Lymphatic Brain Drain Withers in Aging, Worsens Disease

In recent years, scientists have characterized a hidden set of lymphatic vessels in the meninges, the membranes that line the skull and envelope the brain (Oct 2017 news). These meningeal lymphatic vessels carry waste from the cerebrospinal and interstitial fluids to the deep cervical lymph nodes for disposal. Might these vessels weaken with age and play a role in disease? Yes, suggest scientists led by Jonathan Kipnis, University of Virginia, Charlottesville, in the July 25 Nature. They manipulated drainage through these vessels in young, aging, and transgenic mouse models and found cerebrospinal fluid flow slowed, animals had poorer memories, and they accumulated more Aβ. The results suggest the lymph is an important route of waste clearance from the brain, and contributes to both aging and disease if damaged.

  • Lymphatic vessels in the meninges clear waste from the brain.
  • These vessels drain less effectively in aged mice.
  • Interfering with their function exacerbates Aβ deposition.

“Collectively, these experiments suggest that drainage of brain ISF [interstitial fluid] and CSF [cerebrospinal fluid] by the meningeal lymphatics is necessary for proper cognitive function,” wrote Berislav Zlokovic and Melanie Sweeney, University of Southern California, Los Angeles, in an accompanying News and Views (see also comment below). “The finding has implications for normal aging and disorders such as Alzheimer’s disease.”

First author Sandro Da Mesquita and colleagues used several techniques to hinder drainage through the meningeal lymphatic vessels. They genetically inhibited lymphatic development in wild-type mice, gave three-month-old WT animals a drug that damaged the lymphatic vessels around the brain, or surgically tied vessels off so they couldn’t drain to the deep cervical lymph nodes. In all cases, the scientists found that perfusion of CSF through the brain slowed, as did drainage of macromolecules from ISF. CSF normally flows through the brain via the glymphatic system that runs alongside blood vessels in the brain, and scientists think the fluid eventually reaches the lymph system (Aug 2012 news). “These lymphatic vessels in the meninges are not physically connected to the brain perivascular routes, yet are able to regulate a process that takes place alongside the brain vasculature,” wrote Da Mesquita to Alzforum. “We show that there is a meningeal lymphatic-glymphatic connection, which seems to play important roles in rodent models of aging and Alzheimer’s disease.”

From Bad to Worse.

Aβ plaques (red) accumulated more readily in transgenic mice lacking functioning lymph vessels around the brain (right) than in those with intact vessels (left). Neurons are labeled cyan. [Courtesy of Da Mesquita et al., 2018. Nature.]

After a few weeks of impaired drainage, young mice had trouble remembering a sound associated with a slight foot shock. They took longer than intact animals to find the submerged platform in the Morris water maze, either when it was in a familiar location or had been moved, and they spent much less time searching the correct location when the platform was removed altogether.

Would aging similarly impair drainage? In 20-month-old animals, vessel diameter and vessel coverage in the meninges paled compared with that in young mice, and the CSF filtered more slowly through the brain tissue and into the lymph nodes. The authors were able to correct some of this by giving vascular endothelial growth factor C (VEGF-C) to the older mice. This increased vessel diameter, restored CSF flow, and improved their ability to find the platform in the water maze.

Would VEGF-C help remove Aβ from the brain? Da Mesquita and colleagues treated six- to seven-month-old J20 mice with the growth factor to no avail. VEGF-C had no effect on Aβ levels in the CSF or the amyloid burden in the hippocampus, and was not able to calm hyperactivity in these animals. In fact, by following a CSF tracer in the J20s and in three- to four-month-old 5xFAD mice, the researchers concluded that the morphology and expanse of the lymphatic vessels in these animals was no different from that in controls.

What about in older animals? To mimic aging of the vasculature, Da Mesquita and colleagues wiped out meningeal vessels in two-month-old 5xFAD mice and in six- to seven-month-old J20s. During the next six to 12 weeks, both strains accumulated more plaques in the hippocampus than did controls with intact lymph vessels (see image above). In addition, lymph ablation caused Aβ to build up in the meninges of the mice. When the authors examined postmortem brain tissue from nine AD patients, they found Aβ deposited in their cortical meninges, as well.

“As the authors speculate, it will be extremely interesting to see whether aged animals of less aggressive AD models show deposition of Aβ in the lymphatic vessels or meninges, as postmortem human AD brains in this study,” wrote Tarja Malm and Heikki Tanila from the A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio (see full comment below).

Together, the results hint at another way for waste to find its way out of the brain. “In sporadic AD, researchers think that the main problem is getting rid of amyloid,” explained Costantino Iadecola, Weill Cornell Medical College, New York, who was not involved in the study. “Inasmuch as this pathway is used in clearing the amyloid, coupled with the aging effect on the lymphatics that they’ve shown, one can presume there may be a link between dysfunction of this system and the accumulation of Aβ in the brain.”—Gwyneth Dickey Zakaib

Comments

  1. Previous work has shown that the cerebral blood vessel system and the blood-brain barrier play a major role in regulating the composition of brain interstitial fluid and the distribution of molecules within the CNS, which is required for proper neuronal and synaptic functioning. Da Mesquita et al. importantly show that the meningeal lymphatic system takes a share in clearance of molecules from brain ISF and CSF. Their data indicate that lymphatic vessels interact with the blood vessels to regulate brain proteostasis, which is important for maintaining cognitive functions and could play a role in the pathogenesis of Alzheimer’s disease. It remains to be determined whether treatments directed at meningeal lymphatics can also improve the impaired function of blood vessels with age, and whether enhancing clearance at the BBB can improve lymphatic drainage. Whether the meningeal lymphatic system can also influence immune responses to modulate AD pathology remains to be seen.

    This study was not primarily concerned with blood vessels and the BBB, as the authors selectively targeted and damaged lymphatic vessels, leaving blood vessels functional with an intact BBB, as their data show. Therefore in their experiments, when they injected tracers in brain ISF, transport of tracers occurred along blood vessels to reach CSF.

    And vice versa, when they injected high concentrations of tracers into CSF, tracers were diffusing back into the brain along blood vessels. But the changes in blood vessels in terms of expression of different receptors, transporters, and other proteins have not been studied, leaving this an important topic for future studies, as we have emphasized in News and Views.

    View all comments by Berislav Zlokovic

  2. The extensive and elegant study by Kipnis et al. sheds light on the impact of aging and brain amyloidosis in ISF-CSF circulation. This work extends earlier findings of peripheral lymphatic vessel dysfunction in aging to that of dural lymphatic vessels.

    An exciting finding of the study is the impact of aging on the lymphatic cell phenotype and functions in mice, and that the morphological changes were accompanied by changes in the gene expression level. Moreover, the authors show that viral transduction into cisterna magna, and local meningeal application of VEGF-C into aged mice, increased the diameter of the meningeal lymphatic vessels and partially corrected the transport of the dye into the deep cervical lymph nodes. These are encouraging findings demonstrating that age-related degeneration in brain lymphatic vessels can be potentially pharmacologically targeted.

    The authors also tested the effect of VEGF-C treatment on two AD mouse models, APPswe(Ind)J20 (six to seven months of age) and 5xFAD (three to four months of age). The treatment failed to have any observable effects on meningeal lymphatic vessels, CSF Aβ levels, amyloid deposition in the hippocampus, or cognitive deficits. On the other hand, neither one of the employed AD models showed significant deficits in the lymphatics compared to wild-types. These findings further emphasize the important contribution of aging on Alzheimer phenotype also in mouse models.

    As the authors speculate, it will be extremely interesting to see whether aged animals of less aggressive AD models show deposition of Aβ in the lymphatic vessels or meninges, as postmortem human AD brains in this study.

    In addition, it would be interesting to see the impact of VEGF-C in aged mouse models of amyloidosis. Interestingly, no amyloid deposition was detected in the meninges of the 5xFAD mice, whereas mice with ablated meningeal lymphatics with photoconversion showed amyloid deposits. It remains to be investigated whether photoconversion induces a local inflammatory reaction, which in turn promotes amyloid deposition.

    Finally, it will be extremely interesting to see the interplay between the various CSF-ISF drainage pathways, dural lymphatics, glymphatics, nasal lymphatics, intramural periarterial drainage pathway, as well as the drainage via perineural routes, and whether these are impaired upon aging.

    View all comments by Tarja Malm View all comments by Heikki Tanila
    • User Profile Image Roxana Carare
      University of Southampton School of Medicine
    • Posted:

    I am impressed with the variety of tools used in this study, which highlight that the dural lymphatics are affected by age and related functionally to drainage of both CSF and ISF. In examining the drainage of interstitial fluid, the authors focused on quantifying the tracers that remain in the brain one hour after injection. Clearance of the tracers was reduced at this timepoint in the absence of dural lymphatic function.

    This opens up an avenue for future questions that investigate the exact anatomical pathways between the capillary/arterial basement membranes, which are the sites of intramural periarterial drainage (IPAD), and dural lymphatics. Multiple mechanisms of clearance are working within one hour, including LRP1, IPAD, perivascular macrophages, microglia, so it will be interesting to define how exactly each mechanism is affected by the lack of dural lymphatic drainage.

    View all comments by Roxana Carare

  4. This study is important for a couple of reasons. First, somewhat unexpectedly, the authors observed that impairment of lymphatic drainage impaired glymphatic exchange of CSF and interstitial solutes. This suggests that the processes governing exchange between the CSF and interstitial compartments, and between the CSF and lymphatic drainage, are in some way functionally linked. It will be important to figure out what the basis for this functional connection is. It is possible that disruption of lymphatic function promotes reactive gliosis, which could account for the changes in glymphatic function, although no differences in blood-brain barrier permeability were noted. This needs to be evaluated. It could also be that disruption of lymphatic drainage pathways alters cisternal CSF flow pathways or pressure gradients that support CSF-ISF exchange.

    It is also important that the authors observe that impaired lymphatic drainage results in neurocognitive deficits while amyloid deposition within parenchyma is exacerbated in mouse models of amyloidosis. This suggests that meningeal lymphatic function can modulate interstitial amyloid dynamics.

    However, when meningeal lymphatic function was increased in two different rodent models of amyloidosis, no effect on amyloid deposition was observed. This raises the question of whether age- or disease-related impairment of lymphatic function contribute to the development of amyloid plaques. While it is possible that these fast-developing rodent models may not be the appropriate model to evaluate this question, it will be important to evaluate this for its relevance to human Alzheimer’s disease to be fully understood.

    View all comments by Jeffrey Iliff
    • User Profile Image Daniel Reich
      National Institute of Neurological Disorders and Stroke
    • Posted:

    It’s exciting that several of the methods that were used in this paper to show the effects of diminishing or enhancing the function of meningeal lymphatic vessels can be translated directly to humans. These include measuring the diameter of the vessels themselves, which our group demonstrated is possible through a variety of MRI techniques, as well as tracking the flow of MRI contrast dye injected into the cerebrospinal fluid. These methods might therefore provide proof-of-concept outcome measures for early phase testing of drugs that modulate the function of dural lymphatics, accelerating the pathway toward discovery of new therapies for dementia. 

    View all comments by Daniel Reich

  6. We share the authors' enthusiasm regarding the possible role of CNS lymphatic clearance in Alzheimer’s disease. With novel pharmaceutical approaches or lifestyle changes, one might even be able to stem the decline in lymphatic function that occurs with aging and, perhaps, during the disease itself. This may improve the homeostasis of the brain tissue to help prevent aggregation of amyloid plaques, even if lymphatic vessels may not themselves be responsible for the clearance of the Aβ.

    That said, we must object to the authors’ concept of the recently rediscovered meningeal lymphatic vessels draining a significant proportion of cerebrospinal fluid (CSF). In our recently published paper, we clearly demonstrated using fluorescence imaging that the major routes of CSF outflow in mice were through the cribriform plate and along cranial nerves to reach lymphatic vessels outside the skull, not via the meningeal lymphatic vessels (Ma et al., 2017). This finding was consistent with dozens of other reports in many species going back 150 years (Schwalbe et al., 1869; Bradbury and Cserr, 1985; Koh et al, 2005). The authors have appeared to ignore any evidence that argues against the importance of the meningeal lymphatic vessels, to the point of not citing relevant literature in their present report.

    It is evident that more work is needed to clarify the exact roles of the lymphatic system in CSF clearance and in the pathology of Alzheimer’s disease.

    References:

    Ma Q, Ineichen BV, Detmar M, Proulx ST. Outflow of cerebrospinal fluid is predominantly through lymphatic vessels and is reduced in aged mice. Nat Commun. 2017 Nov 10;8(1):1434. PubMed.

    Schwalbe G. Die Arachnoidalraum ein Lymphraum und sein Zusammenhang mit den Perichorioidalraum. [The arachnoidal space as a lymphatic space with connection to the perichoroidal compartment.]. . Zbl. Med. Wiss. 1869; 7, 465–467.

    Bradbury MW, Cserr HF, Johnston MG. Experimental Biology of the Lymphatic Circulation. Elsevier, Amsterdam, 1985

    Koh L, Zakharov A, Johnston M. 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 Steven T. Proulx

  7. The role of the lymphatic system in the central nervous system and in Alzheimer’s disease, a succinct historical perspective.

    The role of the brain lymphatic system in neurologic disease in general and in Alzheimer’s disease (AD) in particular, has largely been ignored. Da Mesquita et al. showed that by interfering with the integrity of the lymphatic system, in a transgenic model of AD amyloidosis, the mice bearing the abnormal lymphatic system accumulated more amyloid than the control mice with intact lymphatic vessels. I would like to congratulate the investigators for the findings and for bringing attention to a generally disregarded topic. However, in recognition of original work conducted by many other investigators, I would like to highlight a few overlooked landmark studies, germane to this area of research.

    As mentioned in another commentary, the existence of lymphatic vessels associated with the dura mater of the brain has originally been recorded in the 1700s (Vasorum lymphaticorum corporis humani historia et ichnographia, accessed August 5, 2018) and further studied by German authors in the 1800s (Schwalbe G. Zbl. Med. Wiss. 1869; 7, 465–467; cited in Lohrberg and Wilting, 2016). In “Gray’s Anatomy,” as well as other classic anatomy textbooks, we can find the description of meningeal lymphatics vessels. Research in the peer-reviewed literature, however, reported meningeal lymphatic channels in the skull base of dogs 50 years ago (Foldi et al., 1966) and in several other sites (such as the spinal meninges) (Reshetilov, 1980). Thirty years ago, lymphatic vessels around the wall of the sagittal sinus were recognized in regions of confluence of sinuses in juxtaposition to the subdural vasculature of the dural tissues (Andres et al., 1987). Two decades ago, Miura et al. studied the structural organization of the epidural lymphatic channels and lymphatic drainage of CSF from the meninges of Macaca fuscata and concluded that meningeal lymphatic vessels function as an absorptive pathway for the CSF.

    More recently, Ma et al. reported that clearance of CSF in mice occurs predominantly through lymphatic vessels, and is reduced with aging. In this important contribution, the authors hypothesized that the lymphatic system may represent a therapeutic target for age-associated neurological conditions like AD. Work by Iliff et al. (2012; 2013), Weller et al. (2009; 2009), and Carare et al. demonstrated that CSF enters the parenchyma flowing along intracerebral paravascular spaces and that this clearance pathway may also contribute to cerebral lymphatic drainage (Iliff et al., 2012; 2013). In 2014, Pappolla et al. demonstrated, for the first time, that Aβ derived from the brain is indeed cleared into the peripheral lymph nodes in a transgenic model of AD amyloidosis. Using novel cellular markers, not available to the talented microscopists of bygone years, investigators confirmed the presence of functional lymphatic vessels lining the dural sinuses in 2015 (Louveau et al., 2015). 

    Pertaining to amyloid clearance, one should keep in mind that in addition to meningeal lymphatics in the dura, there are other pathways for CSF lymphatic clearance (Ma et al., 2017; Louveau et al., 2015; Hladky and Barrand, 2014). Currently, several studies support the concept that multiple mechanisms of clearance are simultaneously operational (Ma et al., 2017; Louveau et al., 2015; Hladky and Barrand, 2014). The efficiency of each route of lymphatic clearance (such as pathways along cranial nerves, spinal nerves, the cribriform plate, etc.) varies according to the molecular weight and size of the substances to be cleared and are also different between species (Hladky and Barrand, 2014). 

    More efforts are necessary to better understand the cross-talk between the cerebral and the peripheral lymphatic systems with the goal of identifying novel therapies for AD. In this regard, we recently showed that lymphatic clearance of Aβ can be enhanced by administering melatonin to AD transgenic mice (Pappolla et al., 2018). In another interesting study supporting the role of lymphatics in AD, Scholtzova et al. showed that stimulation of the innate immune system can reduce both Aβ and tau pathology (Scholtzova, et al., 2017; 2014). 

    Future questions should examine which cellular transport mechanisms are in play, particularly during certain pathological conditions (such as reactivation of dormant infection agents in the CNS and in other neuro-inflammatory conditions). For example, the murine PirB (paired immunoglobulin-like receptor B) and its human ortholog LilrB2 (leukocyte immunoglobulin-like receptor B2), present in human brain, are receptors for Aβ peptides (Kim et al., 2013). Interestingly, LilrB2 are members of the immunoglobulin superfamily and are found in dendritic cells. These cells are known to “travel” between brain and peripheral lymph nodes, particularly during neuroinflammation (Hatterer et al., 2008). 

    In sporadic AD, several genes that associate with increased risk have been discovered in the last few years. Because the products of many of these genes are involved in immune functions, the study of the lymphatic system highlights a new perspective in AD pathogenesis that warrants a fresh reassessment of our conventional thinking.

    References:

    Da Mesquita S, Louveau A, Vaccari A, Smirnov I, Cornelison RC, Kingsmore KM, Contarino C, Onengut-Gumuscu S, Farber E, Raper D, Viar KE, Powell RD, Baker W, Dabhi N, Bai R, Cao R, Hu S, Rich SS, Munson JM, Lopes MB, Overall CC, Acton ST, Kipnis J. Functional aspects of meningeal lymphatics in ageing and Alzheimer's disease. Nature. 2018 Aug;560(7717):185-191. Epub 2018 Jul 25 PubMed.

    Lohrberg M, Wilting J. The lymphatic vascular system of the mouse head. Cell Tissue Res. 2016 Dec;366(3):667-677. Epub 2016 Sep 6 PubMed.

    Földi M, Gellért A, Kozma M, Poberai M, Zoltán OT, Csanda E. New contributions to the anatomical connections of the brain and the lymphatic system. Acta Anat (Basel). 1966;64(4):498-505. PubMed.

    Reshetilov VI. [Presence of lymphatic vessels in the human spinal dura mater]. Zh Vopr Neirokhir Im N N Burdenko. 1980 Jul-Aug;(4):43-6. PubMed.

    Andres KH, von Düring M, Muszynski K, Schmidt RF. Nerve fibres and their terminals of the dura mater encephali of the rat. Anat Embryol (Berl). 1987;175(3):289-301. PubMed.

    Miura M, Kato S, von Lüdinghausen M. Lymphatic drainage of the cerebrospinal fluid from monkey spinal meninges with special reference to the distribution of the epidural lymphatics. Arch Histol Cytol. 1998 Aug;61(3):277-86. PubMed.

    Ma Q, Ineichen BV, Detmar M, Proulx ST. Outflow of cerebrospinal fluid is predominantly through lymphatic vessels and is reduced in aged mice. Nat Commun. 2017 Nov 10;8(1):1434. PubMed.

    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.

    Iliff JJ, Lee H, Yu M, Feng T, Logan J, Nedergaard M, Benveniste H. Brain-wide pathway for waste clearance captured by contrast-enhanced MRI. J Clin Invest. 2013 Mar 1;123(3):1299-309. PubMed.

    Weller RO, Djuanda E, Yow HY, Carare RO. Lymphatic drainage of the brain and the pathophysiology of neurological disease. Acta Neuropathol. 2009 Jan;117(1):1-14. PubMed.

    Weller RO, Preston SD, Subash M, Carare RO. Cerebral amyloid angiopathy in the aetiology and immunotherapy of Alzheimer disease. Alzheimers Res Ther. 2009;1(2):6. PubMed.

    Carare RO, Bernardes-Silva M, Newman TA, Page AM, Nicoll JA, Perry VH, Weller RO. Solutes, but not cells, drain from the brain parenchyma along basement membranes of capillaries and arteries: significance for cerebral amyloid angiopathy and neuroimmunology. Neuropathol Appl Neurobiol. 2008 Apr;34(2):131-44. Epub 2008 Jan 16 PubMed.

    Pappolla M, Sambamurti K, Vidal R, Pacheco-Quinto J, Poeggeler B, Matsubara E. Evidence for lymphatic Aβ clearance in Alzheimer's transgenic mice. Neurobiol Dis. 2014 Nov;71:215-9. Epub 2014 Aug 4 PubMed.

    Louveau A, Smirnov I, Keyes TJ, Eccles JD, Rouhani SJ, Peske JD, Derecki NC, Castle D, Mandell JW, Lee KS, Harris TH, Kipnis J. Structural and functional features of central nervous system lymphatic vessels. Nature. 2015 Jul 16;523(7560):337-41. Epub 2015 Jun 1 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.

    Pappolla MA, Matsubara E, Vidal R, Pacheco-Quinto J, Poeggeler B, Zagorski M, Sambamurti K. Melatonin Treatment Enhances Aβ Lymphatic Clearance in a Transgenic Mouse Model of Amyloidosis. Curr Alzheimer Res. 2018;15(7):637-642. PubMed.

    Scholtzova H, Chianchiano P, Pan J, Sun Y, Goñi F, Mehta PD, Wisniewski T. Amyloid β and Tau Alzheimer's disease related pathology is reduced by Toll-like receptor 9 stimulation. Acta Neuropathol Commun. 2014 Sep 2;2:101. PubMed.

    Kim T, Vidal GS, Djurisic M, William CM, Birnbaum ME, Garcia KC, Hyman BT, Shatz CJ. Human LilrB2 is a β-amyloid receptor and its murine homolog PirB regulates synaptic plasticity in an Alzheimer's model. Science. 2013 Sep 20;341(6152):1399-404. PubMed.

    View all comments by Mike Miguel Pappolla

  8. Great historical perspective!

    View all comments by Kumar Sambamurti

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References

News Citations

  1. Lymphatic Vessels Found in Human Brain
  2. Brain Drain—“Glymphatic” Pathway Clears Aβ, Requires Water Channel

Research Models Citations

  1. J20 (PDGF-APPSw,Ind)
  2. 5xFAD (B6SJL)

Further Reading

Primary Papers

  1. Da Mesquita S, Louveau A, Vaccari A, Smirnov I, Cornelison RC, Kingsmore KM, Contarino C, Onengut-Gumuscu S, Farber E, Raper D, Viar KE, Powell RD, Baker W, Dabhi N, Bai R, Cao R, Hu S, Rich SS, Munson JM, Lopes MB, Overall CC, Acton ST, Kipnis J. Functional aspects of meningeal lymphatics in ageing and Alzheimer's disease. Nature. 2018 Aug;560(7717):185-191. Epub 2018 Jul 25 PubMed.

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