In Mice, CSF Caught Draining Via Lymphatic Vessels, Not Veins

The choroid plexus produces about a half-liter of cerebrospinal fluid daily, meaning this much must be drained from the cranial cavity into the bloodstream. A new study led by Steven Proulx, Swiss Federal Institute of Technology in Zurich, suggests that, in mice at least, this happens primarily through the lymphatic system. Using near-infrared fluorescent markers, the researchers traced the fluid’s exit routes from lymph vessels running alongside cranial nerves, to lymph nodes, and finally into peripheral blood. The study appears in the November 10 Nature Communications.

  • Mouse study claims CSF clears through lymphatic vessels, not veins.
  • CSF tracers reach lymph nodes quickly via vessels surrounding cranial nerves exiting the skull.
  • CSF outflow slows in old mice.

"The elegant techniques are a step forward in resolving some of the problems associated with the dynamics of CSF drainage,” wrote Roxana Carare and Roy Weller, University of Southampton, U.K. (full comment below).

Scientists recognize two main paths for CSF exiting the head (Raper et al., 2016). CSF can seep out through swellings of the arachnoid mater, the middle meningeal membrane that surrounds the brain and spine, flowing into channels connected to veins. Alternatively, it can leave through lymphatic vessels apposed to nerve sheaths at their sites of exit from the skull. New reports of an active lymphatic system in the outermost meninges, the dura mater, have prompted the suggestion that these vessels may clear CSF as well (Aspelund et al., 2015; Louveau et al., 2015; Absinta et al., 2017). However, the relative contributions of these various paths and their exact routes have remained uncertain. 

Exit Route.

Cerebrospinal fluid drains into lymphatic vessels (green lines) to reach lymph nodes. By 30 minutes after infusion into a lateral ventricle, CSF tracers have accumulated in the nodes of two- (left), but not 18-month-old (right) mice. [Courtesy of Ma et al., Nature Communications.]

To get a better view of CSF drainage, first author Qiaoli Ma labeled both the CSF and the lymphatic vessels of mice. She infused a dye that fluoresces in the near-infrared coupled to a 40 kDa poly(ethylene) glycol molecule, P40D680, into the lateral ventricles of Prox1-GFP mice, which express GFP in the lymphatic vasculature (Proulx et al., 2013; Choi et al., 2011). The near-infrared dye allowed the researchers to see far into tissues with low background noise. 

Surprisingly, the authors found the tracer in deep cervical lymph nodes at the earliest time they checked, 10 minutes after infusion. Looking for tracer along previously suggested exit paths, they spotted it at sites where cranial nerves, especially the olfactory and optic nerves, leave the skull. Interestingly, the recently described dural lymph vessels (Oct 17 news) lacked detectable tracer. Proulx suspects this may be because tight junctions between arachnoid mater cells isolate the CSF from the dura mater.

To follow P40D680 beyond the lymphatic system, the authors looked for near-infrared signal in veins. While lymph vessels lit up on average 11 minutes after intraventricular infusion, and lymph nodes on average 16 minutes after, the saphenous and posterior facial veins took approximately 25 minutes to light up. This indicates CSF flows first into the lymphatic system and later enters the bloodstream.

Previous studies in sheep and rabbits indicated that up to 50 percent of CSF might be cleared through lymphatic vessels (Bradbury and Cole, 1980; Boulton et al., 1998). “It’s much higher than 50 percent in our system,” Proulx told Alzforum. “We saw no early signal in blood.” Even small tracers—Evans blue, IRDye680CW, and a 3 kDa dextran coupled to AlexaFluor680—failed to transit rapidly into blood, following lymphatics-first routes similar to those of P40D680.

Wondering if the flow might change with age, the researchers compared P40D680’s travels in two- versus 18-month-old mice. Whereas the tracer reached the blood in approximately 24 minutes in the former, it took about 38 minutes in the latter.

Will the results apply to people? Weller and Carare are unsure. They noted that in mice, the arachnoid projections through which CSF is thought to clear into the bloodstream are fewer, smaller, and simpler than their counterparts in humans (Kida et al., 1993; Upton and Weller, 1985). 

Proulx plans to examine CSF outflow in mouse models of Alzheimer’s disease and test stimulators of lymphatic function. A recent PET study in humans showed that CSF clearance is abnormal in AD (de Leon et al., 2017). 

Carare and Weller suggested using the tracers to study how brain interstitial fluid, rather than CSF, drains along the basement membranes of cerebral capillaries and arteries, a generally accepted Aβ clearance path. They welcomed the possibility of applying the technology to humans. Proulx thinks this is feasible. “The tracers are inert … and are cleared readily through the kidney. To analyze a tracer in the bloodstream, after an injection into CSF, one need only monitor the fluorescent [near-infrared] signal of a blood vessel on the surface of the skin,” he noted. “What is missing at this point is the clinical approval to use these tracers in humans.”—Marina Chicurel

Comments

  1. In this paper, Ma et al. report that the outflow of cerebrospinal fluid in mice is predominantly through lymphatic vessels, and is reduced with aging. They hypothesize that the lymphatic system may represent a target for age-associated neurological conditions.

    The presence of lymphatics in human dura mater was first described by Mascagni in 1787 in his famous book “Vasorum lymphaticorum corporis humani historia et ichonographia.” Since then, his observations, in human subjects, were confirmed by a number of authors who studied connections between subarachnoid space and cervical lymph nodes (Schwalbe 1869; Key and Retzius, 1875; Zwillinger, 1912; Weed, 1914). In 1953, Lecco reported that, during a histological examination of the dura mater of 30 humans, lymphatic structures were found in only four of them. He concluded that lymphatics probably develop in the dura mater of humans for some unknown functional reason and independent of age. In 1964, Földi et al. reported that in human meninges, vessels with the characteristic morphology of lymphatics appeared only in the dura, particularly in regions close to the jugular foramen. In 2015, Aspelund et al. reported that in mice, dural lymphatics were more abundant in the basal than in the apical region.

    Currently, we lack: 

    • Comparative and phylogenetic research on the presence and regional distribution of lymphatics of dura mater, with particular attention to the role that the bipodalic evolution could have determined (e.g., what, if any, role could gravity have played in the localization of lymphatics in dura mater?).
    • Studies in large human populations of the presence and distribution of lymphatics in human dura, with particular attention to a statistical relationship with age, sex, work, lifestyle, morbidities, and other biological parameters that could have an influence.

    We want to stress that the present report of Ma et al. did not confute the dogma that lymphatics are absent from in the central nervous system. In fact, dura mater is not, from both an anatomical and embryological point of view, a component of the neuraxis but one of its covers, along with the other meningeal layers.

    Moreover, the blood–brain barrier prevents the formation of transudate (interstitial fluid) in the nervous tissue, thus preventing any change in the volume of these structures that would interfere with the functioning of neurons. Since transudate does not form, it is not necessary to have any lymphatic vessel here, as we already reported (Bucchieri et al., 2015). 

    References:

    Schwalbe G. Der Arachnoidealraum, ein Lymphraum und sein Zusammenhang mit dem Perichorioidealraum. Z med Wiss 7, 465- (1869).

    Key A, Retzius G. Studien in der Anatomie des Nervensystems und des Bindegewebes. Samson und Wallin, 1875

    Zwillinger H. Die Lymphbahnen des oberen Nasalschnittes und deren Beziehungen zu den perimeningealen Lymphraumen. Arch Laryngol und Rhinol 26, 66-78 (1912).

    Weed LH. Studies on Cerebro-Spinal Fluid. No. IV : The dual Source of Cerebro-Spinal Fluid. J Med Res. 1914 Sep;31(1):93-118.11. PubMed.

    LECCO V. [Probable modification of the lymphatic fissures of the walls of the venous sinuses of the dura mater]. Arch Ital Otol Rinol Laringol. 1953 May-Jun;64(3):287-96. 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.

    Aspelund A, Antila S, Proulx ST, Karlsen TV, Karaman S, Detmar M, Wiig H, Alitalo K. 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.

    Bucchieri F, Farina F, Zummo G, Cappello F. Lymphatic vessels of the dura mater: a new discovery?. J Anat. 2015 Nov;227(5):702-3. Epub 2015 Sep 18 PubMed.

    View all comments by Fabio Bucchieri View all comments by Francesco Cappello
    • User Profile Image Roxana Carare
      University of Southampton School of Medicine
    • User Profile Image Roy Weller
      University of Southampton School of Medicine
    • Posted:

    This is a detailed study of CSF drainage. Its novel techniques include pegylated and small-molecule near-infrared (NIR) tracers and lymphatic-specific reporter mice, combined with stereo microscopy and quantitative measurements of tracers in venous blood. From their studies in mice, the authors suggest that the major route for CSF drainage is by perineural pathways, including the olfactory nerves. They found no drainage of tracer along lymphatics in the dura and detected no drainage of CSF directly into venous blood through arachnoid villi. Furthermore, the authors detected reduced clearance of CSF in aged mice, which may have relevance for Alzheimer's disease.

    The elegant techniques are a step forward in resolving some of the problems associated with the dynamics of CSF drainage. The authors confirmed many findings from previous studies, and the imaging and quantitation of tracers in venous blood improve our understanding of the physiology of CSF drainage that complements the anatomical studies.

    One primary hypothesis tested was that CSF does not flow directly into the venous blood via arachnoid villi. The authors’ results suggest that this is the case in the mouse, but it remains to be determined whether this applies to larger mammals, especially humans. Arachnoid villi in rodents are few in number, small and simple in structure (Kida et al., 1993) compared with arachnoid villi and granulations in humans (Upton and Weller, 1985). It will be interesting to see whether applying the techniques used in this paper can resolve the question of how much CSF drains into the blood through arachnoid villi and granulations in humans, and under what circumstances.

    Using intraventricular and cisternal infusions, the authors found that CSF outflow into lymphatic vessels was significantly slower in aged than in young mice. Evidence that this also occurs in humans comes from a study by Mony de Leon et al. (de Leon et al., 2017), who showed by PET that CSF clearance is reduced in Alzheimer's.

    The relevance of CSF drainage to Alzheimer's disease, however, still needs to be determined. As shown in experimental studies by Helen Cserr et al in the 1980s (Szentistvanyi et al., 1984) and extended more recently by Carare et al. (Carare et al., 2008, 2013; Weller et al., 2015), interstitial fluid and solutes, including Aβ, drain from the brain parenchyma along narrow, 100–150nm-wide basement membranes in the walls of cerebral arteries and capillaries. This pathway constitutes the major lymphatic drainage pathway from the brain parenchyma.

    Failure of the intramural periarterial drainage (IPAD) pathway with age is associated with cerebral amyloid angiopathy (CAA) and Alzheimer's disease. The extremely small size of the IPAD pathways means that they are difficult to resolve with current imaging techniques. We hope that applying similar pegylated and small-molecule NIR tracer techniques used in the present study might further resolve the dynamics of IPAD. Clinical demonstration of IPAD in human patients would greatly assist in monitoring impairment of Aβ elimination from the brain in the elderly, thereby improving the diagnosis and management of CAA and Alzheimer's disease.

    References:

    Kida S, Pantazis A, Weller RO. CSF drains directly from the subarachnoid space into nasal lymphatics in the rat. Anatomy, histology and immunological significance. Neuropathol Appl Neurobiol. 1993 Dec;19(6):480-8. PubMed.

    Upton ML, Weller RO. The morphology of cerebrospinal fluid drainage pathways in human arachnoid granulations. J Neurosurg. 1985 Dec;63(6):867-75. PubMed.

    de Leon MJ, Li Y, Okamura N, Tsui WH, Saint-Louis LA, Glodzik L, Osorio RS, Fortea J, Butler T, Pirraglia E, Fossati S, Kim HJ, Carare RO, Nedergaard M, Benveniste H, Rusinek H. Cerebrospinal Fluid Clearance in Alzheimer Disease Measured with Dynamic PET. J Nucl Med. 2017 Sep;58(9):1471-1476. Epub 2017 Mar 16 PubMed.

    Szentistványi I, Patlak CS, Ellis RA, Cserr HF. Drainage of interstitial fluid from different regions of rat brain. Am J Physiol. 1984 Jun;246(6 Pt 2):F835-44. 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.

    Carare RO, Hawkes CA, Jeffrey M, Kalaria RN, Weller RO. Cerebral amyloid angiopathy, Prion angiopathy, CADASIL and the spectrum of Protein Elimination-Failure Angiopathies (PEFA) in neurodegenerative disease with a focus on therapy. Neuropathol Appl Neurobiol. 2013 Mar 13; PubMed.

    Weller RO, Hawkes CA, Carare RO, Hardy J. Does the difference between PART and Alzheimer's disease lie in the age-related changes in cerebral arteries that trigger the accumulation of Aβ and propagation of tau?. Acta Neuropathol. 2015 May;129(5):763-6. Epub 2015 Mar 27 PubMed.

    View all comments by Roxana Carare View all comments by Roy Weller

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References

News Citations

  1. Lymphatic Vessels Found in Human Brain

Paper Citations

  1. Raper D, Louveau A, Kipnis J. How Do Meningeal Lymphatic Vessels Drain the CNS?. Trends Neurosci. 2016 Sep;39(9):581-586. Epub 2016 Jul 25 PubMed.
  2. Aspelund A, Antila S, Proulx ST, Karlsen TV, Karaman S, Detmar M, Wiig H, Alitalo K. 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.
  3. 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.
  4. Absinta M, Ha SK, Nair G, Sati P, Luciano NJ, Palisoc M, Louveau A, Zaghloul KA, Pittaluga S, Kipnis J, Reich DS. Human and nonhuman primate meninges harbor lymphatic vessels that can be visualized noninvasively by MRI. Elife. 2017 Oct 3;6 PubMed.
  5. Proulx ST, Luciani P, Christiansen A, Karaman S, Blum KS, Rinderknecht M, Leroux JC, Detmar M. Use of a PEG-conjugated bright near-infrared dye for functional imaging of rerouting of tumor lymphatic drainage after sentinel lymph node metastasis. Biomaterials. 2013 Jul;34(21):5128-37. Epub 2013 Apr 6 PubMed.
  6. Choi I, Chung HK, Ramu S, Lee HN, Kim KE, Lee S, Yoo J, Choi D, Lee YS, Aguilar B, Hong YK. Visualization of lymphatic vessels by Prox1-promoter directed GFP reporter in a bacterial artificial chromosome-based transgenic mouse. Blood. 2011 Jan 6;117(1):362-5. Epub 2010 Oct 20 PubMed.
  7. Bradbury MW, Cole DF. The role of the lymphatic system in drainage of cerebrospinal fluid and aqueous humour. J Physiol. 1980 Feb;299:353-65. PubMed.
  8. Boulton M, Flessner M, Armstrong D, Hay J, Johnston M. Determination of volumetric cerebrospinal fluid absorption into extracranial lymphatics in sheep. Am J Physiol. 1998 Jan;274(1 Pt 2):R88-96. PubMed.
  9. Kida S, Pantazis A, Weller RO. CSF drains directly from the subarachnoid space into nasal lymphatics in the rat. Anatomy, histology and immunological significance. Neuropathol Appl Neurobiol. 1993 Dec;19(6):480-8. PubMed.
  10. Upton ML, Weller RO. The morphology of cerebrospinal fluid drainage pathways in human arachnoid granulations. J Neurosurg. 1985 Dec;63(6):867-75. PubMed.
  11. de Leon MJ, Li Y, Okamura N, Tsui WH, Saint-Louis LA, Glodzik L, Osorio RS, Fortea J, Butler T, Pirraglia E, Fossati S, Kim HJ, Carare RO, Nedergaard M, Benveniste H, Rusinek H. Cerebrospinal Fluid Clearance in Alzheimer Disease Measured with Dynamic PET. J Nucl Med. 2017 Sep;58(9):1471-1476. Epub 2017 Mar 16 PubMed.

Further Reading

Papers

  1. Proulx ST, Ma Q, Andina D, Leroux JC, Detmar M. Quantitative measurement of lymphatic function in mice by noninvasive near-infrared imaging of a peripheral vein. JCI Insight. 2017 Jan 12;2(1):e90861. PubMed.

Primary Papers

  1. 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.

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