may not be ruled out. Fluorescent VE-cadherin tagging could show the position of the arachnoid mater, but it could not help precisely locate the barrier cell layer (Mapunda et al., 2023). Interestingly, the appropriate demonstration of arachnoid layers was also missing in Møllgård et al. 's in vivo optical imaging of the injected tracer in Prox1-GFP transgenic mice. It is quite relevant to mention that using a reporter mouse for the ABC layer and Prox1 in tracer-based studies may be a way to examine the presence of SLYM as an independent leptomeningeal layer and compartmentalization of SAS.

Other than these studies, multiple e-letters in the original journal of publication (Møllgård et al., 2023) and comments on the science media platforms (ALZFORUM, 2023.) also questioned the existence of SLYM. The commenters generally believed that tracers appeared above the Prox1 + cells in Møllgård et al. 's experiments because the tracer was injected in the subdural space. Hence, they showed a subdural space created by the experimental manipulations, not a separate subarachnoid space.

The objections raised by the commenters seem coherent, as Møllgård et al. did not use any marker to indicate the location of the ABC layer during optical imaging. Although the tracer they injected into the cisterna magna appeared beneath and did not cross through the Prox1 + layer, appropriate localization of the ABC layer has been a valid concern. The outer compartment they have shown seems to extend up to the dura mater. The absence of arachnoid mater in optical imaging strengthens the view of commentators. Moreover, they also do not precisely describe if the tracer was injected into the inner space beneath the Prox1 + layer of cisterna magna. This becomes even more relevant when considering that the compartmentalization of SAS by the SLYM layer should also extend into the cisterna magna.

In a recent article, Plá et al. presented further immunohistological evidence in postmortem mouse brains to corroborate Møllgård et al. 's findings (Plá et al., 2023). Notably, both studies have a shared team of co-authors. Plá et al. precisely differentiated the SLYM from the arachnoid barrier cell (ABC) layer. The compartmentalization of SAS into the inner and outer spaces, including in the cisterns around the brain stem, was also demonstrated. However, the SLYM was shown to adhere to the ABC layer in the mice's spinal cord. Of note, the authors only examined the upper cervical segments of the spinal cord (Plá et al., 2023).

Plá et al. suggested that the collapse of the SAS during postmortem tissue processing may be a likely reason that the later studies that examined the existence of SLYM cannot show an intervening space between the arachnoid barrier and Prox1 + cells. The authors performed high-resolution (9 Tesla) serial MRI imaging of the postmortem mice brains to substantiate the SAS's collapse during tissue processing (Plá et al., 2023).

However, neither Møllgård et al. nor Plá et al. precisely demonstrated the trabeculae between the leptomeningeal layers. This fails to completely dispel the readers' apprehension that the SAS compartments are not artifactually created due to the mechanical separation of the arachnoid cell layers during postmortem tissue processing. Alternative fixation methods that need shorter fixation time and immunostaining of fresh frozen sections may better preserve the SAS compartments and trabeculae between the leptomeningeal layers. Moreover, exploring the SLYM in larger animals, including humans, where SAS will be more voluminous, will help precisely differentiate it from the other leptomeningeal layers.

Surprisingly, Møllgård et al. 's and Plá et al. 's histological images show that the blood vessels are primarily limited to the inner space. This phenomenon is peculiar and anatomically challenging to explain, considering the established anatomy of the head–neck vessels. In the latter article, Plá et al. referred to the ultrastructural and light microscopic images from the earlier studies in the mammalian brain by Nabeshima et al. (1975) and Orlin et al. (1991) to support the compartmentalization of SAS by an intermediate leptomeningeal layer (Figure 3).

Interestingly, the light microscopic image by Orlin et al., 1991 clearly shows the presence of blood vessels along the complete width of SAS. Also, the blood vessels in the annotated outer compartment in the image appear of a higher caliber than the inner compartment, substantiated by the narrower width of the latter (Figure 3b). Due to its high evolutionary incentives, explaining whether the mouse subarachnoid blood vessels have a different arrangement than the other mammals is difficult yet equally crucial.

TEM-based validation of the SLYM and exploration of its detailed ultrastructure remains a concern. Although only a few TEM studies that examined this are currently available, none explicitly supported SLYM's independent existence (Grubb, 2023; Mapunda et al., 2023; Pietilä et al., 2023). Grubb recently analyzed publicly available electron microscopy datasets for mice brains (Grubb, 2023). The author described the arachnoid mater as composed of at least four different cell types: the dural border cells, ABCs, reticular cells, and inner arachnoid fibroblasts. The arachnoid mater comprises an inner layer of fibroblasts that attach to and are indistinguishable from pia mater fibroblasts and resemble the BFB2 cells identified by Pietilä et al. Above that is a layer of reticular cells, which resemble the BFB3 fibroblasts identified by Pietilä et al. The authors speculated that the reticular cell layer may be the SLYM reported by Møllgård et al. They further noted that reticular cells are fenestrated and are loosely attached among themselves and to ABCs with adherence and gap junctions. Additionally, they observed that the reticular cells have a large amount of rough endoplasmic reticulum and large mitochondria. They often have more than one nucleus and appear darker (more electron-dense) than the other cells in the arachnoid mater. Outer to the reticular cell layer lies the ABC layer. These cells are often multinuclear (up to 4 nuclei), form a tight barrier, and are attached by long stretches of adherence and gap junctions with slim and electron-dense intracellular spaces (Grubb, 2023). The author opined that the inter-cellular junctions of the reticular cell layer (with each other and with the ABC layer) could be VE-cadherin instead of E-cadherin, as indicated in the study by Mapunda et al. (Grubb, 2023; Mapunda et al., 2023). Grubb's view is supported by all contesting studies, which showed E-cadherin located explicitly in the ABC layer (Grubb, 2023; Mapunda et al., 2023; Møllgård et al., 2023; Pietilä et al., 2023).

The possible reasons for the variance of opinion about the intermediate leptomeningeal layer in TEM studies must be resolved. Notably, TEM imaging requires a tiny sample. In little brain samples, leptomeningeal layers may get lost during tissue processing, thus confounding the interpretation of the ultrastructural images. The other factors, such as postmortem delay, type of fixative, and fixation duration, may also impact the salvage of the cellular layers during histological processing for TEM. Moreover, the intermediate leptomeningeal layer may have an irregular placement across the SAS; hence, in ultrastructural imaging, it may be confused with the obliquely placed arachnoid trabeculae. Notably, horizontally placed trabecular sheets are also described in previous ultrastructural studies, augmenting the possibility of an alternative explanation of the results reported by Møllgård et al.

4.5 Hidden indications in published SEM data

A comprehensive review of the prior dissection-based or microscopic studies in animal/human anatomy gives no clear indication of the presence of SLYM. Despite being a macroscopic structure, how it escaped from the observations in the scanning electron microscopy (SEM) studies is astonishing. As a limited stance, Alcolado et al. in 1988 indicated the existence of sheet-like trabeculae in the SAS of the brain, which consisted of collagen bundles and leptomeningeal cells connected by desmosomes and gap junctions (Alcolado et al., 1988). However, they gave no indications of any intermediate leptomeningeal layer. In the same year, Nicholas and Weller described the presence of an intermediate meningeal layer between the arachnoid and pia mater in the human spinal cord using SEM (Nicholas & Weller, 1988). However, they did not demonstrate its presence in the brain. Unfortunately, their discovery was not adequately followed up in later studies.

A review of the published data hints that escaping identification by SEM could have been caused by an error in annotations in the absence of factual knowledge about this layer. Below, we will discuss the SEM images of CNS components from the published literature in mammals (Saboori, 2021; Saboori & Sadegh, 2015; Weller et al., 2018). Figures 4 and 5 show the structural components of the SAS in the brain, Figure 6 in the spinal cord, and Figure 7 around the optic nerve. In Figure 4, the membranous structure annotated as the pia mater by the original authors could be the new meningeal layer, as the SAS can be observed on both sides of this structure. The pia mater is visible as the membrane firmly adhered to the brain tissue. The intermediate layer is seen to give attachment to the trabeculae (more evident in the side of the arachnoid mater). This layer is much thinner than the arachnoid mater but thicker than the pia mater and appears to have a fibrous component. The SAS is visible on both aspects of the intermediate layer.

Similarly, in Figure 5, an intermediate layer is observable in a part of the SAS. However, at other places, it seems confounded by the presence of arachnoid trabeculae. The blood vessels seem to be chiefly present in the inner compartment of SAS, limited by the intermediate layer and pia mater. Figure 6 presents a reannotation of a published SEM image of the cross-section of the dog's spinal cord and covering meninges (Merchant & Low, 1979). Our re-annotation indicates that the pia mater is firmly adherent to the spinal cord and dipped into the spinal fissures (Figure 6a, indicated by yellow arrows). There is another membranous structure underneath the arachnoid mater. This is likely the new meningeal layer, free from neural surfaces and passing over the spinal cord fissures. It divides the SAS into two compartments (Figure 6a, indicated by red arrowheads). The presence of CSF-filled spaces and trabeculae can be marked in both aspects (Figure 6b, inset image). Interestingly, this membranous structure encloses the anterior spinal vessels, sending a septum into the anterior median fissure (Figure 6a, indicated by red arrowheads).

In Figure 7, the membranous structure shown with red arrowheads is likely the new meningeal layer. This membranous layer divides the subarachnoid space (SAS) into two compartments. The outer compartment is visible and contains prominent trabeculae and septae (shown with white arrows). The inner compartment is not so obvious; however, a fine trabecular meshwork is observable inside it. Although the original authors described this layer as the pia mater (Killer et al., 2003), it should not bear a trabeculated space beneath to be considered so. In our view, the actual pia mater is indicated by a thin white sheath closely encircling the nerve (shown with yellow arrows). These observations suggest a revised interpretation of the SEM images of SAS in light of the demonstration of the intermediate leptomeningeal layer in the brain by Møllgård et al. (2023). Apart from the lack of factual information, there could be multiple reasons why the SEM studies missed this macroscopic structure. A thin membrane lacking robust fibrous support could be at a high risk of getting damaged during tissue processing. Moreover, its identification could be prone to confusion and misinterpretation because it is sandwiched between arachnoid trabeculae and possibly is unevenly placed in the collapsed SAS. Specifically, the flattened and obliquely placed trabeculae can confound its identification. As its presence in the brain is established, it can be more explicitly targeted in SEM imaging. Following it along the complete length of SAS may help differentiate it from trabeculae.

4.6 Possible regional differences in SLYM structure

Møllgård et al. 's description of SLYM in mice and humans is limited to only the cortex and brain stem. SLYM has not yet been shown in the spinal cord or around the optic nerve. Both structures are covered with the meninges, showing the continuity of SAS with the brain. The rare description of the epipial tissue layer (Millen & Woollam, 1961) or intermediate leptomeningeal layer (Nicholas & Weller, 1988) between arachnoid and pia mater in the human spinal cord varies significantly from that by Møllgård et al. (2023) in the brain. Millen and Woollam (1961), as well as Nicholas and Weller, described a fenestrated membrane that can allow the CSF content exchange (Nicholas & Weller, 1988) (Figure 8). In contrast, Møllgård et al. showed that SLYM is an impermeable structure that does not allow the passage of moieties more than one μm and three kilodaltons (Møllgård et al., 2023).

Although the studies by Millen and Woollam (1961), Nicholas and Weller (1988), and Møllgård et al. (2023) used different methods, the mismatch of the conclusions is evident. This hints at possible regional differences in the architecture of the SLYM. It will be necessary to investigate its architecture along the entire length of SAS, especially along the spinal cord and optic nerve, to understand its impact on CSF dynamics and brain barrier functions.

4.7 Hidden indications in neuroimaging

Among the commonly practiced neuroimaging methods, ultrasonography (USG) and computed tomography (CT) scans are mainly limited to detecting the dura mater. Magnetic resonance imaging (MRI), the most sensitive imaging modality for viewing the meninges, can see all three meningeal layers (Cinnamon et al., 1994; Kirmi et al., 2009; Meltzer et al., 1996; Patel & Kirmi, 2009; Sze, 1993; Trinh & Massoud, 2023). The leptomeningeal layers, specifically the pia mater, may be difficult to appreciate due to their thinness. The leptomeningeal layers show enhancement in MRI images owing to their high vascularity, mainly when contrast is used. T2-weighted MRI sequences can be more suitable for viewing the leptomeningeal layers as they appear hypointense compared with hyperintense CSF sandwiched between them (Cinnamon et al., 1994; Kirmi et al., 2009; Meltzer et al., 1996; Patel & Kirmi, 2009; Sze, 1993; Trinh & Massoud, 2023). The arachnoid mater and pia mater are more clearly differentiated at the sulci, fissures, and cisterns owing to the increased gap. SAS gets deeper at these locations with ample CSF in-between, which can help differentiate the leptomeningeal layers (Standring, 2021).

Analyzing a typical MRI image from an adult human brain does not indicate an intermediate structure dividing SAS. Using T2 weighted, FLAIR, contrast-enhanced images or applying ultra-high magnetic fields (such as 7 Tesla) seems to provide no help. However, a thin hypointense line can be seen traversing longitudinally through the middle of SAS in T2 weighted images of the spinal cord (Figure 9). This line is observed bilaterally in the complete length of SAS and hints at a fascial structure (Figure 9). Further, the line can be traced upward over the cisterna magna (Figure 9). Being a continuous line, and the presence of CSF on both aspects indicate it to be a membranous structure, most likely the SLYM.

The non-visibility of any such streak in the brain SAS in T2 weighted MRI images of the normal brain can be explained by the narrow width of the SAS. This view is supported by the faint visibility of an intermediate hypointense line in the T2 weighted brain MRI images of the cases of benign enlargement of SAS (BESS) in infants (Figure 10). Wide SAS may help to differentiate the identification of the intermediate hypointense structure against the backdrop of hyperintense CSF. This middle hypointense line in SAS in BESS cases can be seen in the full extent of the brain (Figure 10).

Optical coherence tomography (OCT), an optical imaging modality used for high-resolution cross-sectional imaging, can potentially visualize SAS components (Benko et al., 2020; Hartmann et al., 2019). It can be used in vivo in neurosurgery patients undergoing craniotomy or in situ postmortem brains after opening the cranial vault. We re-annotate a published OCT image of SAS in fresh frozen cadaveric human brains, indicating its plausible compartmentalization by SLYM (Figure 11) (Benko et al., 2020).

4.8 Limitations and future directions

Møllgård et al. 's SLYM description is limited to its identification as a layer-like structure (Møllgård et al., 2023), which may or may not be a distinct layer per se. Very little is known about its structure, especially its macroscopic appearance and ultrastructural details. The controversy surrounding its existence in recent studies (Mapunda et al., 2023; Pietilä et al., 2023) must be resolved.

Although SLYM has to be a macroscopically visible structure, approaching it in the animal or human brain remains challenging, limiting its neurosurgical application and anatomical demonstration during medical teaching. Further, its topographical arrangement around the brain surfaces—particularly at sulci and cisterns, vascular relations, contribution to the blood-CSF/brain barriers, and CSF circulation, including secretion and absorption through the compartments, are poorly understood. Also, the CSF contents in the outer and inner compartments should likely vary. Moreover, no other significant purpose of the compartmentalization of SAS is clear except for creating an immunogenic barrier. These aspects must be extensively studied to understand the complete physiological and clinical significance of the compartmentalization of the SAS.

The presence of the SLYM also necessitates a rethinking of the anatomical recognition of some meningeal structures which are considered modifications of the pia mater, such as tela choroidea in the brain, and ligamentum denticulatum, linea splendens, and filum terminale in the spinal cord. These meningeal modifications are seen just beneath the arachnoid mater. Hence, keeping with traditional description, it is likely that their identification might have been erroneously imparted to the pia mater (Mizutani & Rodesch, 2020).

Despite frequent previous mentions of the intermediate leptomeningeal layer in brain and spinal SAS, it has been little appreciated in the current literature. Available descriptions in the literature are inconsistent and use varying names, which can explain this obscurity. Its thorough exploration along the complete length of the central nervous system will be desirable to establish it as a distinct or independent meningeal layer. Moreover, devising the neurosurgical approaches for cadaveric dissection and in vivo operative procedures and using standard terminology in line with the existing meningeal layers will be essential to popularize the newly discovered meningeal layer in medical education and clinical practice. Also, opinions from evolutionary neuroanatomists cannot be underappreciated in this case as they might help us understand/appreciate embryology in ways that could radically modify our understanding of the CNS.

4.9 Concluding remarks

Occasional studies described the presence of an intermediate meningeal layer in the SAS of the brain or spinal cord, which closely resembles the newly discovered meningeal layer. However, unequivocal descriptions for this layer all along the CNS are scarce. Its detection in neuroanatomical and imaging-based studies, especially in the brain, could have been missed for various reasons, such as extreme thinness, structural loss and collapse during tissue processing, and confounding with the arachnoid trabeculae and blood vessels in the SAS. The discovery of the SLYM is a landmark addition to the neuroanatomy and will likely redefine the subject in many ways. It is expected to change the existing concepts about the protective barriers and the dynamic circulation of CSF around the CNS.

Consequently, established conceptions about the pathogenesis of many neurodegenerative diseases are also likely to change. This discovery can potentially revolutionize drug delivery approaches to the CNS. It seems to open a new avenue in neuroscience research, and future studies are likely to unravel its detailed structure and role in brain health and disease. Although prior descriptions in literature corroborate SLYM, controversy persists about its distinct existence. A potential caveat with SLYM discovery is that it has not yet been established in ultrastructural studies. Its detailed structure and function also remain to be established. Future studies may hopefully fill these gaps.

A recent update in literature: During the peer review period, an interesting update was added to the discussion on SLYM. Eide and Ringstad examined a cohort of 75 human subjects (28 males and 47 females) (Eide & Ringstad, 2024). The authors conducted a series of repeated MRI acquisitions during the first 2 h after intrathecal contrast administration of a CSF tracer (gadobuterol). The MRI examination showed a time-dependent perivascular pattern of enrichment of the CSF tracer antegrade along the major cerebral arteries that created a perivascular hallo, giving rise to a unique donut-shaped appearance in the cross-sectional images. Their observations suggested the existence of a compartmentalized SAS around the arteries. Their finding provides a firm indication of a perivascular semipermeable sheath limiting the spread of the CSF tracer. Moreover, this study also garners strong evidence for the glymphatic system-based clearance of brain waste along the course of cerebral arteries. The perivascular sheath could not be distinctively demonstrated along the cerebral veins.

Eide and Ringstad discussed the possibility of the SLYM contributing to the formation of the perivascular sheath in SAS; however, they did not examine their immunophenotypic similarity (Eide & Ringstad, 2024). Of note, a perivascular sheath around the profiles of the cortical arteries sharing immunophenotypic similarity to the SLYM is evident in Møllgård et al. 's and Plá et al. 's papers (Møllgård et al., 2023; Plá et al., 2023). However, these papers did not explore this aspect in exquisite detail. A detailed study of the immunophenotypic characteristics of perivascular sheath around cerebral arteries will be necessary to draw a valid inference regarding their derivation from SLYM.

AUTHOR CONTRIBUTIONS

Ashutosh Kumar: Conceptualization; investigation; methodology; writing – original draft; writing – review and editing; project administration; formal analysis; data curation; supervision; validation. Rajesh Kumar: Investigation; data curation; writing – review and editing. Ravi K. Narayan: Investigation; data curation; writing – review and editing. Banshi Nath: Writing – review and editing; visualization. Ashok K. Datusalia: Writing – review and editing. Ashok K. Rastogi: Investigation; writing – review and editing. Rakesh K. Jha: Investigation; data curation. Pankaj Kumar: Writing – review and editing. Vikas Pareek: Writing – review and editing. Pranav Prasoon: Writing – review and editing. Muneeb A. Faiq: Writing – review and editing. Prabhat Agrawal: Writing – review and editing. Surya Nandan Prasad: Writing – review and editing. Chiman Kumari: Writing – review and editing; supervision. Adil Asghar: Supervision; writing – review and editing.