. Meningeal lymphatic vessels at the skull base drain cerebrospinal fluid. Nature. 2019 Jul 24; PubMed.

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  1. This is a beautiful paper, convincingly demonstrating that meningeal lymphatic vessels are indeed draining the CSF, whereas the perineural lymphatics are not. These results completely recapitulate our earlier findings (Louveau et al., 2015; Louveau et al., 2018; Da Mesquita et al., 2018) regarding the role of meningeal lymphatics in draining CSF solutes, brain ISF, and immune cells.

    The authors elegantly demonstrate that lymphatic hot spots, sprout-like structures reminiscent of initial lymphatic vasculature, which we have previously described (Louveau et al., 2018), are indeed the access points for CSF drainage. The authors also present evidence that basal lymphatics are more active than dorsal lymphatics in CSF drainage and that this system dysfunctions with aging (as we showed in Da Mesquita et al., 2018).

    Overall, the results reproduce our and others’ (Aspelund et al., 2015) findings on the role of meningeal lymphatics, put the role of perineural lymphatics in CSF drainage to rest, expand on the meningeal lymphatic network through basal lymphatics (which are a continuation of dorsal meningeal lymphatics), recapitulate the importance of lymphatic hot spots, and demonstrate their dysfunction in aging.

    Whilst the major route for CSF solutes may be through the basal lymphatics, the dural sinus is heavily populated by immune cells and their trafficking is happening throughout the entire meningeal lymphatic network, including dorsal sites.

    Targeting meningeal lymphatics alters Alzheimer’s disease progression (Da Mesquita et al., 2018). However, we do not know whether this is through a direct effect on CSF/β-amyloid drainage (happening through meningeal lymphatic network, presumably mainly through its basal part), via regulation of meningeal immunity (presumably mainly by dorsal lymphatics), or indeed both. These questions will need to be addressed to better understand how the meningeal lymphatic system, in its entirety, modulates CNS function and dysfunction. Step by step, the field is recognizing the full potential of the meningeal lymphatic vasculature as a novel and exciting therapeutic target for age-related diseases of the CNS.

    References:

    . Structural and functional features of central nervous system lymphatic vessels. Nature. 2015 Jul 16;523(7560):337-41. Epub 2015 Jun 1 PubMed.

    . CNS lymphatic drainage and neuroinflammation are regulated by meningeal lymphatic vasculature. Nat Neurosci. 2018 Oct;21(10):1380-1391. Epub 2018 Sep 17 PubMed.

    . Functional aspects of meningeal lymphatics in ageing and Alzheimer's disease. Nature. 2018 Aug;560(7717):185-191. Epub 2018 Jul 25 PubMed.

    . A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules. J Exp Med. 2015 Jun 29;212(7):991-9. Epub 2015 Jun 15 PubMed.

    View all comments by Sandro Da Mesquita
  2. The paper describes a lymphatic pathway for CSF clearance located at the base of the skull. It is remarkable that these vessels have the classical morphology and molecular signature of lymphatic vessels elsewhere in the body. The findings, in concert with the previously described dorsal lymphatic pathway along the sagittal sinus, further debunk the myth that the brain does not have functional lymphatic vessels.

    Several CSF clearance pathways have been described in rodents, including one along the olfactory nerve rootlets and cribriform plate, perineural pathways along the cranial nerves, and the dorsal and basal pathways discussed in this paper. However, several issues remain to be addressed: Do these pathways exist in humans? What is their relative contribution to CSF clearance, i.e. is one pathway more important than another? Is the dorsal pathway, which has been confirmed in humans, draining only the meninges or also the CSF? 

    CSF production and clearance are reduced in aging, which has been implicated in the accumulation of toxic proteins in the brain (Aβ, tau, synuclein etc.). Which of these pathways is the most relevant to the disturbance in proteostasis caused by the CSF clearance deficit in aging remains to be established. Perhaps advanced imaging approaches could be used to probe this question in humans, and studies along these lines would provide much-needed clarity in this burgeoning field of research.​

    View all comments by Costantino Iadecola
  3. This is a very elegant anatomical and physiological study. It demonstrates the balance of the active meningeal lymphatics draining the CSF. It is particularly relevant, as rodent experimental models are the current model of choice for studies of drainage of interstitial and cerebrospinal fluids (Weller et al., 2018).

    There are still aspects of the anatomy of the subarachnoid space and movement of CSF that are under investigation and are relevant to early diagnosis in Alzheimer’s disease (de Leon et al., 2017). It remains to be seen to what extent the basal meningeal lymphatics have a role in drainage of interstitial fluid of the brain along the intramural periarterial drainage (IPAD) pathways, especially as IPAD is impaired with vascular dysfunction that accompanies ageing (Sweeney et al., 2019; Hawkes et al., 2013).

    References:

    . The meninges as barriers and facilitators for the movement of fluid, cells and pathogens related to the rodent and human CNS. Acta Neuropathol. 2018 Mar;135(3):363-385. Epub 2018 Jan 24 PubMed.

    . Cerebrospinal Fluid Clearance in Alzheimer Disease Measured with Dynamic PET. J Nucl Med. 2017 Sep;58(9):1471-1476. Epub 2017 Mar 16 PubMed.

    . Vascular dysfunction-The disregarded partner of Alzheimer's disease. Alzheimers Dement. 2019 Jan;15(1):158-167. PubMed.

    . Regional differences in the morphological and functional effects of aging on cerebral basement membranes and perivascular drainage of amyloid-β from the mouse brain. Aging Cell. 2013 Apr;12(2):224-36. PubMed.

    View all comments by Roy Weller
  4. This is an extremely interesting study that addresses a controversial and fundamental topic at the moment—how the cerebrospinal fluid (CSF) drains from the spaces around the brain and spinal cord. The textbook knowledge is that CSF drains directly into veins, however, this is primarily based on studies carried out over a century ago and has never been directly shown in an in vivo physiological study. In the last few years, work performed by a few groups including our own (Ma et al., 2017; Nov 2017 news) has highlighted the forgotten role that the lymphatics might be playing in this process. We showed that in mice, CSF clears predominantly through the lymphatic system rather than directly into blood. However, the anatomical route(s) that the fluid takes to reach lymphatic vessels are still controversial.

    Ahn et al. propose that the meningeal lymphatic vessels located at the base of the skull (in the outermost dura mater layer of the linings of the central nervous system—not the brain itself) are the major route for CSF clearance. This is contradictory to the work performed by Jonathan Kipnis’ group at the University of Virginia, who have proposed that the meningeal lymphatic vessels on the dorsal side (top) of the skull are the most important. In agreement with the current study, our MRI data also support bulk CSF flow mostly in the basal, rather than dorsal, region (Ma et al., 2019). 

    In addition, our own group, backed up by dozens of historical studies (Koh et al., 2005), has shown that CSF outflow can also occur through routes along cranial nerves, especially in the nasal or optical regions, only reaching lymphatic vessels once these nerves have left the skull (Ma et al., 2017). These particular routes were not assessed in the current paper, so the question remains open which routes are the most important.

    References:

    . Outflow of cerebrospinal fluid is predominantly through lymphatic vessels and is reduced in aged mice. Nat Commun. 2017 Nov 10;8(1):1434. PubMed.

    . Rapid lymphatic efflux limits cerebrospinal fluid flow to the brain. Acta Neuropathol. 2019 Jan;137(1):151-165. Epub 2018 Oct 10 PubMed.

    . Integration of the subarachnoid space and lymphatics: is it time to embrace a new concept of cerebrospinal fluid absorption?. Cerebrospinal Fluid Res. 2005 Sep 20;2:6. PubMed.

    View all comments by Miriam Ries
  5. This important study clarifies the role of meningeal lymphatic vessels in clearance of important molecules and immune cells from the brain to the periphery. This paper, and other work, have described how these molecules and cells go from the base of the brain to lymphatic meningeal vessels (Sweeney and Zlokovic, 2018). The lymphatic clearance pathway may be important in the pathogenesis of neurodegenerative diseases and other conditions. As Costantino Iadecola pointed out, other pathways may also be important, such as the olfactory nerve and the cribriform plate.

    We must consider the full course of the passage of material from the brain to the reticuloendothelial system through the meningeal lymphatic vessels. As Ahn et al. propose, lymphatic clearance from the brain goes to the deep cervical lymph nodes. This process will likely be influenced by the oral, nasopharyngeal and laryngeal microbiota. We already know that the gut microbiota influences systemic and cerebral immune processes in profound ways (Lathrop et al., 2011; Rothhammer et al, 2018; Abdel-Haq et al., 2019). It is likely that microbiota in the head will similarly influence immune functions in the cervical lymph nodes to influence clearance processes. This may have important implications for stroke, vascular cognitive impairment, Alzheimer's disease, and idiopathic intracranial hypertension.

    These relationships can be addressed in many ways in humans, including MRI and tonsillar biopsies, as well as studies of the relationship of CSF immune markers and oral bacteria.

    References:

    . Microbiome-microglia connections via the gut-brain axis. J Exp Med. 2019 Jan 7;216(1):41-59. Epub 2018 Nov 1 PubMed.

    . Microglial control of astrocytes in response to microbial metabolites. Nature. 2018 May;557(7707):724-728. Epub 2018 May 16 PubMed.

    . A lymphatic waste-disposal system implicated in Alzheimer's disease. Nature. 2018 Aug;560(7717):172-174. PubMed.

    . Peripheral education of the immune system by colonic commensal microbiota. Nature. 2011 Sep 21;478(7368):250-4. PubMed.

    View all comments by Robert Friedland
  6. We would like to correct a comment made by Dr. Proulx and his colleague, which we consider misleading. The comment states:

    “Ahn et al. propose that the meningeal lymphatic vessels located at the base of the skull (in the outermost dura mater layer of the linings of the central nervous system—not the brain itself) are the major route for CSF clearance. This is contradictory to the work performed by Jonathan Kipnis’ group at the University of Virginia, who have proposed that the meningeal lymphatic vessels on the dorsal side (top) of the skull are the most important. In agreement with the current study, our MRI data also support bulk CSF flow mostly in the basal, rather than dorsal, region (Ma et al., 2019).”

    Since our rediscovery of the meningeal lymphatic vasculature in 2015, we have never divided it into “most important’ or “less important” zones. Supplemental Figure 1 of our recent paper (Louveau et al., Nat Neurosci 2018,) clearly demonstrates the role of basal lymphatics in CSF drainage. We have focused on the dorsal part of meningeal lymphatics because of its comparatively easier accessibility.

    The work of Ahn et al., has recapitulated original findings from our lab (Louveau et al., Nature 2015; Louveau et al., Nat Neurosci 2018; Da Mesquita et al., 2018) and from the lab of Kari Alitalo (Aspelund et al, JEM 2015). It nicely showed that the basal part of the system, which is where dorsal vessels converge and drain into, is supposedly better equipped for CSF drainage than its dorsal part.

    Interestingly, the previous work from Proulx’ lab (Ma et. al., Nat Comm 2017) claimed that the meningeal lymphatic vasculature has no role in CSF drainage, and that CSF is drained entirely through perineural routes. The current work by Ahn et al., clearly demonstrates that meningeal lymphatics indeed play a critical role in CSF drainage, as we and others have previously suggested.

    The goal now is to focus our efforts on developing therapeutics to target meningeal lymphatic vessels in various neurological diseases.

    View all comments by Sandro Da Mesquita
  7. We appreciate the new comment from Dr. Kipnis and colleagues and their willingness to engage in discussion on this controversial topic. We also feel the need to clarify some of the statements that have been made by these authors in their comments to this forum and to further put into context the exciting new findings by Ahn et al., 2019.

    Dr. Kipnis and colleagues are correct that they may have never explicitly stated that the dorsal meningeal lymphatic vessels are “most important” for CSF clearance. However, it is difficult to interpret their recent data any other way. In the study by Louveau et al., 2018, after providing evidence supporting the functionality of the vessels, the authors utilized a photoablation technique that targeted the lymphatic vessels under the dorsal skull (as well as an additional small network of lymphatic vessels located in the dura near the cerebellum). As this method involved shining light through the intact skull to ablate the vessels, the procedure presumably left unaffected both the basal meningeal lymphatic vessels and the perineural outflow routes. The authors then proceeded to show dramatic reductions of CSF transport to draining lymph nodes of fluorescently labeled beads and T cells injected into the cisterna magna. They concluded from this data that the (ablated) meningeal lymphatic vessels are the “main route for immune cell and macromolecule drainage from the CSF.” The authors have indeed shown immunostainings of lymphatic vessels in the basal region of the skull in Supplemental Figure 2. However, they did not demonstrate functionality of these vessels and instead argued against this:

    “After i.c.m. injection, some of the fluorescent particles are seen accumulating around the sides of the skull, close to lymphatic vessels (Supplementary Fig. 2j). These vessels, however, seemed to have lymphatic valves, as previously described (Aspelund et al., 2015), suggesting they were collecting lymphatics, which are usually less accessible by macromolecules.”

    The current report by Ahn et al. has elegantly clarified the morphology of the lymphatic vessel network in the basal region, demonstrating the existence of blind-ended vessels and a patterning of the junctions between cells that would be more indicative of initial or precollecting lymphatic vessel network capable of draining fluid and solutes. Ahn et al. have also confirmed findings from our group that have indicated that the dorsal lymphatic network was discontinuous in nature and did not appear to participate in CSF tracer clearance (Ma et al., 2017). Thus, it is not immediately apparent how Dr. Kipnis and colleagues have come to the conclusion that the current report has “completely recapitulated” their earlier findings.

    Regarding the claims made in our previous report, we invite Dr. Kipnis and colleagues to closely reread this paper (Ma et al., 2017). We have never claimed that the meningeal lymphatic vessels (we prefer the term dural lymphatic vessels, as a consensus has been reached that they are localized to the dura mater) (Mastorakos and McGavern, 2019) do not have a role in CSF drainage or that this fluid is drained entirely through perineural routes. In fact, in the discussion of this paper (Ma et al., 2017) we have written:

    “Interestingly, anatomical studies have demonstrated lymphatic vessels in the dura around the optic and facial nerves and at the cribriform plate and jugular foramen, indicating that lymphatic uptake of CSF may occur as the cranial nerves exit the skull (Aspelund et al., 2015; Földi et al., 1966; Furukawa et al., 2008; Killer et al., 1999). Clearly, more research is needed to determine the importance of the dural lymphatic route for CSF outflow in comparison to the more established perineural pathways to reach extracranial lymphatic vessels.”

    The current report by Ahn et al. has provided beautiful images that support data that we have published in collaboration with the group of Prof. Kari Alitalo at the University of Helsinki (Aspelund et al., 2015), in which tracers were detected within the dural lymphatic vessels at the base of the skull. We have also published data supporting an outflow of CSF-injected tracers from both the stylomastoid (where the facial nerve exits the skull) and jugular foramina (where the glossopharyngeal, vagus, and accessory nerves exit the skull), two of the routes identified in the current paper as outflow sites of dural lymphatic vessels from the cranium (Ma et al., 2017). The exact mechanism of fluid and solute uptake into the lymphatic network at these locations is still not clear. Ahn et al. have claimed that the arachnoid barrier layer is not intact in these regions, which could allow access of CSF to the interstitium of the dura and, thus, to the lymphatics of this tissue. This is a highly interesting finding that awaits further confirmation.  

    Finally, we feel the need to address the comments made by Dr. Kipnis and colleagues in their initial comment on July 26. The authors claim that the report by Ahn et al. has shown “meningeal lymphatic vessels are indeed draining the CSF, whereas the perineural lymphatics are not” and has “put the role of perineural lymphatics in CSF drainage to rest.” By “perineural lymphatics,” we presume that the authors are referring to CSF outflow routes along cranial nerves as they exit the skull. If this presumption is correct, then these statements have no basis in fact. The new report did not assess these routes, as we have pointed out in our initial comment. The evidence in the literature for CSF egress in several species through foramina at the cribriform plate (where olfactory nerves exit the skull) and through the optic foramina (where the optic nerve exits) is very strong. While the exact access points along these routes to lymphatic vessels remains a point of contention, tracers injected into the CSF can be routinely demonstrated by imaging techniques to be within collecting lymphatic vessels outside the skull of these regions (e.g., Ma et al., 2017).       

    This area of research is currently very active, with several independent groups pursuing studies that aim to elucidate the mechanisms of CSF flow and clearance from the central nervous system. Let’s all avoid making premature conclusions before all the evidence can be weighed. We can take strides to engage in an open-minded and constructive discourse between labs. This will facilitate progress toward our shared goal of translating these exciting new discoveries into therapeutic targets for the treatment of neurodegenerative disorders and other CNS diseases. 

    References:

    . A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules. J Exp Med. 2015 Jun 29;212(7):991-9. Epub 2015 Jun 15 PubMed.

    . New contributions to the anatomical connections of the brain and the lymphatic system. Acta Anat (Basel). 1966;64(4):498-505. PubMed.

    . Topographic study on nerve-associated lymphatic vessels in the murine craniofacial region by immunohistochemistry and electron microscopy. Biomed Res. 2008 Dec;29(6):289-96. PubMed.

    . Production and circulation of cerebrospinal fluid with respect to the subarachnoid space of the optic nerve. J Glaucoma. 2013 Jun-Jul;22 Suppl 5:S8-10. PubMed.

    . CNS lymphatic drainage and neuroinflammation are regulated by meningeal lymphatic vasculature. Nat Neurosci. 2018 Oct;21(10):1380-1391. Epub 2018 Sep 17 PubMed.

    . Outflow of cerebrospinal fluid is predominantly through lymphatic vessels and is reduced in aged mice. Nat Commun. 2017 Nov 10;8(1):1434. PubMed.

    . The anatomy and immunology of vasculature in the central nervous system. Sci Immunol. 2019 Jul 12;4(37) PubMed.

    View all comments by Steven Proulx

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