The Brain’s Glymphatic System: Current Controversies

Abstract

Several global models of brain fluid transport have been proposed. When assessing these models, it is imperative that they are based on observations in live animals. Cerebrospinal fluid (CSF) tracer distribution in histological sections mostly reflects nonphysiological events triggered after death.The literature suggests that diffusion and convective flow both contribute to clearance of CNS solutes. Experiments that are aimed at defining the relative contribution of diffusion versus convection are difficult to interpret because minor changes in physiological variables, such as body posture or respiratory rate, can significantly affect both pathways. Invasive procedures, such as tracer injection, will primarily suppress convective flow.The glymphatic system drives CSF into the brain along periarterial spaces and interstitial fluid (ISF) out along perivenous spaces. Aquaporin-4 (AQP4) water channels, densely expressed at the vascular endfeet of astrocytes, facilitate glymphatic transport, based on all studies on this topic (with one exception).Glymphatic–lymphatic efflux of amyloid-β contributes to diurnal variations in amyloid-β concentration in murine Alzheimer’s disease models and represents a potential therapeutic target for Alzheimer’s disease. The glymphatic concept along with the discovery of meningeal lymphatic vessels have, in recent years, highlighted that fluid is directionally transported within the central nervous system (CNS). Imaging studies, as well as manipulations of fluid transport, point to a key role of the glymphatic–lymphatic system in clearance of amyloid-β and other proteins. As such, the glymphatic–lymphatic system represents a new target in combating neurodegenerative diseases. Not unexpectedly, introduction of a new plumbing system in the brain has stirred controversies. This opinion article will highlight what we know about the brain’s fluid transport systems, where experimental data are lacking, and what is still debated. The glymphatic concept along with the discovery of meningeal lymphatic vessels have, in recent years, highlighted that fluid is directionally transported within the central nervous system (CNS). Imaging studies, as well as manipulations of fluid transport, point to a key role of the glymphatic–lymphatic system in clearance of amyloid-β and other proteins. As such, the glymphatic–lymphatic system represents a new target in combating neurodegenerative diseases. Not unexpectedly, introduction of a new plumbing system in the brain has stirred controversies. This opinion article will highlight what we know about the brain’s fluid transport systems, where experimental data are lacking, and what is still debated. The recent discoveries of the brain’s glymphatic (glial–lymphatic) (see Glossary) system and the meningeal lymphatic vessels have stirred considerable debate [1.Iliff J.J. et al.A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid β.Sci. Transl. Med. 2012; 4147ra111Crossref PubMed Scopus (2332) Google Scholar, 2.Louveau A. et al.Structural and functional features of central nervous system lymphatic vessels.Nature. 2015; 523: 337-341Crossref PubMed Scopus (2203) Google Scholar, 3.Aspelund A. et al.A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules.J. Exp. Med. 2015; 212: 991-999Crossref PubMed Scopus (1035) Google Scholar]. An obvious question is why the existence of a brain-wide fluid transport system and the presence of lymphatic vessels in the meningeal layers remained under the radar until now? Before addressing this question, we will here discuss several of the most controversial points. The debates reflect, in part, the fact that critical sets of data have yet to be collected. Other points of controversy are based on debatable assumptions and misunderstandings of the fluid dynamics within the brain. In particular, it is important to stress that the analysis of tracer distribution in histological sections is prone to artifacts. Cardiac arrest or stroke abruptly initiate a pathological influx of cerebrospinal fluid (CSF) [4.Ma Q. et al.Rapid lymphatic efflux limits cerebrospinal fluid flow to the brain.Acta Neuropathol. 2019; 137: 151-165Crossref PubMed Scopus (74) Google Scholar,5.Mestre H. et al.Cerebrospinal fluid influx drives acute ischemic tissue swelling.Science. 2020; 367eaax7171Crossref PubMed Scopus (61) Google Scholar]. As a result, the microscopic localization of CSF tracers in postmortem tissue will inevitably reflect, in part, CSF movements that occurred after death. We will here outline recent developments and add our points to the ongoing debate. The glymphatic system consists of periarterial CSF inflow running in the same direction as blood flow, propelled by the pulsatility of the arterial wall [6.Iliff J.J. et al.Cerebral arterial pulsation drives paravascular CSF–interstitial fluid exchange in the murine brain.J. Neurosci. 2013; 33: 18190-18199Crossref PubMed Scopus (558) Google Scholar,7.Mestre H. et al.Flow of cerebrospinal fluid is driven by arterial pulsations and is reduced in hypertension.Nat. Commun. 2018; 9: 4878Crossref PubMed Scopus (258) Google Scholar]. From here, CSF mixes with interstitial fluid (ISF) in a process facilitated by aquaporin-4 (AQP4) water channels densely present at the vascular astrocytic endfeet [8.Mestre H. et al.Aquaporin-4-dependent glymphatic solute transport in the rodent brain.Elife. 2018; 7e40070Crossref PubMed Scopus (189) Google Scholar]. Influx of fluid across the blood–brain barrier (BBB) or extrachoroidal sources of CSF may also contribute to glymphatic flow [9.Klarica M. et al.The movement of cerebrospinal fluid and its relationship with substances behavior in cerebrospinal and interstitial fluid.Neuroscience. 2019; 414: 28-48Crossref PubMed Scopus (23) Google Scholar, 10.Oreskovic D. et al.Cerebrospinal fluid secretion by the choroid plexus?.Physiol. Rev. 2016; 96: 1661-1662Crossref PubMed Scopus (14) Google Scholar, 11.Cserr H.F. Role of secretion and bulk flow of brain interstitial fluid in brain volume regulation.Ann. N. Y. Acad. Sci. 1988; 529: 9-20Crossref PubMed Scopus (64) Google Scholar, 12.Abbott N.J. Evidence for bulk flow of brain interstitial fluid: significance for physiology and pathology.Neurochem. Int. 2004; 45: 545-552Crossref PubMed Scopus (585) Google Scholar]. The mixture of CSF and ISF leaves the brain via the perivenous space and along cranial and spinal nerves. This fluid is eventually transported out of the CNS by traditional lymphatic vessels located in meninges and in the soft tissue surrounding the skull. Meningeal lymphatic vessels were first described in 2015 and represent a major efflux route for CSF [2.Louveau A. et al.Structural and functional features of central nervous system lymphatic vessels.Nature. 2015; 523: 337-341Crossref PubMed Scopus (2203) Google Scholar,3.Aspelund A. et al.A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules.J. Exp. Med. 2015; 212: 991-999Crossref PubMed Scopus (1035) Google Scholar]. The meningeal lymphatic vessels are especially well developed around the venous sinuses and at the base of the skull [13.Da Mesquita S. et al.Functional aspects of meningeal lymphatics in ageing and Alzheimer's disease.Nature. 2018; 560: 185-191Crossref PubMed Scopus (452) Google Scholar,14.Ahn J.H. et al.Meningeal lymphatic vessels at the skull base drain cerebrospinal fluid.Nature. 2019; 572: 62-66Crossref PubMed Scopus (222) Google Scholar]. Drainage by the meningeal and cervical lymphatic vessels is relatively fast, since tracers injected into brain or CSF accumulate in the cervical lymph nodes within minutes [15.Plog B.A. et al.Biomarkers of traumatic injury are transported from brain to blood via the glymphatic system.J. Neurosci. 2015; 35: 518-526Crossref PubMed Scopus (270) Google Scholar]. Our current model of glymphatic–lymphatic fluid transport is depicted in Figure 1. The model is based on analyses of how tracers distribute after they are delivered into either cisterna magna or directly into the brain. The model is supported by data collected using multiple complementary methodologies, including optical and magnetic resonance imaging (MRI) and dispersion of radiolabeled tracers [16.Louveau A. et al.Understanding the functions and relationships of the glymphatic system and meningeal lymphatics.J. Clin. Invest. 2017; 127: 3210-3219Crossref PubMed Scopus (278) Google Scholar,17.Rasmussen M.K. et al.The glymphatic pathway in neurological disorders.Lancet Neurol. 2018; 17: 1016-1024Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar]. However, it is important to note that several of the processes that guide the highly polarized fluid transport system still need to be defined. In particular, the mechanisms by which AQP4 supports transport of extracellular tracers and the interlinkage of ISF clearance with meningeal and cervical lymphatic vessels are poorly understood. We will here discuss a number of issues that have sparked debate: (i) the loss of the fluid-filled perivascular space in histological sections, (ii) the direction of flow along the periarterial space, (iii) role of AQP4 water channels, (iv) parenchymal convective flow, (v) intracranial pressure (ICP) changes induced by cisternal tracer injections, and (vi) diurnal changes in amyloid-β and tau concentration. In vivo imaging studies in mice have recently documented that pial arteries and penetrating arterioles are surrounded by large periarterial spaces [18.Achariyar T.M. et al.Glymphatic distribution of CSF-derived apoE into brain is isoform specific and suppressed during sleep deprivation.Mol. 2016; PubMed Scopus Google Scholar, et facilitates transport and in the 2013; PubMed Scopus Google Scholar, et in Commun. 2015; PubMed Scopus Google Scholar]. In the of the perivascular space that of the pial The existence of large periarterial spaces to in vivo since these spaces are in during imaging that the periarterial spaces are the As the perivascular space and eventually CSF tracers are into the surrounding and the H. et al.Flow of cerebrospinal fluid is driven by arterial pulsations and is reduced in hypertension.Nat. Commun. 2018; 9: 4878Crossref PubMed Scopus (258) Google observations directly in to the fluid-filled in The discovery of the based on which that large fluid-filled spaces the is into surrounding the same tissue vivo after et and distribution of in 2018; PubMed Scopus Google Scholar]. The important of this is that tracer distribution in histological sections reflects of the tracer after death. postmortem tissue be to models of brain fluid An of such is the periarterial The concept that the of tracers in the and in the in histological sections suggests that these layers can as efflux pathway for in vivo studies have for the model based on in vivo of tracers to penetrating arterioles et as a for paravascular clearance in the 2020; Full Text Full Text PDF PubMed Scopus Google M. et fluid is in ischemic stroke and Alzheimer's disease Neuropathol. 2013; PubMed Scopus Google Scholar]. one of the studies a large volume of fluid the other of the of the We will that the perivascular spaces are the space that can inflow of large of and that a of the vascular wall be to physiological fluid transport, as it pressure along the fluid of its are especially in the studies because with to pathological of the it will be to to such as vascular or the CSF influx documented under physiological H. et al.Flow of cerebrospinal fluid is driven by arterial pulsations and is reduced in hypertension.Nat. Commun. 2018; 9: 4878Crossref PubMed Scopus (258) Google et spaces at the brain for cerebrospinal fluid 2018; PubMed Scopus Google Scholar]. important to the and of the perivascular models that the perivascular space is the of depicted in histological sections the large fluid-filled perivascular space present in vivo H. et al.Flow of cerebrospinal fluid is driven by arterial pulsations and is reduced in hypertension.Nat. Commun. 2018; 9: 4878Crossref PubMed Scopus (258) Google The of the perivascular space in live to CSF flow why several models have that arterial wall pulsatility convective flow M. et al.Glymphatic solute transport bulk 2016; PubMed Scopus Google M. flow in brain parenchyma and perivascular a model 2018; PubMed Scopus Google Scholar]. to this is that the perivascular space is the in most In the perivascular spaces are and recent that the in can be a et of periarterial spaces in the 2019; PubMed Scopus Google Scholar]. the perivascular spaces to to An obvious question the loss of the large fluid-filled perivascular space in histological sections the studies of the glymphatic system based on vivo analysis of tracer We will that vivo tracer analysis is as as its is to the of tracer influx of animals. This is by of in vivo and vivo tracer influx in the same a B.A. et optical imaging a pathway for the of to the 2018; PubMed Scopus Google et glymphatic influx is with and in mice under 2019; PubMed Scopus Google Scholar]. to the from postmortem it is important to in that can be from with to the via which tracer transport N.J. et tracers injected into the CSF and the brain along periarterial Neuropathol. 2018; PubMed Scopus Google Scholar]. The physiological role of is to facilitate water transport across the In brain and spinal AQP4 is primarily expressed by astrocytic endfeet the The highly polarized on endfeet the and the fact that brain are of water channels suggests that AQP4 facilitates CSF inflow into the et of perivascular localization of aquaporin-4 with and disease in Neurol. 2017; PubMed Scopus Google Scholar]. The based on the that both CSF influx and ISF efflux were reduced in mice AQP4 water channels with [1.Iliff J.J. et al.A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid β.Sci. Transl. Med. 2012; 4147ra111Crossref PubMed Scopus (2332) Google Scholar]. the of AQP4 in glymphatic transport by that of AQP4 affect CSF tracer it affect dispersion of tracers injected into et of the and solute transport in rodent brain 2017; PubMed Scopus Google Scholar]. were in a with data from that of AQP4 reduced inflow of CSF tracers in AQP4 mice including the one in the [8.Mestre H. et al.Aquaporin-4-dependent glymphatic solute transport in the rodent brain.Elife. 2018; 7e40070Crossref PubMed Scopus (189) Google Scholar]. The data from et of the and solute transport in rodent brain 2017; PubMed Scopus Google can be by such as the of that are to suppress glymphatic [8.Mestre H. et al.Aquaporin-4-dependent glymphatic solute transport in the rodent brain.Elife. 2018; 7e40070Crossref PubMed Scopus (189) Google a et glymphatic influx is with and in mice under 2019; PubMed Scopus Google and of mice of that the of the brain’s plumbing system in et of paravascular clearance in the Neurol. PubMed Scopus Google it is imperative to mice within a to potential that tracers of were from brain at the same a in diffusion H.F. et of radiolabeled and from Google Scholar]. the existence of convective flow in the perivascular space documented based on H. et al.Flow of cerebrospinal fluid is driven by arterial pulsations and is reduced in hypertension.Nat. Commun. 2018; 9: 4878Crossref PubMed Scopus (258) Google et spaces at the brain for cerebrospinal fluid 2018; PubMed Scopus Google and H. et al.Cerebrospinal fluid influx drives acute ischemic tissue swelling.Science. 2020; 367eaax7171Crossref PubMed Scopus (61) Google B.A. et optical imaging a pathway for the of to the 2018; PubMed Scopus Google et is for of the glymphatic 2019; Full Text Full Text PDF PubMed Scopus Google Scholar]. studies all the dispersion of CSF tracers by cisterna magna injection, which is relatively it controversial convective flow within the parenchymal tracer transport in the live brain is since into the through cranial or of the the pressure that glymphatic transport [8.Mestre H. et al.Aquaporin-4-dependent glymphatic solute transport in the rodent brain.Elife. 2018; 7e40070Crossref PubMed Scopus (189) Google Scholar]. However, documented in solute within the brain and that convective flow with diffusion S. et transport with and diffusion driven solute transport in the glymphatic 2020; PubMed Scopus Google Scholar]. This is in with a recent that the that convection and diffusion in solute they in the same as solutes, into the perivascular space, they are transported which in a concentration that supports the diffusion of the the perivascular spaces J.H. dynamics of cerebrospinal fluid flow in perivascular 2019; PubMed Scopus Google Scholar]. We that convection and diffusion most in the the of these processes the experimental body of and of and it be to on transport, which is for which transport of glymphatic–lymphatic studies is that of tracers into cisterna magna is to in In other it is that brain tracer influx is and induced by in This is based on physiological changes in during changes in body or the changes in that have been during the of CSF tracers physiological pressure changes are to physiological CSF transport by CSF in cisterna magna is that the tracer its distribution in the rodent a tracer this the CSF tracer will distribute in the at the base of the brain. the of the or the tracers into the the is As in Figure parenchymal tracer influx first after has to contribute to the parenchymal tracer distribution using glymphatic The same point is in where all the same of tracers the brain et glymphatic influx is with and in mice under 2019; PubMed Scopus Google Scholar]. of the that tracer directly affect CSF in recent studies, CSF flow in the perivascular space a in H. et al.Flow of cerebrospinal fluid is driven by arterial pulsations and is reduced in hypertension.Nat. Commun. 2018; 9: 4878Crossref PubMed Scopus (258) Google et spaces at the brain for cerebrospinal fluid 2018; PubMed Scopus Google Scholar]. The et spaces at the brain for cerebrospinal fluid 2018; PubMed Scopus Google Scholar]. the flow of the on or in H. et al.Flow of cerebrospinal fluid is driven by arterial pulsations and is reduced in hypertension.Nat. Commun. 2018; 9: 4878Crossref PubMed Scopus (258) Google This is for into with and such as the cisterna However, this is in the of where the volume and of the have a considerable on tracer distribution within the studies have that several contribute to physiological CSF transport, including the and which all the movement of fluid within the brain et magnetic resonance of physiological brain glymphatic pulsation 2016; PubMed Scopus Google Scholar]. An of has that the concentration of amyloid-β and tau in the interstitial space and CSF a diurnal amyloid-β and tau are during and during sleep in both et dynamics are by and the PubMed Scopus Google et al.The brain interstitial fluid tau in mice and CSF tau in 2019; PubMed Scopus Google Scholar]. to one a of sleep in a in amyloid-β et in the brain after one of sleep Acad. Sci. S. A. 2018; PubMed Scopus Google Scholar]. This of of may that glymphatic clearance is on during sleep and during et al.Meningeal and glymphatic in mice with Neurosci. 2020; PubMed Scopus Google Scholar]. In delivered amyloid-β is in versus mice et drives clearance from the 2013; PubMed Scopus Google Scholar]. However, it has also been that clearance may be for the diurnal in amyloid-β and An important at in the of studies, is that the diurnal changes in were in mice amyloid-β under the and tau under the of the et and amyloid in PubMed Scopus Google Scholar, et CNS and in mice amyloid Full Text PDF PubMed Scopus Google Scholar, Y. et loss and in a Full Text Full Text PDF PubMed Scopus Google Scholar]. are and under or the most of these is that the concentration of amyloid-β and tau in the CSF and ISF the of and variations in CSF rate, ISF ISF rate, and glymphatic is most for the diurnal of amyloid-β and in brain fluid transport has in recent and we about this of brain that of the glymphatic–lymphatic were described the documented the existence of convective clearance H.F. et of radiolabeled and from Google and the that CSF is transported along perivascular spaces et al.Rapid solute transport the brain via paravascular fluid Neurol. Google Scholar]. is the of meningeal lymphatic vessels by the S. et al.A of the brain lymphatic Med. 2019; PubMed Scopus Google Scholar]. The for the current of is that the glymphatic–lymphatic concept has a of the transport system by its role in amyloid-β clearance [1.Iliff J.J. et al.A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid β.Sci. Transl. Med. 2012; 4147ra111Crossref PubMed Scopus (2332) Google Scholar]. The glymphatic–lymphatic system key in of glymphatic–lymphatic transport and in models of Alzheimer’s traumatic brain and disease [13.Da Mesquita S. et al.Functional aspects of meningeal lymphatics in ageing and Alzheimer's disease.Nature. 2018; 560: 185-191Crossref PubMed Scopus (452) Google et of aquaporin-4 in mice brain and 2015; PubMed Scopus (189) Google Scholar, J.J. et of glymphatic pathway tau after traumatic brain Neurosci. PubMed Scopus Google Scholar, et meningeal lymphatic in mice 2019; PubMed Scopus Google Scholar]. to these for most brain fluid transport as The in brain fluid transport also with the of the Alzheimer’s is for to suppress through a what about what 2018; PubMed Scopus Google Scholar]. the brain’s system is because are in bulk the of specific on a clearance pathway that is it as points to amyloid-β as directly loss and in Alzheimer’s disease et and CSF and in Alzheimer's Transl. Med. 2016; PubMed Scopus Google Scholar]. points to with tau and et vivo of tau with in Alzheimer's Neurol. 2019; PubMed Scopus Google Scholar]. and important is that glymphatic clearance is primarily during sleep is for clearance of that in the the we after a sleep et and CSF and in Alzheimer's Transl. Med. 2016; PubMed Scopus Google Scholar]. As such, the glymphatic clearance model of the for the to model by process model of sleep Google Scholar]. of sleep is to a of and that are also with reduced glymphatic clearance et al.Meningeal and glymphatic in mice with Neurosci. 2020; PubMed Scopus Google and the of from and to and Full Text Full Text PDF PubMed Scopus Google Scholar]. the need for sleep across multiple at in part, the need for glymphatic The that glymphatic is highly on brain also has for of are to yet a literature that CSF is out of the CNS in the [4.Ma Q. et al.Rapid lymphatic efflux limits cerebrospinal fluid flow to the brain.Acta Neuropathol. 2019; 137: 151-165Crossref PubMed Scopus (74) Google Scholar]. have been to brain-wide and studies the of brain into B.A. et optical imaging a pathway for the of to the 2018; PubMed Scopus Google et glymphatic brain of 2019; PubMed Scopus Google Scholar]. the glymphatic–lymphatic concept represents a for the to new that can the of As glymphatic–lymphatic fluid transport the of and the studies have documented the existence of the glymphatic system and meningeal lymphatic vessels in brain et al.Glymphatic in pressure 2017; PubMed Scopus Google M. et and meninges lymphatic vessels that can be by 2017; PubMed Google Scholar]. We are of that is of assessing the functional of on a Several are under et magnetic resonance of physiological brain glymphatic pulsation 2016; PubMed Scopus Google et imaging of brain clearance via of perivascular fluid movement with diffusion 2018; PubMed Scopus Google and can be with functional imaging that recent the dynamics of CSF flow with the by functional The that large of CSF inflow with changes in et and cerebrospinal fluid in 2019; PubMed Scopus Google Scholar]. the reflects in imaging of the functional of and fluid transport by (see astrocytic channels, from a role in exchange during a or in are the relative of meningeal cranial and to changes in brain affect imaging and models our of the CNS interstitial space that flow of ISF clearance are brain that have clearance and this to CSF influx directly ISF or are fluid sources that clearance of clearance for the of amyloid-β along arteries in Alzheimer’s disease and amyloid the of amyloid-β along arteries the fact that are highly and prone to of ISF clearance be using brain-wide imaging can of fluid movements as a for of glymphatic in the In the will a be to the for will such a be in during astrocytic channels, from a role in exchange during a or in are the relative of meningeal cranial and to changes in brain affect imaging and models our of the CNS interstitial space that flow of ISF clearance are brain that have clearance and this to CSF influx directly ISF or are fluid sources that clearance of clearance for the of amyloid-β along arteries in Alzheimer’s disease and amyloid the of amyloid-β along arteries the fact that are highly and prone to of ISF clearance be using brain-wide imaging can of fluid movements as a for of glymphatic in the In the will a be to the for will such a be in during We for with the This is supported by the of and and the on of and the number to of and and the and number The and in this opinion article are of the and be as the either expressed or of the of or the The is to and distribute for water isoform primarily expressed by in the AQP4 is expressed the astrocytic is highly polarized to the perivascular endfeet that the brain AQP4 the of the water to into and out of the during a fluid that is in the system and the space surrounding the brain. CSF is at the choroid is still debate about extrachoroidal CSF are to blood and it a role in and to the CSF has also been to as a for in the of CSF into the brain along perivascular spaces surrounding arteries and This process is facilitated by sleep and that arterial pulsations CSF bulk flow the brain in the direction of blood This process has been to be highly on the and AQP4 transport of CSF and ISF that from the interstitial space of the brain parenchyma primarily during sleep and of This process a in the of ISF from the brain parenchyma CSF or clearance This clearance is to at perivenous spaces and This process has been by several to be by a bulk flow and is highly on and AQP4 lymphatic vessels in the the venous sinuses and at the base of the skull. vessels have been to drain CSF solute to cervical lymph nodes and a role in in the as the that and where along the they are is still a of debate. are spaces surrounding blood vessels in the brain that as flow for CSF and

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