March 2019 - Update on CSF and Cerebral Lymphatic Circulation





https://doi.org/10.1016/j.neuron.2018.09.022

Perspective| Volume 100, ISSUE 2, P375-388, October 24, 2018

The Meningeal Lymphatic System: A New Player in Neurophysiology









The nature of fluid dynamics within the brain parenchyma is a focus of intensive research. Of particular relevance is its participation in diseases associated with protein accumulation and aggregation in the brain, such as Alzheimer’s disease (AD). The meningeal lymphatic vessels have recently been recognized as an important player in the complex circulation and exchange of soluble contents between the cerebrospinal fluid (CSF) and the interstitial fluid (ISF). In aging mammals, for example, impaired functioning of the meningeal lymphatic vessels can lead to accelerated accumulation of toxic amyloid beta protein in the brain parenchyma, thus aggravating AD-related pathology. Given that meningeal lymphatic vessels are functionally linked to paravascular influx/efflux of the CSF/ISF, and in light of recent findings that certain cytokines, classically perceived as immune molecules, exert neuromodulatory effects, it is reasonable to suggest that the activity of meningeal lymphatics could alter the accessibility of CSF-borne immune neuromodulators to the brain parenchyma, thereby altering their effects on the brain. Accordingly, in this Perspective we propose that the meningeal lymphatic system can be viewed as a novel player in neurophysiology.

Main Text

 Introduction

Cerebrospinal fluid (CSF) recirculation within the CNS happens through numerous different pathways. Recent revelations about a previously unappreciated meningeal lymphatic system of the CNS (
,
) have prompted a fresh look at some basic ideas about fluid, molecular, and cellular exchanges between different brain compartments, as well as a reassessment of the importance of these CNS-draining lymphatics for CNS homeostasis (
,
,
,
). After a brief historical summary, we begin by introducing several new concepts and findings concerning the development and function of the meningeal lymphatic system, and its role in CSF drainage and as a modulator of paravascular mechanisms for macromolecular exchange (through the glymphatic route). We then discuss the relevance of the meningeal lymphatic system for aging-associated brain dysfunction, amyloid clearance in Alzheimer’s disease (AD), and cytokine signaling in the brain.

 History of the Discovery and Rediscovery of Meningeal Lymphatic Vessels

The existence of lymphatic vessels in the brain meninges was first mentioned toward the end of the 18th century by Paolo Mascagni, an Italian physician known for his unparalleled anatomical knowledge (
). Despite the exceptional anatomical precision of Mascagni’s wax models of human body parts and organs (on display at the Josephinum Medical Museum in Vienna), his claim that lymphatic vessels are present in the brain meninges was discredited and evidently forgotten (
). Almost two centuries later, another Italian scientist reported Mascagni’s “discovery” of lymphatic vessels after inspecting samples of human dura (
), and in the 1960s, Csanda and colleagues (
) described the existence of a lymphatic connection between the CNS and the periphery that was actually involved in drainage of CNS molecules. Those works were also met with skepticism by their contemporaries. At the end of the last century, Jicheng Li and colleagues, using the more robust scientific technique of scanning electron microscopy, claimed discovery of meningeal lymphatic vessels, which they named cerebral meningeal stomata, and suggested to be part of the cerebral prelymphatic capillary system (
). However, given the available methodology, they could not be certain whether the round to oval stomata, which were localized between the mesothelial cells of the cerebral meninges (
), were in fact lymphatic vessels.
It thus took more than 200 years, and the eventual application of state-of-the-art techniques, for Mascagni’s initial observations to be confirmed by a sufficiently detailed structural and functional characterization of meningeal lymphatic vessels (
). This odyssey serves as a good example of the rejection of new paradigms by the contemporary community simply because they do not fit the prevailing dogma—which, in the present case, was based on the universally held conception of the CNS as an “immune-privileged” organ having no direct communication or interaction with the immune system, at least under healthy conditions. Parenthetically, this begs an intriguing question: how many other rejected discoveries are awaiting “rebirth”?
The bona fide lymphatic vessels existing in the meninges of the CNS express the classic markers of lymphatic endothelial cells (LECs), namely, vascular endothelial growth factor receptor 3 (VEGFR3), prospero homeobox protein 1 (Prox1), podoplanin, lymphatic vessel endothelial hyaluronan receptor 1, C-C motif chemokine ligand 21, and CD31, and can efficiently drain both molecules and immune cells from the subarachnoid space into the cervical lymph nodes (
,
). Ever since their characterization in mice, publications on the subject have reported that meningeal lymphatic vessels are evolutionarily conserved and are found also in fish, rats, non-human primates, and humans (
,
,
). In the developing embryo, peripheral lymphatic vessels sprout from venous vasculature, by differentiation of venous endothelial cells into LECs, through Prox1- and VEGFR3-dependent mechanisms (
,
). On the contrary, meningeal lymphatics (in rodents) develop and mature after birth and respond to vascular endothelial growth factor C (VEGF-C), but not to VEGF-D (
).

BG: 
that meningeal lymphatics do not develop prenatally (at least in rodents), suggests in the first 6mths postnatally, neuroinflammatory events such as triggered by vaccination are particularly perillous.

Why? because of reduced capacity to remove inflammatory signaling molecules, waste, and cytokines.




However, as seen in peripheral lymphatics (
,
), blocking of the interaction of VEGF-C with one of its receptors (particularly VEGFR3) impairs the development of brain meningeal lymphatic vessels (
). In peripheral tissues, cells and molecules are first guided into smaller caliber initial lymphatic vessel capillaries (initial lymphatics) and then into precollector and larger collector lymphatic vessels, which are equipped with valves (specialized structures expressing integrin-α9) that prevent backflow of lymph constituents. Initial lymphatics are permeable to cells and debris/molecules due to the existence of flap-like openings between the single layer of LECs, which is made possible due to discontinuous button-like junctions between LECs (
,
,
). When compared to peripheral lymphatics, meningeal lymphatics are composed of a less ramified network of thin-walled initial lymphatic vessels (without integrin-α9-expressing valves) that converge and exit the cranium along particular anatomical structures, namely, along the retroglenoid vein and sigmoid sinus and along the meningeal portions of the pterygopalatine artery (
,
). In adult mice, diffusible solutes (in this case Evans blue) injected into the CSF that fills the cisterna magna were shown to drain first into the deep cervical lymph nodes (dCLNs) and later into superficial cervical lymph nodes (sCLNs) (
). Molecular tracers injected into the mouse brain parenchyma were first detected only in the draining dCLNs, but not in the sCLNs (
), suggesting that interstitial fluid (ISF) and CSF molecules in mice are initially drained into dCLNs and only later into sCLNs. Evans blue injected into the nasal mucosa, however, could not be detected in the dCLNs 30 min later (
), pointing to slower drainage by the nasal lymphatics and/or a possible alternative drainage route. Still lacking, however, is a comprehensive characterization of the cellular players in meningeal lymphatic system formation and maintenance, and of the lymphatic network that connects the CNS-draining initial lymphatics to the CLNs. We also need to broaden our knowledge of meningeal lymphatic network complexity in rodents, non-human primates, and humans (
). It will be interesting to evaluate whether this network of lymphatics is more complex in organisms that have more convoluted brains and higher cortical neuronal density and that display multifaceted cognitive behaviors (
,
).

 CSF Homeostasis

The main components of the CSF, which fills the brain ventricles and the subarachnoid spaces, are produced by the choroid plexus (
). Besides modulating CSF composition, this highly vascularized epithelium, which is present in all four brain ventricles, constitutes the blood-CSF barrier, one of the interface barriers of the CNS (
,
). Epithelial cells of the choroid plexus secrete some of the main CSF proteins, such as transthyretin (
), and express different ionic and water transporters, such as aquaporin-1, both at the basolateral membranes (on the blood side) and at the apical membranes (on the CSF side), thus tightly regulating CSF ionic and hydrostatic properties (
,
,
,
). Owing to size-restricted filtration at the brain’s barriers, the protein contents of the CSF and the ISF are lower than those of plasma; nevertheless, some blood-borne ions and molecules (such as the hormone leptin) are able to reach both the CSF and the ISF via selective transport mechanisms present in epithelial cells of the choroid plexus and in endothelial cells of the blood-brain barrier (BBB) (
,
,
,
). The composition of the CSF can also be modulated by brain parenchymal cells, which secrete molecules that can be transported from the ISF into the CSF sink through a paravascular route (
,
), also termed the glymphatic route (discussed below).
The total CSF volume is renewed about 11 times a day in young-adult rats and about 4 times a day in healthy humans (
,
). CSF flow within the ventricles is pulsatile, being influenced by respiratory and cardiac pulsation (
,
), and directionality of the ventricular CSF flow is influenced by the beating of cilia from ependymal cells that ensheathe the ventricular wall (
). According to the traditional view of CSF homeostasis, the molecular contents of the CSF are cleared via three routes: via arachnoid granulations into the meningeal sinus (based on evidence from ex vivo experiments using dogs and non-human primates;
,
), via lymphatics of the nasal mucosa into the CLNs (cervical lymph nodes - after crossing the cribriform plate and following a poorly characterized route along the olfactory nerves;
,
,
), and via transporters and receptors present on the apical side of the choroid plexus epithelium (
,
,
,
). This traditional concept of CSF homeostasis has now been challenged, however, by recently acquired knowledge about meningeal lymphatic vessels, specifically, that they have access to the CSF and, under healthy conditions, continuously drain its molecular and cellular contents (Figure 1) into the CLNs (
,
,
). Thus, contrary to the proposed direct system for venous CSF outflow through arachnoid granulations (
,
), recent studies (using in vivo imaging techniques) show that molecular tracers present in the CSF are largely drained by the lymphatic vessels of the meninges (THIS IS MICE...NOT confirmed with primates) (
,
,
,
), highlighting the importance of this draining lymphatic route for the clearance of molecules from the brain. However, to further reinforce the observations obtained using the mouse model, it would be interesting to use larger mammals such as dogs or sheep (where the meningeal arachnoid granulations are actually detectable) to measure the differential contribution of the meningeal lymphatic vessels and of the arachnoid granulations-venous sinus route to CSF outflow from the CNS.









Figure thumbnail gr1
Figure 1Cytoarchitecture of the Meninges, Brain Vasculature, and Pathways of Paravascular Recirculation



Figure 1. Cytoarchitecture of the Meninges,
Brain Vasculature, and Pathways of
Paravascular Recirculation
A schematic representation of the brain meninges
constituted by dura, arachnoid, and pia layers.
Lymphatic vessels that are present in the menin-
geal dura drain components of the cerebrospinal
fluid (CSF) that fills the subarachnoid space.

Arising from the brain surface, cerebral arteries
extend into pial and then subpial arteries. Higher-
caliber pial arteries extend into smaller caliber
arterioles (both wrapped by smooth muscle
cells) that dive into the brain parenchyma. Clearly
defined paravascular spaces of about 50–100 nm,
the Virchow-Robin spaces, are filled with CSF that
flows into deeper brain regions, along the arteri-
oles and capillaries, and diffuses through the glia
limitans into the parenchyma. Efflux of interstitial
fluid (ISF) happens through paravenous spaces
back into the subarachnoid CSF.


Changes in CSF composition and renewal are involved in several key aspects of CNS physiology, including intracranial pressure equilibrium (through counterbalance of CSF formation and drainage;
,
), removal of waste metabolites from brain-cell activity, such as amyloid beta (Aβ), which ends up in the CSF sink (
,
,
,
), and neuroinflammation (
,
). Changes in CSF composition also affect neural cell development and function (
,
), for example, by modulating the proliferation of neuronal progenitors during brain development (
), or neurogenesis in the adult brain (
). Decrease in CSF production and clearance is considered to be a serious aggravating factor in different models of neuropathology such as hydrocephalus and ischemia (
,
) and contributes to the decay of brain function in aging and in aging-associated neurodegenerative diseases such as AD (
,
,
,
,
,
). In patients with late-onset AD, faulty clearance of Aβ from the ISF/CSF (rather than its increased production) is closely related to its accumulation in the brain (
,
). The protein content of the CSF increases with aging (
,
) and was shown to be even higher in patients with aging-associated dementia (
). Both in rodents and in humans the CSF turnover rate declines with age; the total CSF volume is twice as high in the elderly as in young adults, mostly because of age-related atrophy of the brain parenchyma (
).
It is well accepted that alterations in CSF homeostasis, either acute or chronic, are closely associated with changes in brain function. Below, we discuss the importance of the paravascular glymphatic route, as well as the contribution of meningeal lymphatic drainage to CSF/ISF recirculation and to the maintenance of CNS fluid composition. Finally, we address the impact of aging-associated meningeal lymphatic dysfunction on the manifestation of behavioral deficits, as well as on the development of neurodegenerative disorders, with particular focus on AD.

 Glymphatic Route and Neurophysiology

The CNS parenchyma presents a complex network of blood vessels, which provide oxygen and nutrients to neural cells (
) but is devoid of lymphatic vessels (
). In contrast, all peripheral organs possess lymphatic vasculatures that are critical for the maintenance of tissue homeostasis (
). In the CNS parenchyma, drainage of cellular debris and waste products from cell metabolism, which in the periphery is usually attributed to the conventional lymphatic vasculature, is in part performed by a paravascular route through which interchange between the CSF and the ISF takes place (
,
,
,
,
). Classic studies performed in the 1970s showed that molecular tracers injected into the brain parenchyma circulate within the interstitium and end up in paravascular spaces (
,
). On the other hand, a study performed in the 1980s showed that molecules injected into the subarachnoid CSF could follow a paravascular route and enter the brain in under 10 min, providing one of the first indications of a continuous interchange between the CSF and the ISF (
). It was not until recently, however, that more light could be shed on this route of communication between the subarachnoid CSF and the parenchymal ISF, by the use of fluorescent molecular tracers and in vivo two-photon laser scanning microscopy (
).
To understand how the interchange between the CSF and ISF takes place, we first need to examine the cytoarchitecture of the brain vasculature (Figure 1). Cerebral arteries at the cortical surface extend into pial and then subpial arteries, all of which are wrapped by smooth muscle cells and are in direct contact with the subarachnoid CSF (
,
). Higher-caliber pial arteries then branch into lower-caliber arterioles that penetrate the brain parenchyma (
), forming clearly defined CSF-filled Virchow-Robin spaces (of about 50−100 nm) between the arterial basement membrane and the glia limitans composed by the astrocytic endfeet (
). The Virchow-Robin (or paravascular) spaces exist along the arterioles and capillaries that dive into deeper brain regions (
,
,
). The arterial capillaries then converge into enlarged venules, which are still ensheathed by the glia limitans and also present a paravascular CSF/ISF-filled space (
). The venules converge into larger subcortical and cortical veins, which in turn converge into sinus networks (such as the meningeal superior sagittal sinus) that exit the CNS to drain into the jugular veins (
,
). Pericytes, essential for maintenance of the BBB integrity and function (
,
,
), are found in close contact both with the basal lamina extracellular matrix (ECM) of arteries and capillaries and with the CSF/ISF that fills the paravascular space (
,
). Importantly, the basal lamina ECM of brain capillaries and venules is composed mainly of laminin, fibronectin, type IV collagen, and heparan sulfate proteoglycans (
) and therefore provides only minimal resistance to the paravascular fluid exchange and recirculation.
This paravascular route was proposed as a mechanism for small-molecule exchange between the CSF and the ISF (
,
). Briefly, subarachnoid CSF flows along the Virchow-Robin spaces deep into the brain (paravascular influx), leaves the periarterial space to interchange with ISF within the parenchyma, and then exits along perivenous spaces (paravascular efflux) back into the subarachnoid space. Molecules of 100 kDa or less (such as horseradish peroxidase or ovalbumin, both ∼45 kDa) encounter no major resistance to entering or leaving the brain parenchyma through the endfeet clefts of the glia limitans (
,
). Also in disagreement with this paravascular pathway theory (
,
), it was initially hypothesized that interstitial molecules such as Aβ would leave the brain exclusively through the periarterial spaces, which in the case of Aβ peptides would lead to its aggregation and accumulation around the walls of brain arteries (
,
). However, it is still debatable whether blood vessel capillaries (which account for the vast majority of the blood vessel length in the brain;
) have a perivascular space filled with CSF/ISF or if this space is at some point fully replaced by basal lamina ECM content. Additional studies (using ultrastructural imaging techniques) should be performed to fully address this aspect of brain vascular cytoarchitecture. Ultimately, an improved knowledge of the extent of perivascular spaces in the brain would allow us to better understand the phenomenon of macromolecule recirculation through the glymphatic pathway. Although some controversy remains about the mechanisms that modulate the paravascular CSF/ISF recirculation (
,
), one of the features of this route is the crucial role of aquaporin 4 (AQP4), which is expressed in the astrocytic endfeet that ensheathe the brain’s blood vasculature (
,
). Furthermore, in addition to its contribution to paravascular fluid and macromolecule exchange, astrocytic AQP4 was shown to be important for the preservation of glia limitans integrity at the BBB (
). Cerebral arterial pulsation was proposed as another essential mechanism for promoting the paravascular flow of CSF into the brain (
), although recent studies have claimed otherwise (
,
). Interestingly, it was shown that paravascular solute influx/efflux mechanisms become more efficient under anesthesia or sleep state (
), contributing to increased brain ISF metabolite (like lactate and Aβ) clearance (
,
,
).
Decreased brain-wide paravascular solute influx/efflux, with subsequently impaired clearance of ISF waste, has been associated with poor outcome in different models of brain disease and pathology. Decreased paravascular fluid influx/efflux was observed in rodent models of traumatic brain injury (a risk factor for early-onset dementia;
), of cortical spreading depression (an animal model of migraine aura;
), and of multiple microinfarcts (closely associated with the development of vascular dementia;
,
). In the traumatic brain injury model, decreased paravascular efflux of tau protein results in augmented pathology (
), which might have repercussions for frontotemporal dementia and AD, two diseases with raised levels of intraneuronal neurofibrillary tangles and extracellular tau in the brain interstitium (
,
,
). Importantly, aged mice demonstrate a marked decrease in glymphatic function (
,
), which might represent an aggravating factor for inherent or existing pathology and contribute to aging-related brain dysfunction and cognitive deficits. In AD, toxic Aβ peptides are present in the extracellular ISF (
,
) and can be excreted into the blood at the BBB by receptor-mediated transcytosis (by low-density lipoprotein receptor-related protein 1 [
,
,
] in a process mediated by phosphatidylinositol binding clathrin assembly protein or PICALM [
]), be internalized and degraded by brain phagocytes (
,
,
), or be transported back into the CSF through the glymphatic route (
) (Figure 2). Excretion of Aβ from the ISF into the CSF is dependent on the fitness of the paravascular route and is markedly decreased when the ISF efflux is dampened by AQP4 deficiency (
). Consistently, brain amyloid pathology is significantly worsened in AD transgenic mice that are crossed with mice on an Aqp4-null background (
). Moreover, decreased paravascular CSF influx/ISF efflux, possibly due to early Aβ accumulation in both perivenous and periarterial spaces (
,
), precedes amyloid plaque deposition in the brain parenchyma of AD transgenic mice (
).









Figure thumbnail gr2
Figure 2Mechanisms of Amyloid Beta Clearance from the Brain

Me: 
3 paths for eliminating toxic Amyloid beta peptides (Ab)

1.  Receptor-mediated transcytosis across BBB directly into vasculature.
2.  Aggregated Ab phagocytosis within brain parenchyma.
3.  CSF Paravascular "Glymphatic" flow into sub arachnoid CSF sink

Contingencies/Dependencies

-  cerebral arterial flow rate
(decreased by reduced cardiac output, atherosclerosis
(increased with inc. cognitive activity
 
-  venous flow rate 
(decreased with higher central venous pressure, transverse venous stenosis

- CSF production rate 
choroid plexus patency  decreased with scarring
cerebral blood pressure/csf pressure differential 

- CSF flow rate through ventricles and spinal cord
decreased with 
   stenosis of apertures, aqueducts, foramina, spinal cord 
   spinal leaks
   IC-HTN and spinal-HTN
   saggy brain

- CSF flow rate through parenchyma
(higher with REM sleep)
(if parenchymal metabolic rate is elevated, then arterial flow will increase, and CSF flow rate will likely be impeded in paravascular spaces (Virchow-Robin Spaces).

- CSF flow rate into sinuses
(csf pressure, arachnoid granulation patency, venous sinus pressure)

- CSF/ISF solutes.  does plasma glucose and peptide level influence CSF concentrations, and thereby waste removal efficiency.

- parenchymal metabolic rate 
(inc. production of m.waste with inc. cognition.) 


------------------------------------------------------------------------------------------------------
Perivascular Spaces (Robin-Virchow Spaces)  & Perivascular Cuffs

A perivascular space, also known as a Virchow–Robin space, is a fluid-filled space surrounding certain blood vessels in several organs, potentially having an immunological function, but more broadly a dispersive role for neural and blood-derived messengers.[1] The brain pia mater is reflected from the surface of the brain onto the surface of blood vessels in the subarachnoid space. In the brain, perivascular cuffs are regions of leukocyte aggregation in the perivascular spaces, usually found in patients with viral encephalitis.
Perivascular spaces vary in dimension according to the type of blood vessel. In the brain where most capillaries have an imperceptible perivascular space, select structures of the brain, such as the circumventricular organs, are notable for having large perivascular spaces surrounding highly permeable capillaries, as observed by microscopy. The median eminence, a brain structure at the base of the hypothalamus, contains capillaries with wide perivascular spaces.[2]
In humans, perivascular spaces surround arteries and veins can usually be seen as areas of dilatation on MRI images. While many normal brains will show a few dilated spaces, an increase in these spaces may correlate with the incidence of several neurodegenerative diseases, making the spaces a topic of research.[3]



-------------------------------------------------------------------------------------------------------------------- 


Despite substantial research efforts, the cellular and molecular culprits for the age-related weakening of glymphatic function remain poorly understood. Furthermore, longitudinal and mechanistic experiments will be needed in order to address, in greater detail, the implications of decreased brain paravascular fluid circulation for specific aspects of brain physiology, both under healthy conditions and in models of aging and AD.

 The Meningeal Lymphatics-Glymphatics Connection in Aging

Based on current knowledge about the exchange of solutes between CNS compartments, it can reasonably be hypothesized that solutes present in the CSF may either travel via the paravascular route to reach the brain parenchyma or be drained by meningeal lymphatics into the periphery. However, until recently, it was unknown whether these two systems, the meningeal lymphatics and the paravascular flow (glymphatics), worked in tandem to regulate CSF/ISF homeostasis. Recent work from our lab demonstrated that the fitness of glymphatics (paravascular CSF influx and ISF efflux of macromolecules) is modulated by meningeal lymphatic function (
), suggesting a direct linkage between the two systems via brain fluids without any obvious anatomical connection (Figure 3). Using pharmacological, surgical, and genetic models (
,
,
,
), we showed that decreased drainage by meningeal lymphatic vessels results in impaired influx of CSF molecules into the brain (
). Notably, 1 month after pharmacological ablation of brain meningeal lymphatics in young-adult mice, learning and memory deficits were elicited without any detectable side effects on blood vasculature (
).









Figure thumbnail gr3
Figure 3Aging Diminishes Meningeal Lymphatic Drainage and Paravascular Recirculation of CSF Macromolecules
Aging is known to negatively affect the function of lymphatic vessels in peripheral organs (
,
,
). Likewise, in the CNS, aging was shown to be associated with impaired functioning of meningeal lymphatic vessels (
,
) (Figure 3). Sequencing of meningeal LECs from young-adult and aged mice revealed significant differences between the two groups in gene expression, pointing to impaired immune-related function, cytoarchitecture, and morphology (along with differences in ECM) and response to growth factors in the older mice (
). Changes in meningeal LEC transcriptome in old mice were further supported by substantial alterations in the morphology and complexity of lymphatic vessels and their decreased capacity for drainage of CSF solutes into dCLNs (
). In a concomitant recent publication, it was demonstrated that CSF solute outflow into the sCLNs is decreased in old mice (
). Notably, using different strategies for delivery of VEGF-C, a growth factor that can act on both peripheral and CNS-draining lymphatics (
,
,
), into the meningeal milieu of old mice, we were able to enhance both lymphatic drainage into the dCLNs and brain paravascular CSF influx; moreover, old mice treated with VEGF-C showed improved performance in behavioral tests that assess learning and memory, namely, the novel location recognition test and Morris water maze (
). The existence of a meningeal lymphatic-glymphatic connection with implications to brain function in aging raised other interesting questions that should be addressed in future studies. For example, despite the connection between the two systems, it is still unclear whether aging’s terrible toll acts first on meningeal lymphatic function or on glymphatics (or even on the components of the neurovascular unit that is indissociably linked to the glymphatic route) or if both systems become independently impaired. Alongside a more detailed longitudinal characterization of changes in meningeal lymphatic and glymphatic function with aging, it would be essential to explore possible connections with age-dependent alterations in CSF/ISF composition, molecular exchange mechanisms at the BBB (and with decreased barrier integrity with aging), and ultimately with the function of neural cells, particularly of microglia (Figure 2).

 Meningeal Lymphatics and AD

The major risk factor for late-onset AD is age (
,
). Aging is accompanied by a progressive deterioration of brain vascular function, which in AD is further aggravated owing to cerebral amyloid angiopathy (
,
,
,
). Several of the genetic risk factors for late-onset AD, such as the apolipoprotein E4 (ApoE4) gene variant (
), or presence of single nucleotide polymorphisms in the genes that encode the proteins clusterin and PICALM (
), are associated with BBB dysfunction and impaired transvascular clearance of Aβ (
,
,
,
). Clearance mechanisms at the BBB are responsible for ∼75% of Aβ excretion from the brain (
,
). However, recognition of the glymphatic system as an alternative route of Aβ recirculation from brain ISF into the CSF sink (
,
,
) and detection of Aβ in the CLNs of AD transgenic mice (
) led to the hypothesis that impaired meningeal lymphatic drainage of CSF would affect Aβ clearance and hence exacerbate the brain’s amyloid burden in AD (
). Besides describing the close relationship between meningeal lymphatic drainage and glymphatic function as well as the decline in meningeal lymphatic function with age (
), we have now also shown that ablation of meningeal lymphatic drainage in young-adult AD transgenic mice (both J20 and 5xFAD models) leads to more severe brain amyloid pathology. Surprisingly, we also found significant deposition of Aβ in the meninges of AD transgenic mice with ablated meningeal lymphatics, a feature not observed in their counterparts with intact meningeal lymphatics (
). Both the meningeal amyloid deposition and the recruitment of local macrophages around Aβ deposits that we observed in AD transgenic mice after lymphatic ablation were similar to what was observed in human dura from AD patients (
).
Taken together, our most recent observations suggest that the decreased meningeal lymphatic function in aging might exacerbate brain and meningeal amyloid pathology (Figure 3) and eventually precipitate the appearance of cognitive deficits in AD. However, there is still much to learn about the consequences of this age-related decrease in meningeal lymphatic drainage. Regulation of meningeal immunity and of inflammatory cytokine levels in the CSF by meningeal lymphatic function (
), together with their ability to modulate brain paravascular CSF influx/ISF efflux mechanisms, might represent a novel and unexplored mechanism to be manipulated for the benefit of the brain. This novel potential role for the meningeal lymphatics is discussed below.

 Meningeal Lymphatics-Glymphatics and Neuromodulation by Cytokines

Neurons, by communicating with each other through electrical and chemical synapses, serve as the primary functional building units of the CNS (
,
), with glia playing vital support roles in neuronal function. Chemical transmission occurs when one or more types of neurotransmitters, in response to an action potential, are released from presynaptic buttons and bind with postsynaptic receptors (ionotropic or metabotropic), thereby inducing downstream changes in resting potential and/or activation of intracellular signaling transducing pathways that can influence gene expression (
,
,
). Depending on their different postsynaptic downstream effects, neurotransmitters can be divided into two main categories, fast-acting and slow-acting (
,
). Foremost among the fast-acting neurotransmitters are glutamate and gamma-aminobutyric acid (GABA). Upon their release at the synaptic cleft, they bind with postsynaptic ionotropic receptors to induce, within milliseconds, excitatory and inhibitory responses, respectively (
,
,
). Examples of slow-acting neurotransmitters/neuromodulators are monoamines (such as dopamine, serotonin, norepinephrine, and histamine), acetylcholine, purines (such as ATP), and gasotransmitters (such as nitric oxide and carbon monoxide) (
,
,
). The term “slow-acting” neuromodulator refers to their ability to modulate the neuronal response to fast-acting neurotransmitters upon being released separately or together with them (
,
,
,
,
). After binding to metabotropic postsynaptic receptors, the slow-acting neuromodulators do not directly induce neuronal membrane depolarization but rather influence postsynaptic neuronal function by activating intracellular second-messenger signaling pathways (
,
,
).
In the peripheral nervous system, neurotransmitters and neuromodulators can modulate the immune response and inflammatory disease outcome at the level of peripheral organs, such as liver, lung, or intestine (
,
,
,
,
,
). For example, vagal nerve stimulation (via release of acetylcholine [ACh]) attenuates systemic inflammation by inhibiting the production and release of tumor necrosis factor (TNF) by the liver, through a mechanism that is dependent on expression of the nicotinic ACh receptor alpha 7 subunit by macrophages (
,
). On the other hand, activation of dopaminergic neurons of the ventral tegmental area can boost innate and adaptive immunity (
). Notably, at mucosal surfaces the peripheral nervous system produces neuromedin U to promote antimicrobial responses by type 2 innate lymphoid cells, boosting the production of inflammatory and tissue repair cytokines (
,
,
).
Besides relaying electrical and chemical signals and modulating inflammatory responses, neurons also express cytokine receptors, which mediate immune cell-cell communication and are essential players in both innate and adaptive immune responses (
,
,
). Cytokines, together with neurotransmitters, play an important role in synaptic plasticity, a process that is strongly linked to learning and memory. Under physiological conditions, TNF released by astrocytes regulates homeostatic synaptic plasticity (
) and experience-dependent plasticity in the developing visual cortex (
). Increased cytokine production in response to exogenous pathological stimuli was also shown to affect neuronal synaptic plasticity. For example, increased levels of the double-stranded RNA viral mimetic poly(I:C) led to the production of TNF by peripheral monocyte-derived immune cells that caused dendritic spine loss in the mouse primary motor cortex, impairments in learning-dependent dendritic spine formation, and deficits in multiple learning tasks (
). Another pro-inflammatory cytokine, interleukin 1 beta (IL-1β), is also involved in memory formation and maintenance. Long-term potentiation (LTP) in the hippocampus is accompanied by increased expression of Il1b (the gene encoding IL-1β) and incubation of brain slices with IL-1 receptor antagonist induces a reversible impairment of LTP maintenance (
). Increased expression of Il1b in the hippocampus is also induced following fear conditioning training and blockade of IL-1β signaling, either by IL-1 receptor antagonist or lack of IL-1β receptor expression, dampens fear memory as well as spatial reference learning and memory (
,
). These behavioral deficits were associated with enhanced paired-pulse inhibition in the hippocampus, in response to perforant path stimulation and consequent impaired LTP in the dentate gyrus (
). Other cytokines such as IL-4, interferon gamma (IFN-γ), and type I IFNs, when present in the ISF and CSF, can bind to cytokine receptors on neurons, inducing changes in neuronal transmission and consequently affecting higher brain functions such as social and learning behaviors (
,
,
,
,
,
). Performance of a cognitive task increases the numbers of IL-4-producing T cells in the brain meninges (
), and both learning and memory are impaired in T cell-depleted and Il4-null mice (
,
). Those observations were further supported by the finding that IL-4 produced by T cells can be directly recognized by neurons (which express the IL-4 receptor) thereby inhibiting axonal degeneration and improving disease outcome in models of CNS injury or autoimmunity (
,
). Another T cell-derived cytokine, IFN-γ, is needed for synaptic transmission of GABAergic neurons in the prefrontal cortex, which support normal social interactions in mice (
). Recent findings in the nematode Caenorhabditis elegans suggest that neuromodulation by cytokines might be conserved across species. In C. elegans, IL-17 acts directly on RMG interneurons, modulating their responsiveness to input from oxygen sensors (
). In mice, interestingly, fetal exposure to high levels of maternal IL-17 leads to development of behavioral deficits in the offspring (autism spectrum disorder-related behavioral impairments) and abnormal cortical development (
,
), supporting a crucial role for IL-17 in brain development and homeostasis. In view of the wide but highly controlled distribution of cytokines within the CNS and their influence on brain development and function, in some cases through direct signaling on neurons, at least some cytokines could be considered as a new family of neuromodulators (
,
,
).
Although recent studies suggest that cytokines can act as neuromodulators under both physiological and pathological conditions, it is nearly always difficult to clearly identify the specific cellular sources of cytokines and whether or how these cytokines recirculate within the brain. Here, we consider three possible sources of neuromodulatory cytokines (Figure 4). The best-characterized source is that of the brain parenchymal cells, particularly glia. For example, release of TNF by astrocytes is essential for the homeostatic plasticity of neurons during the critical period of their development (
,
). In most cases, however, increased cytokine production in the brain occurs in response to noxious stimuli, such as the release of IL-33 by oligodendrocytes in response to injury (
,
) or the production of TNF, IL-6, and IL-1β by myeloid cells recruited into the brain after infection (
,
) or by microglia, the brain resident phagocytes, in models of AD-related amyloidosis (
,
).
An alternative source of cytokines in the CNS is represented by brain meningeal immune cells, both myeloid and lymphoid (
,
,
). Cytokines produced by meningeal immune cells and released into the CSF may diffuse into the brain via the glymphatic pathway and thus interact with receptor-bearing neurons and glial cells (Figure 4). It will be interesting to examine whether immune cells residing in different locations of the brain meninges produce different cytokines, thereby affecting particular neuronal subpopulations and generating different behavioral responses. It is also possible that the response of meningeal immune cells to different stimuli from the brain may result in their expression of different cytokines.
A third potential source of cytokines in the CNS is the blood. Blood-borne cytokines might act directly on brain endothelial cells (like type I IFNs upon infection [
]), or they might reach the brain parenchyma, especially under brain inflammatory conditions, when BBB permeability is dramatically increased. In this situation, not only macromolecules but also circulating immune cells can readily access paravascular spaces and cross the glia limitans to reach the brain interstitium (
,
,
,
) (Figure 4).
The release of cytokines by different cells, as well as their recirculation within the CNS, is thought to be regulated by circadian rhythm. Circadian control of BBB permeability, CSF production, and cellular cytokine release are all suggestive of a precise spatiotemporal mechanism that controls cytokine levels in the brain and also affects neuronal activity (
,
,
,
,
).
Little is known about possible mechanisms that modulate cytokine recirculation and exchange between the ISF and the CSF, or about the kinetics of cytokine clearance from the brain, all of which might differ depending on the brain region and the time of day. As mentioned above, natural sleep or anesthesia are associated with a 60% increase in the interstitial space and result in a dramatic increase in exchange between CSF and ISF through glymphatics (
,
). In this context, it is important to gain a better understanding of the circadian changes in meningeal lymphatic drainage (and its relationship with the circadian-regulation of glymphatic function) and of the role of the meningeal lymphatics-glymphatics connection, particularly as a mechanism for controlling the paravascular influx of cytokines secreted by meningeal immune cells. Cytokine efflux from the parenchymal ISF might also be affected by aging or by conditions in which glymphatic and meningeal lymphatic functions are diminished or impaired (
,
), possibly resulting in delayed cytokine clearance and prolonged action (protective or noxious) on neural cells (
,
,
,
). It is also possible that altered levels of cytokines in the brain parenchyma or its fluids might modulate the meningeal lymphatic-glymphatic connection and, hence, brain CSF/ISF exchange and CSF drainage. This might happen actively, through direct signaling on the cellular components within each system (for example, on astrocytes along the glymphatic route or on endothelial cells of the meningeal lymphatics), or passively, through indirect regulation of the sleep-wake cycle, which can be profoundly affected by different proinflammatory cytokines (
,
,
).

 Concluding Remarks and Future Directions

Meningeal lymphatic dysfunction, associated, for example, with aging, correlates closely with impaired paravascular CSF influx/ISF efflux of solutes in the brain through the glymphatic route, as well as with the manifestation of cognitive deficits and with more severe amyloid pathology in models of AD (
). However, knowledge about the effect of meningeal lymphatic dysfunction on the properties of the brain neurovascular unit is almost nonexistent. Prolonged or chronic impairment of meningeal lymphatics, through decreased drainage of CSF contents and resulting decrease in CSF/ISF macromolecule renewal through glymphatics (
,
), might induce functional changes in astrocytes, pericytes, smooth muscle cells, and even in brain endothelial cells, which altogether might trigger neurodegeneration (
,
,
). Likewise, it is not known whether genetic risk factors of late-onset AD, such as ApoE4 (
,
,
), have any effect on meningeal lymphatic function.
We hypothesize that age-dependent impaired lymphatic drainage will lead to changes in the frequency of particular populations of meningeal immune cells, to alterations in the accessibility of immune-derived cytokines to the brain parenchyma (by indirectly affecting molecular recirculation through the glymphatic pathway) and will ultimately affect glial and neuronal activity (
,
,
,
,
,
). Recent data also indicate that aging-induced alterations in meningeal lymphatic drainage and immunity might contribute to the buildup of brain amyloid pathology and manifestation of cognitive decline in AD (
,
). Modulation of cytokine exchange between the CSF and the ISF through changes in meningeal lymphatics-glymphatics (with possible fluctuations along the sleep-wake cycle) might serve as another key mechanism for the fine-tuning of neuronal activity (and overall brain homeostasis), as well as for the outcome of brain disease. Conditions with strong neuroinflammatory components, such as CNS autoimmune diseases like multiple sclerosis (
,
), CNS infection or injury (
,
), sickness behavior and inflammaging (
,
), might be influenced by the fitness of the meningeal lymphatic vasculature as well as by brain macromolecule and cellular recirculation through the meningeal lymphatic-glymphatic axis. All of these aspects should be addressed in future studies.
Owing to its effect on brain-wide macromolecule exchange and clearance mechanisms, which are of particular relevance for removal of toxic Aβ from the AD brain and for cytokine signaling within the CNS, we propose that progressive dysfunction of the meningeal lymphatic system should be considered as a risk factor for aging-related brain disorders. We also anticipate the development of better imaging techniques (
,
,
), which will make it possible to employ meningeal lymphatic drainage and glymphatic influx/efflux measurements in humans as diagnostic and/or prognostic tools in patients with neuroinflammatory and neurodegenerative disorders.



 











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https://www.ncbi.nlm.nih.gov/pubmed/30046111

2018 Aug;560(7717):185-191. doi: 10.1038/s41586-018-0368-8. Epub 2018 Jul 25.

Functional aspects of meningeal lymphatics in ageing and Alzheimer's disease.

Abstract
Ageing is a major risk factor for many neurological pathologies, but its mechanisms remain unclear. Unlike other tissues, the parenchyma of the central nervous system (CNS) lacks lymphatic vasculature and waste products are removed partly through a paravascular route. (Re)discovery and characterization of meningeal lymphatic vessels has prompted an assessment of their role in waste clearance from the CNS. Here we show that meningeal lymphatic vessels drain macromolecules from the CNS (cerebrospinal and interstitial fluids) into the cervical lymph nodes in mice. Impairment of meningeal lymphatic function slows paravascular influx of macromolecules into the brain and efflux of macromolecules from the interstitial fluid, and induces cognitive impairment in mice. Treatment of aged mice with vascular endothelial growth factor C enhances meningeal lymphatic drainage of macromolecules from the cerebrospinal fluid, improving brain perfusion and learning and memory performance. Disruption of meningeal lymphatic vessels in transgenic mouse models of Alzheimer's disease promotes amyloid-β deposition in the meninges, which resembles human meningeal pathology, and aggravates parenchymal amyloid-β accumulation. Meningeal lymphatic dysfunction may be an aggravating factor in Alzheimer's disease pathology and in age-associated cognitive decline. Thus, augmentation of meningeal lymphatic function might be a promising therapeutic target for preventing or delaying age-associated neurological diseases.












https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5002390/figure/F1/

2016 Sep;39(9):581-586. doi: 10.1016/j.tins.2016.07.001. Epub 2016 Jul 25.

How Do Meningeal Lymphatic Vessels Drain the CNS?

Raper D#1,2, Louveau A#1,3, Kipnis J1,3.
Abstract
The many interactions between the nervous and the immune systems, which are active in both physiological and pathological states, have recently become more clearly delineated with the discovery of a meningeal lymphatic system capable of carrying fluid, immune cells, and macromolecules from the central nervous system (CNS) to the draining deep cervical lymph nodes. However, the exact localization of the meningeal lymphatic vasculature and the path of drainage from the cerebrospinal fluid (CSF) to the lymphatics remain poorly understood. Here, we discuss the potential differences between peripheral and CNS lymphatic vessels and examine the purported mechanisms of CNS lymphatic drainage, along with how these may fit into established patterns of CSF flow.











2018 Oct;73:34-40. doi: 10.1016/j.bbi.2018.07.020. Epub 2018 Jul 25.
Characterization of dural sinus-associated lymphatic vasculature in human Alzheimer's dementia subjects.
Abstract
Recent reports describing lymphatic vasculature in the meninges have challenged the traditional understanding of interstitial solute clearance from the central nervous system, although the significance of this finding in human neurological disease remains unclear. To begin to define the role of meningeal lymphatic function in the clearance of interstitial amyloid beta (Aβ), and the contribution that its failure may make to the development of Alzheimer's disease (AD), we examined meningeal tissue from a case series including AD and control subjects by confocal microscopy. Our findings confirm the presence of lymphatic vasculature in the human meninges and indicate that, unlike perivascular efflux pathways in the brain parenchyma in subjects with AD, Aβ is not deposited in or around meningeal lymphatic vessels associated with dural sinuses. Our findings demonstrate that while the meningeal lymphatic vasculature may serve as an efflux route for Aβ from the brain and cerebrospinal fluid, Aβ does not deposit in the walls of meningeal lymphatic vessels in the setting of AD.




2018 Nov;17(11):1016-1024. doi: 10.1016/S1474-4422(18)30318-1.

The glymphatic pathway in neurological disorders.

Abstract
BACKGROUND:
The glymphatic (glial-lymphatic) pathway is a fluid-clearance pathway identified in the rodent brain in 2012. This pathway subserves the flow of CSF into the brain along arterial perivascular spaces and subsequently into the brain interstitium, facilitated by aquaporin 4 (AQP4) water channels. The pathway then directs flow towards the venous perivascular and perineuronal spaces, ultimately clearing solutes from the neuropil into meningeal and cervical lymphatic drainage vessels. In rodents, the glymphatic pathway is predominantly active during sleep, when the clearance of harmful metabolites such as amyloid β (Aβ) increases two-fold relative to the waking state. Glymphatic dysfunction, probably related to perturbed AQP4 expression, has been shown in animal models of traumatic brain injury, Alzheimer's disease, and stroke. The recent characterisations of the glymphatic and meningeal lymphatic systems in rodents and in humans call for revaluation of the anatomical routes for CSF-interstitial fluid flow and the physiological role that these pathways play in CNS health.
RECENT DEVELOPMENTS:
Several features of the glymphatic and meningeal lymphatic systems have been shown to be present in humans. MRI scans with intrathecally administered contrast agent show that CSF flows along pathways that closely resemble the glymphatic system outlined in rodents. Furthermore, PET studies have revealed that Aβ accumulates in the healthy brain after a single night of sleep deprivation, suggesting that the human glymphatic pathway might also be primarily active during sleep. Other PET studies have shown that CSF clearance of Aβ and tau tracers is reduced in patients with Alzheimer's disease compared with healthy controls. The observed reduction in CSF clearance was associated with increasing grey-matter concentrations of Aβ in the human brain, consistent with findings in mice showing that decreased glymphatic function leads to Aβ accumulation. Altered AQP4 expression is also evident in brain tissue from patients with Alzheimer's disease or normal pressure hydrocephalus; glymphatic MRI scans of patients with normal pressure hydrocephalus show reduced CSF tracer entry and clearance. WHERE NEXT?: Research is needed to confirm whether specific factors driving glymphatic flow in rodents also apply to humans. Longitudinal imaging studies evaluating human CSF dynamics will determine whether a causal link exists between reduced brain solute clearance and the development of neurodegenerative diseases. Assessment of glymphatic function after stroke or traumatic brain injury could identify whether this function correlates with neurological recovery. New insights into how behaviour and genetics modify glymphatic function, and how this function decompensates in disease, should lead to the development of new preventive and diagnostic tools and novel therapeutic targets.









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 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5803388/



. Author manuscript; available in PMC 2018 Feb 8.
Published in final edited form as:
PMCID: PMC5803388
NIHMSID: NIHMS932029
PMID: 29195051

The glymphatic system in CNS health and disease: past, present and future

Abstract

The central nervous system (CNS) is unique in being the only organ system lacking lymphatic vessels to assist in the removal of interstitial metabolic waste products. Recent work has led to the discovery of the glymphatic system, a glial-dependent perivascular network that subserves a pseudo-lymphatic function in the brain. Within the glymphatic pathway, cerebrospinal fluid (CSF) enters brain via periarterial spaces, passes into the interstitium via perivascular astrocytic aquaporin-4, and then drives the perivenous drainage of interstitial fluid (ISF) and its solute. Here we review the role of the glymphatic pathway in CNS physiology, factors known to regulate glymphatic flow, and pathologic processes where a breakdown of glymphatic CSF-ISF exchange has been implicated in disease initiation and progression. Important areas of future research, including manipulation of glymphatic activity aiming to improve waste clearance and therapeutic agent delivery, will also be discussed.
Keywords: Glymphatic, cerebrospinal fluid, perivascular space, aquaporin-4, amyloid-β, astrocyte

INTRODUCTION

Within the central nervous system (CNS), approximately 60–68% of total water content falls within the intracellular space, while the remaining 32–40% occupies the extracellular compartment (). The extracellular fluid can then be further divided into interstitial fluid (ISF), which surrounds the cells of the parenchyma and represents 12–20% of brain water, and the cerebrospinal fluid (CSF) and blood compartments, each comprising 10% of the intracranial water volume (). In peripheral organs, products of cellular metabolism released into the ISF, as well as colloids and fluid filtered across a fenestrated capillary bed, are cleared to the venous blood through a network of lymphatic vessels that run in parallel to the blood supply (, ). The CNS, however, is the only organ of the body that lacks anatomically defined lymphoid tissues (), and as a result, has developed unique adaptations for achieving fluid balance and interstitial waste removal. In addition to its traditionally identified role providing buoyancy to the brain and thus protecting it from the rigid surrounding skull, the CSF has also been suggested to function as a pseudo-lymphatic system, acting as a sink for brain interstitial solute, particularly high molecular weight substances such as proteins (, ). Consequently, this review will focus on the efforts that have been made to identify the anatomical pathways and physiologic regulation governing the interaction between the CSF and ISF, the role of CSF-ISF exchange in neurophysiology and the promotion of extracellular homeostasis, and how the breakdown of this exchange may result from and contribute to diseases of the CNS, as well as have implications for the diagnosis and treatment of these diseases.

CSF – FORMATION AND CIRCULATION

CSF is formed by the choroid plexuses, protrusions of the ependymal lining of the lateral, third and fourth cerebral ventricles (). The choroid plexuses are highly vascularized tissues characterized by a stroma embedded with fenestrated capillaries and surrounded by a single layer of secretory epithelial cells (, ). The absence of tight junctions between endothelial cells makes the choroid plexuses one of the few places within the CNS devoid of a blood-brain barrier (BBB), and this permits the movement of crystalloids, colloids, and fluid from the blood into the stroma down hydrostatic and osmotic pressure gradients (). The secretion of CSF, however, is selective and regulated due to the presence of tight junctions between epithelial cells, thereby preventing the paracellular movement of most solutes into the ventricular lumen, and dividing the epithelial cell into an apical and basolateral membrane (). To increase the surface area for solute and water transport, the basolateral membrane is highly folded and the epithelial apical membrane consists of a dense brush border of microvilli (). For a comprehensive treatment of choroidal CSF secretion see (); for purposes of this discussion, the major molecular species involved in this process will be briefly reviewed.
The principal ions transported by the choroid plexus are Na+, HCO3, Cl and K+ (, ). Primary active transport by the apical membrane Na+/K+-ATPase (, ), pumping Na+ out of and K+ into the cell up their concentration gradients, generates the requisite energy for all other secondary active transport processes, and inhibition of this enzyme with ouabain has been shown to reduce CSF production 70–80% in dog and rabbit (, ). Due to its low intracellular and high blood concentration, Na+ will enter the epithelial cell via the basolateral Na+-dependent Cl/HCO3 exchanger (NCBE), the Na+/HCO3 co-transporter (NBCn1/2) or the Na+/H+ exchanger (NHE1) (, , ). These transporters, as well as cytoplasmic carbonic anhydrase, will increase intracellular HCO3, which can then move into the ventricular CSF by the apical Na+/HCO3 co-transporter (NBCe2), or can drive basolateral Cl entry through the Cl/HCO3 anion exchanger (AE2) (, , ). Passage of Cl into the ventricular lumen has been described to occur via the apical K+/Cl co-transporter (KCC4), or the electroneutral apical Na+/K+/2Cl co-transporter (NKCC1) (, , ). The net transit of Na+, HCO3 and Cl from the blood to the ventricular lumen establishes an osmotic gradient that will also drive water across the epithelial membrane. The movement of water is facilitated by a high apical, and lower basolateral, expression of aquaporin-1 (AQP1) (, , ). Interestingly, genetic deletion of AQP1 only reduces CSF production by 25% (, ), suggesting alternative mechanisms for water transport, including paracellular and transcellular diffusion, and as a requisite co-transport molecule. As an example, it has been demonstrated that for every turnover of NKCC1, 590 water molecules are transported alongside the four ionic osmolytes (). Additionally, the glucose transporter, GLUT1, is highly expressed in the basolateral membrane of choroidal epithelial cells (), potentially to support the high metabolic rate of this secretory tissue, but it has also been suggested their presence is to facilitate water co-transport, thereby increasing the water permeability of the cell required for CSF secretion (, , ).
Collectively, this molecular machinery produces 500–600 mL of CSF each day in humans (, ). Following production, CSF will flow from the lateral ventricles to the third via the foramina of Monro, continue to the fourth by passing through the cerebral aqueduct, and ultimately enters the subarachnoid space and cisterns via the midline foramen of Magendie and the two lateral foramina of Luschka (). In order to fulfill its posited lymphatic function, subarachnoidal CSF must then be able to enter brain to renew ISF, and ISF and solute must be able to drain back to the CSF to achieve waste removal and volume homeostasis. Consequently, the pathways facilitating these fluid dynamics have been an intense area of study over the past several decades.

PERIVASCULAR SPACES – CONDUITS FOR FLUID MOVEMENT INTO AND OUT OF BRAIN

In early work attempting to elaborate the anatomy of ISF drainage from the CNS, traceable solutes were injected directly into the brain parenchyma and then, after allowing various periods of time to elapse, the distribution of these molecules was evaluated, assuming they would label pathways of ISF exit from the tissue. In the first of these studies, Blue Dextran 2000 was injected to the caudate nucleus of rats, and at both 15 minutes and 24 hours it was observed that this dye did not disperse isotropically from the injection site, but rather seemed to preferentially move in certain directions along what appeared to be cerebral blood vessels (). To more clearly localize the sites of interstitial solute efflux, horse radish peroxidase (HRP) was again injected to the rat striatum, and after allowing 4–8 hours for spread within the extracellular spaces of the brain, it was noted that there was significant appearance of this tracer molecule within perivascular spaces (). It was demonstrated that this perivascular drainage of interstitial fluid and solute was, at least in part, directed to the subarachnoid CSF (, ), and further, that perivascular ISF removal was ubiquitous throughout the brain, occurring in disparate regions beyond the caudate nucleus, including cerebral cortex, midbrain, and inferior colliculus ().
There are several potential mechanisms by which fluids and the solutes contained therein may move within the brain. The first of these is diffusion, a passive process of stochastic Brownian motion that derives its energy not from metabolism, but from the thermal energy of the surrounding environment. At a constant physiologic temperature, diffusion can be thought of as a series of random molecular walks dependent upon the molecular size, the concentration gradient, and the distance over which diffusion is occurring (). Conversely, advection, also referred to as bulk flow, is an active process requiring energy from cellular metabolism to produce hydrostatic, electrical, or chemical gradients that can then drive the bulk movement of a fluid. Whereas small molecules tend to diffuse faster than larger molecules, advection has no molecular size dependence and all molecules are predicted to move at a rate equal to the flow of the fluid body (). When both diffusive and advective processes govern molecular dynamics, this is referred to as convection (). There has been much debate regarding whether the efflux of interstitial fluid, and its constituent solute, from brain are diffusion limited or driven by advection. When different molecular weight tracers, including albumin (69 kDa) and polyethylene glycols (4 kDa and 900 Da), were injected to the caudate nucleus of rats, despite there being an approximately five-fold difference in diffusion coefficients between these molecules, all were cleared with a nearly equivalent half-time of disappearance (, ). From this it was concluded that perivascular ISF drainage occurred by bulk fluid flow, as opposed to diffusion, and the rate of this flow was determined to be 0.1–0.3 μL/g brain/min (, , ).
With compelling evidence that perivascular spaces serve as low resistance channels for ISF egress from brain to CSF, Rennels and colleagues next sought to determine if CSF could move from the subarachnoid compartment into the cerebral interstitial spaces, and if so, to identify the pathway of this influx. Here it was determined that within 4–10 minutes of delivery to the subarachnoid CSF, there was significant appearance of HRP within the perivascular spaces of cerebral blood vessels all the way down to the level of the microvascular basement membranes (, ). As a result, the authors concluded that CSF can penetrate the brain parenchyma using the same perivascular conduits ISF employs for drainage back to the CSF, and that this was likely a bulk flow-mediated process due to the rapidity of influx (, ). Interestingly, this same group found that influx of CSF within these perivascular spaces was significantly impaired within edematous cerebral tissues (), suggesting that under normal conditions there is a pressure differential between the CSF and the tissue which facilitates movement into the brain, and that this can be ablated by increasing tissue water content and pressure. In a later study from Ichimura and colleagues where tracer molecules were micro-injected into the perivascular spaces of surface vessels, it was observed that the direction of flow was variable, with a vector into the brain along one segment of an artery and out of the brain in a more distal segment (), thus challenging this concept of CSF penetrance into brain within perivascular channels.

THE GLYMPHATIC SYSTEM – A PATHWAY FOR CSF-ISF EXCHANGE

Anatomical organization

In a more recent study, fluorescently labeled dextrans were injected into the cisternal CSF of mice and it was observed that within 30 minutes there was robust perivascular labeling (), confirming Rennels’ prior work (, ). Using intravital 2-photon microscopy, these fluorescent CSF tracers rapidly appeared, as early as 5 minutes following injection, within the perivascular spaces of surface arteries, and then, over the subsequent 25 minutes, moved progressively deeper into the parenchyma within the perivascular spaces of penetrating arteries (). In Tie2-GFP:NG2-DsRed double reporter mice, with labeled endothelial and smooth muscle cells, respectively, it was found that fluorescent ovalbumin entered the brain specifically within the periarterial space between the smooth muscle and the astrocyte end-feet of the glial limiting membrane. At 3 hours following cisterna magna injection of the fluorescent ovalbumin, tracer could be identified within the basement membranes of parenchymal capillaries and in the perivascular spaces of large caliber draining veins, including the internal cerebral and caudal rhinal veins (). Thus, not only was perivascular influx of CSF validated, but a directionality to this fluid movement was demonstrated, with CSF entering the brain exclusively within periarterial spaces and ISF leaving the brain within perivenous channels ().

The role of aquaporin-4 water channels

Within the CNS, aquaporin-4 (AQP4) is a water channel predominantly expressed within astrocytic processes forming the subpial and subependymal glial limiting membranes, as well as the perivascular astrocytic end-foot processes circumscribing the entirety of the cerebrovasculature (, ). Tetramers of AQP4 assemble into supramolecular structures referred to as square arrays or orthogonal arrays of particles (OAPs) (, ). The shorter M23 isoform of AQP4, through intermolecular N-terminal interactions, forms the core of these OAPs, while the M1 isoform is restricted to the perimeter of the arrays (). OAPs segregate to the plasma membrane of perivascular end-foot processes due to their association with the dystrophin-associated protein complex (DAPC). AQP4 is anchored to the DAPC through α-syntrophin, and the DAPC is in turn attached to laminin and agrin in the perivascular glial basement membrane via α-dystroglycan (). As a consequence of this complex molecular organization, there is an unusually high density of these water channels positioned at the interface between the perivascular and interstitial spaces of the brain.
It has been posited that this localization of AQP4 channels functions to decrease the resistance to CSF-ISF exchange. Testing this assumption, Iliff and colleagues injected a fluorescent ovalbumin to the cisterna magna of global AQP4 knockout mice and found significantly reduced CSF influx relative to wildtype animals (). Interestingly, compartmental analysis revealed that influx within periarterial spaces was unperturbed in the mice lacking AQP4, however, there was significantly impaired flow of the tracer from these spaces to the surrounding parenchyma (), supporting the idea that these channels facilitate fluid movement between the perivascular and interstitial spaces. Further, intrastriatal injection of radiolabeled mannitol revealed that the rate of fluid and solute clearance from the brain’s interstitial spaces was significantly suppressed in the knockout mice (). Consequently, due to its dependence on the glial AQP4 channel, and pseudo-lymphatic function, Nedergaard’s group named this pathway of periarterial CSF inflow and perivenous ISF and solute drainage, the glial-associated lymphatic pathway, or glymphatic pathway () (Fig. 1).
An external file that holds a picture, illustration, etc. Object name is nihms932029f1.jpg
Overview of the circulation of CSF and ISF through the glymphatic pathway
The bulk flow of CSF into brain specifically within the perivascular spaces of penetrating arteries drives interstitial metabolic waste products toward perivenous spaces, and ultimately from the cranium via several post-glymphatic clearance sites, including arachnoid granulations, meningeal lymphatic vessels, and along cranial and spinal nerve roots. AQP4 water channels densely expressed within astrocyte end-foot processes circumscribing both arteries and veins act to reduce the resistance to CSF movement from periarterial spaces into the interstitium, and from the interstitium into perivenous spaces. Reproduced with permission from ().

Post-glymphatic clearance pathways

Historically, subarachnoid CSF, and the ISF that drains into this compartment, have been thought to leave the cranium via the one-way valve arachnoid granulations, which release CSF into the dural venous sinuses (). It has also been demonstrated, however, that a significant proportion of CSF can exit the cranial vault along the internal carotid artery (), as well as within the perineural spaces of cranial nerves, including the vagal and olfactory nerves (, ). In particular, extensions of the subarachnoid space that follow the olfactory tracts, cross the cribiform plate, and project into the nasal submucosa alongside olfactory nerves, have been shown to be responsible for 15–30% of the removal of CSF solute (). There is a dense lymphatic network within the nasal submucosa that then drains this CSF and solute to the deep cervical lymph nodes (dcLNs) (). This pathway of clearance to the dcLNs is especially important for large molecular weight molecules, as solutes under 5 kDa are capable of passing from CSF to blood directly across the microvascular wall within the nasal submucosa (). Up to 50% of radioiodinated serum albumin (RISA) injected to the caudate nucleus drains via the olfactory-nasal submucosa-dcLN pathway, suggesting that this may be the dominant egress site for interstitial solutes (), however, there is also anatomical variability in the magnitude of ISF clearance to the dcLNs that may be reflective of distance from the olfactory bulbs. Illustrating this point, only 22% and 18% of RISA injected to the internal capsule and the more caudal midbrain, respectively, could be collected in the deep cervical lymph (). Efflux of CSF and ISF to the dcLNs is likely critical for intracranial volume regulation, waste removal, and neuroimmunology, as it has been shown to be evolutionarily conserved across mammalian species ranging from mice to non-human primates to humans (, ).
More recent evidence has challenged the paradigm of the CNS being devoid of lymphatic vessels. In studies by Louveau et al and Aspelund et al, vessels with structural, molecular, and functional similarities to peripheral lymphatic vessels were identified immediately adjacent to dural sinuses, including the superior sagittal sinus and the transverse sinuses, as well as aligning the meningeal vascular supply, such as the middle meningeal artery (, ) (Fig. 2). It was shown that fluorescent tracers, delivered either intracerebroventricular or intraparenchymal, could be identified within the lumen of these dural lymphatics, and that ultimately these vessels drained to the dcLNs (, ). Ligation of the afferent lymphatic vessel to the dcLNs led to dilation of the meningeal vessels, suggesting upstream congestion (), and genetic ablation of the meningeal lymphatics significantly impaired clearance of CSF-based tracer to the dcLNs (). Questions persist, however, regarding whether these vessels are positioned within the dural membrane, or rather lie at the interface between the dura and the subarachnoid space, and further, the mechanism by which CSF and solute can traverse the dura and the wall of these vessels to arrive within the lumen remains to be elaborated ().
An external file that holds a picture, illustration, etc. Object name is nihms932029f2.jpg
Post-glymphatic clearance pathways
Glymphatic convective flow is responsible for the drainage of ISF and its constituent solutes, at least in part, to the subarachnoid CSF via perivenous spaces. These solutes can then be cleared to the peripheral venous blood, ultimately to be eliminated in the liver or kidney, by a number of post-glymphatic clearance sites. CSF and waste can pass directly into the venous blood via arachnoid granulations protruding into dural sinuses, such as the superior sagittal sinus (lower left inset). Additionally, macromolecules contained within the CSF can exit the cranium via lymphatic vessels aligning the dural venous sinuses, or alongside olfactory nerves as they traverse the cribiform plate. Both meningeal lymphatics and those positioned within the olfactory mucosa drain to the deep cervical lymph nodes before returning to the venous blood. Reproduced with permission from ().

Regulation of glymphatic flow

Physiologic regulation of glymphatic pathway function is multifaceted. Iliff and colleagues were able to demonstrate that ligation of the internal carotid artery, dampening cardiac cycle-related pulsatility of cortical penetrating arteries, led to impaired glymphatic CSF tracer influx into cerebral tissues (). Conversely, when dobutamine, an inotropic adrenergic agonist, was systemically given to mice it was found that the penetrating arterial pulsatility index was increased, and that this was associated with significantly more CSF tracer penetrance into brain (). Consequently, the authors concluded their metric of penetrating arterial pulsatility, which integrated changes related to amplitude and frequency of diameter oscillation, was positively associated with CSF influx within the glymphatic pathway (). Interestingly, in a separate study it was found that partial occlusion of the brachiocephalic artery, to eliminate pulsatility while maintaining blood flow in the carotid artery, also led to impaired movement of subarachnoid CSF into brain (). These findings were supported by later work using ultra-fast magnetic resonance encephalography that demonstrated cardiac cycle-related pulsatility was responsible for driving periarterial CSF from the circle of Willis centrifugally toward the dorsal cortical surface (). This same study also identified a role for respiratory cycle-related pulsatility in centripetal perivenous fluid movements, as well as fluid dynamics related to very low frequency vasomotor oscillations ().
It has also been demonstrated that level of arousal plays an important role in governing glymphatic CSF and ISF dynamics. Natural sleep was associated with enhanced periarterial CSF tracer influx and improved interstitial solute clearance, including soluble amyloid-β (Aβ) (). These findings were recapitulated in anesthetized mice, suggesting that changes in glymphatic transport were related to state of consciousness and not circadian rhythms (). Increased glymphatic function in the sleep state was determined to result from an increased interstitial space volume fraction, and this in turn was found to be a consequence of lower locus coeruleus-derived noradrenergic tone (). As a result, here it was concluded that in the transition from wakefulness to sleep, as central norepinephrine levels decline, the extracellular space expands, and the resultant decrease in tissue resistance leads to faster CSF influx and interstitial solute efflux (). In a separate study, it was found that head position during sleep also modifies flow through this pathway. Here, using dynamic-contrast-enhanced MRI, it was found that there was lower interstitial solute retention and improved clearance when mice were placed in the lateral decubitus position compared to either prone or supine positions (). Further, it was demonstrated that there was enhanced fluorescent CSF tracer influx to the cerebrum when mice were placed in the lateral position relative to being prone (). Thus, it is clear that postural or gravitational factors also exert regulatory control over the glymphatic pathway.

Functions of the glymphatic system

Glymphatic CSF-ISF exchange has been demonstrated to perform a number of roles in neurophysiology. Perhaps most central to this pathway’s lymphatic function is its waste clearance capacity. In AQP4 knockout mice with reduced glymphatic function, the clearance of interstitial solutes, including mannitol and Aβ, has been observed to be significantly impaired (). Additionally, it was found that enhanced glymphatic clearance is responsible for the reduced brain lactate levels that accompany the transition from wakefulness to sleep. Here, inhibition of glymphatic clearance in anesthetized mice, either with AQP4 deletion, acetazolamide therapy, cisterna magna puncture, or changes in head position, led to higher brain and lower cervical lymph node lactate levels (). Beyond clearance, this pathway has been shown to be critical for the distribution of nutrients, such as glucose, throughout the brain (), and for the delivery of therapeutic agents, as reduced viral transduction was demonstrated following intracerbroventricular injection of AAV9-GFP in AQP4 knockout mice (). Further, bulk flow through the glymphatic pathway contributes to volume transmission and paracrine signaling. It was found that suppression of glymphatic flow with cisterna magna puncture impaired perivascular lipid transport, and as a consequence spontaneous astrocytic Ca2+ signaling within the awake cortex became more frequent, but with reduced synchronization (). Finally, in a recent study, it was found that fluid shear stress, analogous to that produced by perivascular CSF or ISF dynamics, is capable of mechanically opening NMDA receptors on cultured astrocytes, producing increased Ca2+ current (), and suggesting a role for glymphatic flow in mechanotransduction.

Glymphatic dysfunction in CNS disease

With multiple critical roles in CNS physiology, it is perhaps not surprising that dysfunction of the glymphatic pathway has been implicated in a variety of neurologic diseases, particularly those where accumulation of pathologic solute is a prominent feature. It has been demonstrated that there is an age-associated decline in glymphatic CSF influx, as well as interstitial solute clearance, including Aβ, and this appears to be related to reduced penetrating arterial pulsatility in the aged brain (). In the context of Alzheimer’s disease (AD), young APP/PS1 double transgenic mice, expressing chimeric mouse/human amyloid precursor protein (Mo/HuAPP695swe) and a mutant form of human presenilin-1 (PS1-dE9), were found to have both reduced glymphatic influx and clearance of Aβ, and this was shown to worsen as a function of age (). Further, pretreatment of wildtype mice with Aβ led to significant suppression of CSF tracer influx, suggesting that not only does AD lead to reduced glymphatic clearance and accumulation of Aβ, but that this Aβ aggregation will feed forward and produce further glymphatic slowing (). Decreased glymphatic influx has also been observed secondary to subarachnoid hemorrhage, acute ischemia, and multiple micro-infarction (, ). Interestingly, in the case of multiple small embolic strokes, while glymphatic perfusion spontaneously recovers by 14 days, there is persistent solute trapping within lesion cores, potentially explaining the clinical connection between this disease, Aβ plaque formation, and long-term neurodegeneration (). In the murine ‘Hit & Run’ model of traumatic brain injury (TBI), impairment of glymphatic CSF inflow to brain is seen between 1 and 28 days following injury (Fig. 3), and solute clearance from the cortical interstitium is slowed at 7 days (). When there is a second hit to the glymphatic system, and TBI is provided in AQP4 knockout mice, solute clearance is even further suppressed, showing a significant reduction relative to wildtype TBI mice (). Functionally, post-traumatic glymphatic failure, particularly in Aqp4−/− mice, is associated with significant motor, object memory, and spatial memory deficits (). All of the previously discussed diseases are characterized by astrogliosis, measured by increased glial fibrillary acidic protein (GFAP) expression, and this has been shown to drive a loss of perivascular AQP4 localization, potentially representing a common mechanism of glymphatic dysfunction in these pathologies (, , , ) (Fig. 4). Thus far, type II diabetes mellitus is the only disease process characterized by enhanced glymphatic CSF influx and slowed interstitial solute clearance, and the magnitude of this mismatched inflow and outflow has been correlated with degree of cognitive decline (). Due to its pathophysiologic contribution to such a broad segment of CNS diseases, the glymphatic system represents an important target for therapeutic intervention. As known regulatory elements have yet to yield glymphatic-directed treatment strategies, further work is necessary to uncover novel regulation of this pathway.
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Disruption of glymphatic CSF inflow following traumatic brain injury
(A and B) At 1, 3, 7 and 28 days following lateral impact murine ‘Hit & Run’ TBI, mice received a cisterna magna injection (1 μL/min, 10 min) of AlexFluor647-conjugated ovalbumin (45 kDa, 0.5% m/v in artificial CSF). After 30 min of tracer circulation, mice were perfusion fixed and cerebral tissues collected to evaluate glymphatic CSF influx with ex vivo conventional fluorescence microscopy. (C–G) Between 1 and 28 days following TBI there was a significant reduction in glymphatic CSF influx within the hemisphere ipsilateral to the TBI. Interestingly, at 7 days following TBI there was significant global suppression of glymphatic influx with reduced CSF tracer also seen in the contralateral hemisphere. Reproduced with permission from ().
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Impaired glymphatic function after traumatic brain injury is associated with astrocytosis and AQP4 mislocalization
(A) Schematic diagram of lateral impact murine ‘Hit & Run’ TBI. (B and C) At 28 days following TBI there was a significant reactive astrocytosis, as measured by increased GFAP expression, surrounding the lesion core and infiltrating the ipsilateral hemisphere to the impact. (D–F) At the same time point, the astrocytic inflammatory state also resulted in mislocalization of AQP4 water channels away from a perivascular distribution, potentially offering a common mechanism between injury, inflammation and glymphatic failure. Reproduced with permission from ().

FUTURE DIRECTIONS OF STUDY

Cerebral ISF formation as a novel target for therapeutic regulation of glymphatic function

Whereas in peripheral organs ISF is formed as a product of hydrostatic filtration of blood plasma through a fenestrated capillary bed, owing to the presence of tight junctions between adjacent endothelial cells of the BBB (, ), the same is not true in the CNS, where ISF is instead actively secreted by the cerebrovascular endothelium (). The endothelial layer of brain blood vessels is thought to behave analogously to the epithelium of the choroid plexus, and has been noted to have an increased mitochondrial content relative to peripheral endothelial cells to energetically support this secretory function (). Here, the secretion of ISF will be discussed with respect to only those proteins that have been molecularly and functionally identified and localized to either the luminal or abluminal membrane of the endothelial cell; for a more comprehensive review, see (). Similar to the choroid epithelium, the Na+/K+-ATPase is positioned on the secretory, abluminal surface of the endothelial cell, driving Na+ into the brain’s interstitial space and pumping K+ back into the cell (). The establishment of a low intracellular concentration of Na+ relative to the blood plasma allows Na+ to then enter the cell down this gradient via the luminal Na+/H+ exchangers (NHE1/2) or the Na+/K+/2Cl co-transporter (NKCC1) (). At present, it is not clear how Cl traverses the abluminal membrane to maintain electroneutrality with Na+, however, a role for yet to be identified K+/Cl co-transporters or Cl channels has been posited (). While AQP1 facilitates water transport in capillary endothelial cells in peripheral organs, it is not expressed throughout the brain endothelium (, ), and further, it has been demonstrated that AQP4 channels are specific to the astrocyte end-foot, with no expression within the endothelial layer (, ). Consequently, water transport at the BBB is likely by co-transport alongside the ionic species previously discussed. While the rate of ISF secretion has proven difficult to determine, indirect measurement in choroid plexectomized animals has suggested that approximately 20% of the total CSF volume is secondary to ISF formation (), and consequently, ISF secretion has historically been referred to as extrachoroidal CSF production.
Though ISF is formed in the perivascular and interstitial spaces that make up the glymphatic pathway, and drains in large part to the subarachnoid CSF compartment, surprisingly very little is known about how ISF production affects bulk fluid flow within this system. It has been posited in the literature that the CSF compartment can buffer changes in tissue volume that result from different rates of ISF secretion by the cerebrovascular endothelium (). This model suggests that when ISF secretion declines and brain volume contracts, the bulk flow of CSF into brain is increased as a mechanism of replacing lost volume. In the alternative situation, however, when ISF secretion is increased, it has been proposed that the CSF compartment can act as a sink for excess fluid, and therefore, bulk movement of CSF into brain is reduced (). Consequently, altering the rate of brain ISF secretion, potentially with pharmacology, may be an effective approach for both up and down-regulating glymphatic pathway function. Prior work from our group has demonstrated that changes in noradrenergic tone play a role in regulating glymphatic physiology (). During wakefulness, elevated norepinephrine levels lead to a contraction in the extracellular space volume fraction, and the resultant increased interstitial resistance reduces both CSF influx, as well as ISF and solute efflux from brain (). Locus coeruleus-derived norepinephrine, however, has also been shown to increase BBB water permeability through increased activity of the endothelial abluminal Na+/K+-ATPase (, ), thus effectively increasing ISF secretion, and potentially representing an alternative mechanism for slowed glymphatic kinetics during wakefulness. Consequently, centrally administered adrenergic agonists and antagonists may be an effective way of targeting norepinephrine-mediated ISF production, and thereby modulating glymphatic function. Additionally, the central hormone, arginine vasopressin (AVP), has been found to increase cerebral capillary water permeability and brain water content (, ), and antagonism of the cerebrovascular expressed V1a receptor has been shown to reduce cerebral edema following TBI (). Interestingly, systemic administration of AVP did not reproduce these findings, suggesting that similar to other vasopressin-sensitive membranes, the BBB endothelium is only responsive on a single side, its abluminal secretory surface (). As a result, targeting brain-derived AVP may represent another powerful tool in the control of cerebral ISF secretion and flow within the glymphatic pathway. Further, pharmacologic modulation of ISF secretion potentially allows for evaluation of the effect of high or low glymphatic flow, in the absence of superimposed pathology, on cellular and molecular neurobiology, including neuroinflammatory processes, as well as behavioral function.
These neuromodulatory and hormonal systems also may play a role in CNS pathology through their influence on BBB ISF secretion. For example, locus coeruleus degeneration is a prominent feature in Alzheimer’s disease (). While this would be predicted to lead to decreased norepinephrine levels within the brains of these patients, in fact, noradrenergic tone is elevated (), suggesting that degeneration may disproportionately affect inhibitory interneurons. Consequently, this observation of increased central norepinephrine, and the predicted increase in ISF secretion, may explain the reduced glymphatic influx observed in the APP/PS1 Alzheimer’s disease mice ().

Modulation of ISF production to improve gene therapy and drug delivery within the CNS

While increased ISF production may be useful for improving the clearance of interstitial solutes from brain, low ISF formation, through enhancing glymphatic CSF influx, may represent a potential tool for increasing the transduction of intrathecally-delivered virally-packaged gene therapy, as well as the distribution of drugs, such as anti-neoplastic treatments, to a larger area of brain and to structures not in direct contact with the CSF compartment. In a recent study, it was demonstrated that the glymphatic system is responsible for the brain-wide delivery of an adeno-associated virus (AAV)-GFP construct, and that decreased glymphatic influx in AQP4 knockout mice resulted in reduced viral transduction and GFP expression (). Additionally, it has been reported that AAV-mediated GFP labeling of primary sensory neurons was enhanced with intravenous mannitol pre-treatment prior to intrathecal virus injection (). Consequently, an important area of future study will be in determining whether the mechanism of this improved transduction is through increased glymphatic CSF bulk flow, and if so, whether this can be used to functionally modify diseases with a known genetic etiology.

Elaboration of a three-dimensional glymphatic connectome within the intact CNS

The glymphatic pathway, because of the parallel nature with which it runs to the blood supply, spans the entirety of the CNS and is truly an organ-wide system (, ). Further, the annular perivascular channels and intervening interstitial spaces that constitute this pathway within the CNS are connected to the periphery via a number of post-glymphatic efflux sites, including arachnoid granulations (), meningeal lymphatics (, ), perineural spaces of cranial and spinal nerves (, ), and potentially the soft tissues surrounding large vessels such as the internal carotid artery (). Historically, fluid dynamics within the glymphatic pathway have been studied with either in vivo 2-photon laser scanning microscopy or ex vivo conventional fluorescence and confocal microscopy (, , , , , , , ). While 2-photon imaging is capable of providing dynamic information on perivascular flows and flows between the perivascular and interstitial spaces within the living subject (), a narrow focal field and shallow focal depth precludes assessment of glymphatic function at a brain-wide level and in structures deeper than several hundred microns below the cortical surface. Conversely, ex vivo imaging modalities are better suited for evaluating CSF-ISF exchange simultaneously in disparate brain regions, in anatomical structures deep to the surface of the brain, and for evaluating cellular and molecular contributions to glymphatic function. This approach, however, does not provide any dynamic flow information. Additionally, removal of the brain from the skull dissociates the glymphatic system from post-glymphatic pathways, and sectioning of the cerebrum leads to disruption of glymphatic connections within the brain. Finally, while MRI coupled with intrathecal gadolinium-based contrast agents allows for dynamic, macroscopic imaging of glymphatic function throughout the whole brain, this modality is limited by poor anatomical resolution for micron-scale perivascular spaces and meningeal lymph vessels. Consequently, it is clear that novel technique development is required to study the glymphatic connectome, both at the level of the brain and spinal cord, as well as how this system communicates with peripheral organs throughout the body.

SUMMARY AND CONCLUSIONS

Glymphatic dysfunction characterized by a failure of interstitial solute clearance is a central feature of natural brain aging, as well as a broad segment of CNS diseases including Alzheimer’s disease, TBI, and ischemic and hemorrhagic stroke (). Additionally, in type II diabetes mellitus, an imbalance exists where there is increased glymphatic CSF influx without a concomitant increase in ISF efflux, thus leading to extracellular solute accumulation and cognitive decline (). While much is known about the physiologic regulation of glymphatic pathway function, including the roles of cerebral arterial pulsatility (, , ), state of consciousness (), and even head position (), at present there are no glymphatic-directed therapies to intervene in any of these various disease processes. As a result, the primary goal of future studies will be the identification of a novel target for up or down-regulating CSF-ISF exchange within the glymphatic pathway, ultimately to promote improved solute clearance in diseases where metabolite accumulation is a prominent feature.


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http://www.ajnr.org/content/36/5/831


CSF Flow in the Brain in the Context of Normal Pressure Hydrocephalus

W.G. Bradley
 
Abstract
SUMMARY: CSF normally flows back and forth through the aqueduct during the cardiac cycle. During systole, the brain and intracranial vasculature expand and compress the lateral and third ventricles, forcing CSF craniocaudad. During diastole, they contract and flow through the aqueduct reverses. Hyperdynamic CSF flow through the aqueduct is seen when there is ventricular enlargement without cerebral atrophy. Therefore, patients presenting with clinical normal pressure hydrocephalus who have hyperdynamic CSF flow have been found to respond better to ventriculoperitoneal shunting than those with normal or decreased CSF flow. Patients with normal pressure hydrocephalus have also been found to have larger intracranial volumes than sex-matched controls, suggesting that they may have had benign external hydrocephalus as infants. While their arachnoidal granulations clearly have decreased CSF resorptive capacity, it now appears that this is fixed and that the arachnoidal granulations are not merely immature. Such patients appear to develop a parallel pathway for CSF to exit the ventricles through the extracellular space of the brain and the venous side of the glymphatic system. This pathway remains functional until late adulthood when the patient develops deep white matter ischemia, which is characterized histologically by myelin pallor (ie, loss of lipid). The attraction between the bare myelin protein and the CSF increases resistance to the extracellular outflow of CSF, causing it to back up, resulting in hydrocephalus. Thus idiopathic normal pressure hydrocephalus appears to be a “2 hit” disease: benign external hydrocephalus in infancy followed by deep white matter ischemia in late adulthood.



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