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Cerebrospinal fluid (CSF) flowing through periarterial spaces is integral to the brain's mechanism for clearing metabolic waste products. Experiments that track tracer particles injected into the cisterna magna (CM) of mouse brains have shown evidence of pulsatile CSF flow in perivascular spaces surrounding pial arteries, with a bulk flow in the same direction as blood flow. However, the driving mechanism remains elusive. Several studies have suggested that the bulk flow might be an artifact, driven by the injection itself. Here, we address this hypothesis with new in vivo experiments where tracer particles are injected into the CM using a dual-syringe system, with simultaneous injection and withdrawal of equal amounts of fluid. This method produces no net increase in CSF volume and no significant increase in intracranial pressure. Yet, particle-tracking reveals flows that are consistent in all respects with the flows observed in earlier experiments with single-syringe injection., Competing Interests: AR, AL, JT, HM, TD, MN, JT, DK No competing interests declared, (© 2021, Raghunandan et al.)

The emerging interest in brain fluid transport has prompted a need for techniques that provide an understanding of what factors regulate cerebrospinal fluid (CSF) production. Here, we describe a methodology for direct quantification of CSF production in awake mice. We measure CSF production by placing a catheter in a lateral ventricle, while physically blocking outflow from the 4 th ventricle. Using this methodology, we show that CSF production increases during isoflurane anesthesia, and to a lesser extent with ketamine/xylazine anesthesia, relative to the awake state. Aged mice have reduced CSF production, which is even lower in aged mice overexpressing amyloid-β. Unexpectedly, CSF production in young female mice is 30% higher than in age-matched males. Altogether, the present observations imply that a reduction in CSF production might contribute to the age-related risk of proteinopathies but that the rate of CSF production and glymphatic fluid transport are not directly linked., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The glymphatic system is a network of perivascular spaces that promotes movement of cerebrospinal fluid (CSF) into the brain and clearance of metabolic waste. This fluid transport system is supported by the water channel aquaporin-4 (AQP4) localized to vascular endfeet of astrocytes. The glymphatic system is more effective during sleep, but whether sleep timing promotes glymphatic function remains unknown. We here show glymphatic influx and clearance exhibit endogenous, circadian rhythms peaking during the mid-rest phase of mice. Drainage of CSF from the cisterna magna to the lymph nodes exhibits daily variation opposite to glymphatic influx, suggesting distribution of CSF throughout the animal depends on time-of-day. The perivascular polarization of AQP4 is highest during the rest phase and loss of AQP4 eliminates the day-night difference in both glymphatic influx and drainage to the lymph nodes. We conclude that CSF distribution is under circadian control and that AQP4 supports this rhythm.

The glymphatic system (GS) hypothesis states that advective driven cerebrospinal fluid (CSF) influx from the perivascular spaces into the interstitial fluid space rapidly transport solutes and clear waste from brain. However, the presence of advection in neuropil is contested and solutes are claimed to be transported by diffusion only. To address this controversy, we implemented a regularized version of the optimal mass transport (rOMT) problem, wherein the advection/diffusion equation is the only a priori assumption required. rOMT analysis with a Lagrangian perspective of GS transport revealed that solute speed was faster in CSF compared to grey and white matter. Further, rOMT analysis also demonstrated 2-fold differences in regional solute speed within the brain. Collectively, these results imply that advective transport dominates in CSF while diffusion and advection both contribute to GS transport in parenchyma. In a rat model of cerebral small vessel disease (cSVD), solute transport in the perivascular spaces (PVS) and PVS-to-tissue transfer was slower compared to normal rats. Thus, the analytical framework of rOMT provides novel insights in the local dynamics of GS transport that may have implications for neurodegenerative diseases. Future studies should apply the rOMT analysis approach to confirm GS transport reductions in humans with cSVD.

Background: Periarterial spaces (PASs) are annular channels that surround arteries in the brain and contain cerebrospinal fluid (CSF): a flow of CSF in these channels is thought to be an important part of the brain's system for clearing metabolic wastes. In vivo observations reveal that they are not concentric, circular annuli, however: the outer boundaries are often oblate, and the arteries that form the inner boundaries are often offset from the central axis., Methods: We model PAS cross-sections as circles surrounded by ellipses and vary the radii of the circles, major and minor axes of the ellipses, and two-dimensional eccentricities of the circles with respect to the ellipses. For each shape, we solve the governing Navier-Stokes equation to determine the velocity profile for steady laminar flow and then compute the corresponding hydraulic resistance., Results: We find that the observed shapes of PASs have lower hydraulic resistance than concentric, circular annuli of the same size, and therefore allow faster, more efficient flow of cerebrospinal fluid. We find that the minimum hydraulic resistance (and therefore maximum flow rate) for a given PAS cross-sectional area occurs when the ellipse is elongated and intersects the circle, dividing the PAS into two lobes, as is common around pial arteries. We also find that if both the inner and outer boundaries are nearly circular, the minimum hydraulic resistance occurs when the eccentricity is large, as is common around penetrating arteries., Conclusions: The concentric circular annulus assumed in recent studies is not a good model of the shape of actual PASs observed in vivo, and it greatly overestimates the hydraulic resistance of the PAS. Our parameterization can be used to incorporate more realistic resistances into hydraulic network models of flow of cerebrospinal fluid in the brain. Our results demonstrate that actual shapes observed in vivo are nearly optimal, in the sense of offering the least hydraulic resistance. This optimization may well represent an evolutionary adaptation that maximizes clearance of metabolic waste from the brain.

Flow of cerebrospinal fluid (CSF) through perivascular spaces (PVSs) in the brain is important for clearance of metabolic waste. Arterial pulsations are thought to drive flow, but this has never been quantitatively shown. We used particle tracking to quantify CSF flow velocities in PVSs of live mice. CSF flow is pulsatile and driven primarily by the cardiac cycle. The speed of the arterial wall matches that of the CSF, suggesting arterial wall motion is the principal driving mechanism, via a process known as perivascular pumping. Increasing blood pressure leaves the artery diameter unchanged but changes the pulsations of the arterial wall, increasing backflow and thereby reducing net flow in the PVS. Perfusion-fixation alters the normal flow direction and causes a 10-fold reduction in PVS size. We conclude that particle tracking velocimetry enables the study of CSF flow in unprecedented detail and that studying the PVS in vivo avoids fixation artifacts.

Despite the initial promise of immunotherapy for CNS disease, multiple recent clinical trials have failed. This may be due in part to characteristically low penetration of antibodies to cerebrospinal fluid (CSF) and brain parenchyma, resulting in poor target engagement. We here utilized transcranial macroscopic imaging to noninvasively evaluate in vivo delivery pathways of CSF fluorescent tracers. Tracers in CSF proved to be distributed through a brain-wide network of periarterial spaces, previously denoted as the glymphatic system. CSF tracer entry was enhanced approximately 3-fold by increasing plasma osmolality without disruption of the blood-brain barrier. Further, plasma hyperosmolality overrode the inhibition of glymphatic transport that characterizes the awake state and reversed glymphatic suppression in a mouse model of Alzheimer's disease. Plasma hyperosmolality enhanced the delivery of an amyloid-β (Aβ) antibody, obtaining a 5-fold increase in antibody binding to Aβ plaques. Thus, manipulation of glymphatic activity may represent a novel strategy for improving penetration of therapeutic antibodies to the CNS.

Brain lactate concentration is higher during wakefulness than in sleep. However, it is unknown why arousal is linked to an increase in brain lactate and why lactate declines within minutes of sleep. Here, we show that the glymphatic system is responsible for state-dependent changes in brain lactate concentration. Suppression of glymphatic function via acetazolamide treatment, cisterna magna puncture, aquaporin 4 deletion, or changes in body position reduced the decline in brain lactate normally observed when awake mice transition into sleep or anesthesia. Concurrently, the same manipulations diminished accumulation of lactate in cervical, but not in inguinal lymph nodes when mice were anesthetized. Thus, our study suggests that brain lactate is an excellent biomarker of the sleep-wake cycle and increases further during sleep deprivation, because brain lactate is inversely correlated with glymphatic-lymphatic clearance. This analysis provides fundamental new insight into brain energy metabolism by demonstrating that glucose that is not fully oxidized can be exported as lactate via glymphatic-lymphatic fluid transport.

The glymphatic system has recently been shown to clear brain extracellular solutes and abnormalities in glymphatic clearance system may contribute to both initiation and progression of neurological diseases. Despite that diabetes is known as a risk factor for vascular diseases, little is known how diabetes affects the glymphatic system. The current study is the first investigation of the effect of diabetes on the glymphatic system and the link between alteration of glymphatic clearance and cognitive impairment in Type-2 diabetes mellitus rats. MRI analysis revealed that clearance of cerebrospinal fluid contrast agent Gd-DTPA from the interstitial space was slowed by a factor of three in the hippocampus of Type-2 diabetes mellitus rats compared to the non-DM rats and confirmed by florescence imaging analysis. Cognitive deficits detected by behavioral tests were highly and inversely correlated to the retention of Gd-DTPA contrast and fluorescent tracer in the hippocampus of Type-2 diabetes mellitus rats. Type-2 diabetes mellitus suppresses clearance of interstitial fluid in the hippocampus and hypothalamus, suggesting that an impairment of the glymphatic system contributes to Type-2 diabetes mellitus-induced cognitive deficits. Whole brain MRI provides a sensitive, non-invasive tool to quantitatively evaluate cerebrospinal fluid and interstitial fluid exchange in Type-2 diabetes mellitus and possibly in other neurological disorders, with potential clinical application.

Microinfarcts occur commonly in the aging brain as a consequence of diffuse embolic events and are associated with the development of vascular dementia and Alzheimer's disease. However, the manner in which disperse microscopic lesions reduce global cognitive function and increase the risk for Alzheimer's disease is unclear. The glymphatic system, which is a brain-wide perivascular network that supports the recirculation of CSF through the brain parenchyma, facilitates the clearance of interstitial solutes including amyloid β and tau. We investigated whether glymphatic pathway function is impaired in a murine model of multiple microinfarcts induced by intraarterial injection of cholesterol crystals. The analysis showed that multiple microinfarcts markedly impaired global influx of CSF along the glymphatic pathway. Although suppression of global glymphatic function was transient, resolving within 2 weeks of injury, CSF tracers also accumulated within tissue associated with microinfarcts. The effect of diffuse microinfarcts on global glymphatic pathway function was exacerbated in the mice aged 12 months compared with the 2- to 3-month-old mice. These findings indicate that glymphatic function is focally disrupted around microinfarcts and that the aging brain is more vulnerable to this disruption than the young brain. These observations suggest that microlesions may trap proteins and other interstitial solutes within the brain parenchyma, increasing the risk of amyloid plaque formation. SIGNIFICANCE STATEMENT Microinfarcts, small (<1 mm) ischemic lesions, are strongly associated with age-related dementia. However, how these microscopic lesions affect global cognitive function and predispose to Alzheimer's disease is unclear. The glymphatic system is a brain-wide network of channels surrounding brain blood vessels that allows CSF to exchange with interstitial fluid, clearing away cellular wastes such as amyloid β. We observed that, in mice, microinfarcts impaired global glymphatic function and solutes from the CSF became trapped in tissue associated with microinfarcts. These data suggest that small, disperse ischemic lesions can impair glymphatic function across the brain and trapping of solutes in these lesions may promote protein aggregation and neuroinflammation and eventually lead to neurodegeneration, especially in the aging brain., (Copyright © 2017 the authors 0270-6474/17/372870-08$15.00/0.)

The glymphatic pathway expedites clearance of waste, including soluble amyloid β (Aβ) from the brain. Transport through this pathway is controlled by the brain's arousal level because, during sleep or anesthesia, the brain's interstitial space volume expands (compared with wakefulness), resulting in faster waste removal. Humans, as well as animals, exhibit different body postures during sleep, which may also affect waste removal. Therefore, not only the level of consciousness, but also body posture, might affect CSF-interstitial fluid (ISF) exchange efficiency. We used dynamic-contrast-enhanced MRI and kinetic modeling to quantify CSF-ISF exchange rates in anesthetized rodents' brains in supine, prone, or lateral positions. To validate the MRI data and to assess specifically the influence of body posture on clearance of Aβ, we used fluorescence microscopy and radioactive tracers, respectively. The analysis showed that glymphatic transport was most efficient in the lateral position compared with the supine or prone positions. In the prone position, in which the rat's head was in the most upright position (mimicking posture during the awake state), transport was characterized by "retention" of the tracer, slower clearance, and more CSF efflux along larger caliber cervical vessels. The optical imaging and radiotracer studies confirmed that glymphatic transport and Aβ clearance were superior in the lateral and supine positions. We propose that the most popular sleep posture (lateral) has evolved to optimize waste removal during sleep and that posture must be considered in diagnostic imaging procedures developed in the future to assess CSF-ISF transport in humans., Significance Statement: The rodent brain removes waste better during sleep or anesthesia compared with the awake state. Animals exhibit different body posture during the awake and sleep states, which might affect the brain's waste removal efficiency. We investigated the influence of body posture on brainwide transport of inert tracers of anesthetized rodents. The major finding of our study was that waste, including Aβ, removal was most efficient in the lateral position (compared with the prone position), which mimics the natural resting/sleeping position of rodents. Although our finding awaits testing in humans, we speculate that the lateral position during sleep has advantage with regard to the removal of waste products including Aβ, because clinical studies have shown that sleep drives Aβ clearance from the brain., (Copyright © 2015 the authors 0270-6474/15/3511034-11$15.00/0.)

Traumatic brain injury (TBI) is an established risk factor for the early development of dementia, including Alzheimer's disease, and the post-traumatic brain frequently exhibits neurofibrillary tangles comprised of aggregates of the protein tau. We have recently defined a brain-wide network of paravascular channels, termed the "glymphatic" pathway, along which CSF moves into and through the brain parenchyma, facilitating the clearance of interstitial solutes, including amyloid-β, from the brain. Here we demonstrate in mice that extracellular tau is cleared from the brain along these paravascular pathways. After TBI, glymphatic pathway function was reduced by ∼60%, with this impairment persisting for at least 1 month post injury. Genetic knock-out of the gene encoding the astroglial water channel aquaporin-4, which is importantly involved in paravascular interstitial solute clearance, exacerbated glymphatic pathway dysfunction after TBI and promoted the development of neurofibrillary pathology and neurodegeneration in the post-traumatic brain. These findings suggest that chronic impairment of glymphatic pathway function after TBI may be a key factor that renders the post-traumatic brain vulnerable to tau aggregation and the onset of neurodegeneration., (Copyright © 2014 the authors 0270-6474/14/3416180-14$15.00/0.)

CSF from the subarachnoid space moves rapidly into the brain along paravascular routes surrounding penetrating cerebral arteries, exchanging with brain interstitial fluid (ISF) and facilitating the clearance of interstitial solutes, such as amyloid β, in a pathway that we have termed the "glymphatic" system. Prior reports have suggested that paravascular bulk flow of CSF or ISF may be driven by arterial pulsation. However, cerebral arterial pulsation could not be directly assessed. In the present study, we use in vivo two-photon microscopy in mice to visualize vascular wall pulsatility in penetrating intracortical arteries. We observed that unilateral ligation of the internal carotid artery significantly reduced arterial pulsatility by ~50%, while systemic administration of the adrenergic agonist dobutamine increased pulsatility of penetrating arteries by ~60%. When paravascular CSF-ISF exchange was evaluated in real time using in vivo two-photon and ex vivo fluorescence imaging, we observed that internal carotid artery ligation slowed the rate of paravascular CSF-ISF exchange, while dobutamine increased the rate of paravascular CSF-ISF exchange. These findings demonstrate that cerebral arterial pulsatility is a key driver of paravascular CSF influx into and through the brain parenchyma, and suggest that changes in arterial pulsatility may contribute to accumulation and deposition of toxic solutes, including amyloid β, in the aging brain.

Background: Neurodegenerative diseases such as Alzheimer's are associated with the aggregation of endogenous peptides and proteins that contribute to neuronal dysfunction and loss. The glymphatic system, a brain-wide perivascular pathway along which cerebrospinal fluid (CSF) and interstitial fluid (ISF) rapidly exchange, has recently been identified as a key contributor to the clearance of interstitial solutes from the brain, including amyloid β. These findings suggest that measuring changes in glymphatic pathway function may be an important prognostic for evaluating neurodegenerative disease susceptibility or progression. However, no clinically acceptable approach to evaluate glymphatic pathway function in humans has yet been developed., Methods: Time-sequenced ex vivo fluorescence imaging of coronal rat and mouse brain slices was performed at 30-180 min following intrathecal infusion of CSF tracer (Texas Red- dextran-3, MW 3 kD; FITC- dextran-500, MW 500 kD) into the cisterna magna or lumbar spine. Tracer influx into different brain regions (cortex, white matter, subcortical structures, and hippocampus) in rat was quantified to map the movement of CSF tracer following infusion along both routes, and to determine whether glymphatic pathway function could be evaluated after lumbar intrathecal infusion., Results: Following lumbar intrathecal infusions, small molecular weight TR-d3 entered the brain along perivascular pathways and exchanged broadly with the brain ISF, consistent with the initial characterization of the glymphatic pathway in mice. Large molecular weight FITC-d500 remained confined to the perivascular spaces. Lumbar intrathecal infusions exhibited a reduced and delayed peak parenchymal fluorescence intensity compared to intracisternal infusions., Conclusion: Lumbar intrathecal contrast delivery is a clinically useful approach that could be used in conjunction with dynamic contrast enhanced MRI nuclear imaging to assess glymphatic pathway function in humans.

Histological studies have for decades documented that each of the classical meningeal membranes contains multiple fibroblast layers with distinct cellular morphology. Particularly, the sublayers of the arachnoid membranes have received attention due to their anatomical complexity. Early studies found that tracers injected into the cerebrospinal fluid (CSF) do not distribute freely but are restricted by the innermost sublayer of the arachnoid membrane. The existence of restrictions on CSF movement and the subdivision of the subarachnoid space into several distinct compartments have recently been confirmed by in vivo 2-photon studies of rodents, as well as macroscopic imaging of pigs and magnetic resonance imaging of human brain. Based on in vivo imaging and immunophenotyping characterization, we identified the structural basis for this compartmentalization of the subarachnoid space, which we term 'Subarachnoid lymphatic-like membrane', SLYM. The SLYM layer engages the subarachnoid vasculature as it approaches the brain parenchyma, demarcating a roof over pial perivascular spaces. Functionally, the separation of pial periarterial and perivenous spaces in the larger subarachnoid space is critical for the maintenance of unidirectional glymphatic clearance. In light of its close apposition to the pial surface and to the brain perivascular fluid exit points, the SLYM also provides a primary locus for immune surveillance of the brain. Yet, the introduction of SLYM, in terms of its anatomic distinction and hence functional specialization, has met resistance. Its critics assert that SLYM has been described in the literature by other terms, including the inner arachnoid membrane, the interlaminate membrane, the outer pial layer, the intermediate lamella, the pial membrane, the reticular layer of the arachnoid membrane or, more recently, BFB2-3. We argue that our conception of SLYM as an anatomically and functionally distinct construct is both necessary and warranted since its functional roles are wholly distinct from those of the overlying arachnoid barrier layer. Our terminology also lends clarity to a complex anatomy that has hitherto been ill-described. In that regard, we also note the lack of specificity of DPP4, which has recently been introduced as a 'selected defining marker' of the arachnoid barrier layer. We note that DPP4 labels fibroblasts in all meningeal membranes as well as in the trabecula arachnoides and the vascular adventitial layers, thus obviating its utility in meningeal characterization. Instead, we report a set of glymphatic-associated proteins that serve to accurately specify SLYM and distinguish it from its adjacent yet functionally distinct membranes. [ABSTRACT FROM AUTHOR]

The ocular glymphatic system subserves the bidirectional polarized fluid transport in the optic nerve, whereby cerebrospinal fluid from the brain is directed along periarterial spaces towards the eye, and fluid from the retina is directed along perivenous spaces following upon its axonal transport across the glial lamina. Fluid homeostasis and waste removal are vital for retinal function, making the ocular glymphatic fluid pathway a potential route for targeted manipulation to combat blinding ocular diseases such as age-related macular degeneration, diabetic retinopathy, and glaucoma. Several lines of work investigating the bidirectional ocular glymphatic transport with varying methodologies have developed diverging mechanistic models, which has created some confusion about how ocular glymphatic transport should be defined. In this review, we provide a comprehensive summary of the current understanding of the ocular glymphatic system, aiming to address misconceptions and foster a cohesive understanding of the topic. [ABSTRACT FROM AUTHOR]

Fluid efflux from the brain plays an important role in solute waste clearance. Current experimental approaches provide little spatial information, and data collection is limited due to short duration or low frequency of sampling. One approach shows tracer efflux to be independent of molecular size, indicating bulk flow, yet also decelerating like simple membrane diffusion. In an apparent contradiction to this report, other studies point to tracer efflux acceleration. We here develop a one-dimensional advection-diffusion model to gain insight into brain efflux principles. The model is characterized by nine physiological constants and three efflux parameters for which we quantify prior uncertainty. Using Bayes' rule and the two efflux studies, we validate the model and calculate data-informed parameter distributions. The apparent contradictions in the efflux studies are resolved by brain surface boundaries being bottlenecks for efflux. To critically test the model, a custom MRI efflux assay measuring solute dispersion in tissue and release to cerebrospinal fluid was employed. The model passed the test with tissue bulk flow velocities in the range 60 to 190 μm/h. Dimensional analysis identified three principal determinants of efflux, highlighting brain surfaces as a restricting factor for metabolite solute clearance. [ABSTRACT FROM AUTHOR]

Over the past decade, there has been a tremendously increased interest in understanding the neurophysiology of cerebrospinal fluid (CSF) flow, which plays a crucial role in clearing metabolic waste from the brain. This growing interest was largely initiated by two significant discoveries: the glymphatic system (a pathway for solute exchange between interstitial fluid deep within the brain and the CSF surrounding the brain) and meningeal lymphatic vessels (lymphatic vessels in the layer of tissue surrounding the brain that drains CSF). These two CSF systems work in unison, and their disruption has been implicated in several neurological disorders including Alzheimer's disease, stroke, and traumatic brain injury. Here, we present experimental techniques for in vivo quantification of CSF flow via direct imaging of fluorescent microspheres injected into the CSF. We discuss detailed image processing methods, including registration and masking of stagnant particles, to improve the quality of measurements. We provide guidance for quantifying CSF flow through particle tracking and offer tips for optimizing the process. Additionally, we describe techniques for measuring changes in arterial diameter, which is an hypothesized CSF pumping mechanism. Finally, we outline how these same techniques can be applied to cervical lymphatic vessels, which collect fluid downstream from meningeal lymphatic vessels. We anticipate that these fluid mechanical techniques will prove valuable for future quantitative studies aimed at understanding mechanisms of CSF transport and disruption, as well as for other complex biophysical systems. [ABSTRACT FROM AUTHOR]

Perivascular spaces (PVS) drain brain waste metabolites, but their specific flow paths are debated. Meningeal pia mater reportedly forms the outermost boundary that confines flow around blood vessels. Yet, we show that pia is perforated and permissive to PVS fluid flow. Furthermore, we demonstrate that pia is comprised of vascular and cerebral layers that coalesce in variable patterns along leptomeningeal arteries, often merging around penetrating arterioles. Heterogeneous pial architectures form variable sieve-like structures that differentially influence cerebrospinal fluid (CSF) transport along PVS. The degree of pial coverage correlates with macrophage density and phagocytosis of CSF tracer. In vivo imaging confirms transpial influx of CSF tracer, suggesting a role of pia in CSF filtration, but not flow restriction. Additionally, pial layers atrophy with age. Old mice also exhibit areas of pial denudation that are not observed in young animals, but pia is unexpectedly hypertrophied in a mouse model of Alzheimer's disease. Moreover, pial thickness correlates with improved CSF flow and reduced β-amyloid deposits in PVS of old mice. We show that PVS morphology in mice is variable and that the structure and function of pia suggests a previously unrecognized role in regulating CSF transport and amyloid clearance in aging and disease. The precise boundaries and flow compartments of perivascular spaces in the brain are incompletely understood. Here the authors show that pia is perforated and permissive to CSF flow, forming three types of perivascular spaces that remodel with age, with an abnormal type arising in Alzheimer's disease and correlating with β-amyloid burden and differential macrophage uptake. [ABSTRACT FROM AUTHOR]

Spaceflight-associated neuro-ocular syndrome (SANS) has been well documented in astronauts both during and after long-duration spaceflight and is characterized by the development of optic disc edema, globe flattening, choroidal folds, and hyperopic refractive error shifts. The exact mechanisms underlying these ophthalmic abnormalities remain unclear. New findings regarding spaceflight-associated alterations in cerebrospinal fluid spaces, specifically perivascular spaces, may shed more light on the pathophysiology of SANS. The preliminary results of a recent brain magnetic resonance imaging study show that perivascular spaces enlarge under prolonged microgravity conditions, and that the amount of fluid in perivascular spaces is linked to SANS. The exact pathophysiological mechanisms underlying enlargement of perivascular spaces in space crews are currently unclear. Here, we speculate that the dilation of perivascular spaces observed in long-duration space travelers may result from impaired cerebral venous outflow and compromised cerebrospinal fluid resorption, leading to obstruction of glymphatic perivenous outflow and increased periarterial cerebrospinal fluid inflow, respectively. Further, we provide a possible explanation for how dilated perivascular spaces can be associated with SANS. Given that enlarged perivascular spaces in space crews may be a marker of altered venous hemodynamics and reduced cerebrospinal fluid outflow, at the level of the optic nerve and eye, these disturbances may contribute to SANS. If confirmed by further studies, brain glymphatic dysfunction in space crews could potentially be considered a risk factor for the development of neurodegenerative diseases, such as Alzheimer's disease. Furthermore, long-duration exposure to microgravity might contribute to SANS through dysregulation of the ocular glymphatic system. If prolonged spaceflight exposure causes disruption of the glymphatic systems, this might affect the ability to conduct future exploration missions, for example, to Mars. The considerations outlined in the present paper further stress the crucial need to develop effective long-term countermeasures to mitigate SANS-related physiologic changes during long-duration spaceflight. [ABSTRACT FROM AUTHOR]

The brain harbors a unique ability to, figuratively speaking, shift its gears. During wakefulness, the brain is geared fully toward processing information and behaving, while homeostatic functions predominate during sleep. The bloodbrain barrier establishes a stable environment that is optimal for neuronal function, yet the barrier imposes a physiological problem; transcapillary filtration that forms extracellular fluid in other organs is reduced to a minimum in brain. Consequently, the brain depends on a special fluid [the cerebrospinal fluid (CSF)] that is flushed into brain along the unique perivascular spaces created by astrocytic vascular endfeet. We describe this pathway, coined the term glymphatic system, based on its dependency on astrocytic vascular endfeet and their adluminal expression of aquaporin-4 water channels facing toward CSF-filled perivascular spaces. Glymphatic clearance of potentially harmful metabolic or protein waste products, such as amyloid-β, is primarily active during sleep, when its physiological drivers, the cardiac cycle, respiration, and slow vasomotion, together efficiently propel CSF inflow along periarterial spaces. The brain's extracellular space contains an abundance of proteoglycans and hyaluronan, which provide a low-resistance hydraulic conduit that rapidly can expand and shrink during the sleep-wake cycle. We describe this unique fluid system of the brain, which meets the brain's requisites to maintain homeostasis similar to peripheral organs, considering the blood-brain-barrier and the paths for formation and egress of the CSF. [ABSTRACT FROM AUTHOR]

The physiological underpinnings of the necessity of sleep remain uncertain. Recent evidence suggests that sleep increases the convection of cerebrospinal fluid (CSF) and promotes the export of interstitial solutes, thus providing a framework to explain why all vertebrate species require sleep. Cardiovascular, respiratory and vasomotor brain pulsations have each been shown to drive CSF flow along perivascular spaces, yet it is unknown how such pulsations may change during sleep in humans. To investigate these pulsation phenomena in relation to sleep, we simultaneously recorded fast fMRI, magnetic resonance encephalography (MREG), and electroencephalography (EEG) signals in a group of healthy volunteers. We quantified sleep-related changes in the signal frequency distributions by spectral entropy analysis and calculated the strength of the physiological (vasomotor, respiratory, and cardiac) brain pulsations by power sum analysis in 15 subjects (age 26.5 6 4.2 years, 6 females). Finally, we identified spatial similarities between EEG slow oscillation (0.2-2 Hz) power and MREG pulsations. Compared with wakefulness, nonrapid eye movement (NREM) sleep was characterized by reduced spectral entropy and increased brain pulsation intensity. These effects were most pronounced in posterior brain areas for very low-frequency (£ 0.1 Hz) vasomotor pulsations but were also evident brain-wide for respiratory pulsations, and to a lesser extent for cardiac brain pulsations. There was increased EEG slow oscillation power in brain regions spatially overlapping with those showing sleep-related MREG pulsation changes. We suggest that reduced spectral entropy and enhanced pulsation intensity are characteristic of NREM sleep. With our findings of increased power of slow oscillation, the present results support the proposition that sleep promotes fluid transport in human brain. [ABSTRACT FROM AUTHOR]

Throughout the body, lymphatic fluid movement supports critical functions including clearance of excess fluid and metabolic waste. The glymphatic system is the analog of the lymphatic system in the CNS. As such, the glymphatic system plays a key role in regulating directional interstitial fluid movement, waste clearance, and, potentially, brain immunity. The glymphatic system enables bulk movement of CSF from the subarachnoid space along periarterial spaces, where it mixes with interstitial fluid within the parenchyma before ultimately exiting from the parenchyma via perivenous spaces. This review focuses on important questions about the structure of this system, why the brain needs a fluid transport system, and unexplored aspects of brain fluid transport. We provide evidence that astrocytes and blood vessels determine the shape of the perivascular space, ultimately controlling the movement of perivascular fluid. Glymphatic fluid movement has the potential to alter local as well as global transport of signaling molecules and metabolites. We also highlight the evidence for cross talk among the glymphatic system, cardiovascular system, gastrointestinal tract, and lymphatic system. Much remains to be studied, but we propose that the glymphatic/lymphatic system acts as a cornerstone in signaling between the brain and body. [ABSTRACT FROM AUTHOR]

Cerebrospinal fluid (CSF) flow through the brain parenchyma is facilitated by the astrocytic water channel aquaporin 4 (AQP4). Homeostatically regulated electroencephalographic (EEG) slow waves are a hallmark of deep non–rapid eye movement (NREM) sleep and have been implicated in the regulation of parenchymal CSF flow and brain clearance. The human AQP4 gene harbors several single nucleotide polymorphisms (SNPs) associated with AQP4 expression, brain-water homeostasis, and neurodegenerative diseases. To date, their role in sleep-wake regulation is unknown. To investigate whether functional variants in AQP4 modulate human sleep, nocturnal EEG recordings and cognitive performance were investigated in 123 healthy participants genotyped for a common eight-SNP AQP4-haplotype. We show that this AQP4-haplotype is associated with distinct modulations of NREM slow wave energy, strongest in early sleep and mirrored by changes in sleepiness and reaction times during extended wakefulness. The study provides the first human evidence for a link between AQP4, deep NREM sleep, and cognitive consequences of prolonged wakefulness. Aquaporin 4 (AQP4) in astrocytes facilitates a flow of cerebrospinal fluid between perivascular spaces and the brain parenchyma. This study shows that a common genetic haplotype linked to lower levels of AQP4 is associated with more intense NREM sleep and an improved response to extended wakefulness. [ABSTRACT FROM AUTHOR]

Background: The classical view of cerebrospinal fluid (CSF) production posits the choroid plexus as its major source. Although previous studies indicate that part of CSF production occurs in the subarachnoid space (SAS), the mechanisms underlying extra-choroidal CSF production remain elusive. We here investigated the distributions of aquaporin 1 (AQP1) and Na+/K+/2Cl− cotransporter 1 (NKCC1), key proteins for choroidal CSF production, in the adult rodent brain and spinal cord. Methods: We have accessed AQP1 distribution in the intact brain using uDISCO tissue clearing technique and by Western blot. AQP1 and NKCC1 cellular localization were accessed by immunohistochemistry in brain and spinal cord obtained from adult rodents. Imaging was performed using light-sheet, confocal and bright field light microscopy. Results: We determined that AQP1 is widely distributed in the leptomeningeal vasculature of the intact brain and that its glycosylated isoform is the most prominent in different brain regions. Moreover, AQP1 and NKCC1 show specific distributions in the smooth muscle cell layer of penetrating arterioles and veins in the brain and spinal cord, and in the endothelia of capillaries and venules, restricted to the SAS vasculature. Conclusions: Our results shed light on the molecular framework that may underlie extra-choroidal CSF production and we propose that AQP1 and NKCC1 within the leptomeningeal vasculature, specifically at the capillary level, are poised to play a role in CSF production throughout the central nervous system. [ABSTRACT FROM AUTHOR]

Background: Apolipoprotein E (apoE) is a major carrier of cholesterol and essential for synaptic plasticity. In brain, it's expressed by many cells but highly expressed by the choroid plexus and the predominant apolipoprotein in cerebrospinal fluid (CSF). The role of apoE in the CSF is unclear. Recently, the glymphatic system was described as a clearance system whereby CSF and ISF (interstitial fluid) is exchanged via the peri-arterial space and convective flow of ISF clearance is mediated by aquaporin 4 (AQP4), a water channel. We reasoned that this system also serves to distribute essential molecules in CSF into brain. The aim was to establish whether apoE in CSF, secreted by the choroid plexus, is distributed into brain, and whether this distribution pattern was altered by sleep deprivation. Methods: We used fluorescently labeled lipidated apoE isoforms, lenti-apoE3 delivered to the choroid plexus, immunohistochemistry to map apoE brain distribution, immunolabeled cells and proteins in brain, Western blot analysis and ELISA to determine apoE levels and radiolabeled molecules to quantify CSF inflow into brain and brain clearance in mice. Data were statistically analyzed using ANOVA or Student's t-test. Results: We show that the glymphatic fluid transporting system contributes to the delivery of choroid plexus/CSF-derived human apoE to neurons. CSF-delivered human apoE entered brain via the perivascular space of penetrating arteries and flows radially around arteries, but not veins, in an isoform specific manner (apoE2 > apoE3 > apoE4). Flow of apoE around arteries was facilitated by AQP4, a characteristic feature of the glymphatic system. ApoE3, delivered by lentivirus to the choroid plexus and ependymal layer but not to the parenchymal cells, was present in the CSF, penetrating arteries and neurons. The inflow of CSF, which contains apoE, into brain and its clearance from the interstitium were severely suppressed by sleep deprivation compared to the sleep state. Conclusions: Thus, choroid plexus/CSF provides an additional source of apoE and the glymphatic fluid transporting system delivers it to brain via the periarterial space. By implication, failure in this essential physiological role of the glymphatic fluid flow and ISF clearance may also contribute to apoE isoform-specific disorders in the long term. [ABSTRACT FROM AUTHOR]

In recent years, traumatic brain injury (TBI) has emerged as a rapidly growing public health challenge. Annually, approximately 1.7 million people will sustain a TBI in the USA and WHO has named TBI the leading cause of death and disability in young adults worldwide, predicting it will become the third leading cause of death in the general population by 2020. The medical community currently relies on clinical examination and various neuroimaging modalities for the diagnosis of TBI; however, these methodologies are often confounded by altered patient mental status and are particularly poor at identifying mild-to-moderate injury. Despite decades of basic and clinical research, and the identification of hundreds of biochemical markers, presently there is no blood test to objectively assess TBI severity. Recent work suggests treatment-induced variance in the brain's glymphatic clearance may be responsible for the breakdown between biomarker discovery and clinical translation. [ABSTRACT FROM AUTHOR]

Background: Neurodegenerative diseases such as Alzheimer's are associated with the aggregation of endogenous peptides and proteins that contribute to neuronal dysfunction and loss. The glymphatic system, a brain-wide perivascular pathway along which cerebrospinal fluid (CSF) and interstitial fluid (ISF) rapidly exchange, has recently been identified as a key contributor to the clearance of interstitial solutes from the brain, including amyloid β. These findings suggest that measuring changes in glymphatic pathway function may be an important prognostic for evaluating neurodegenerative disease susceptibility or progression. However, no clinically acceptable approach to evaluate glymphatic pathway function in humans has yet been developed. Methods: Time-sequenced ex vivo fluorescence imaging of coronal rat and mouse brain slices was performed at 30-180 min following intrathecal infusion of CSF tracer (Texas Red- dextran-3, MW 3 kD; FITC- dextran-500, MW 500 kD) into the cisterna magna or lumbar spine. Tracer influx into different brain regions (cortex, white matter, subcortical structures, and hippocampus) in rat was quantified to map the movement of CSF tracer following infusion along both routes, and to determine whether glymphatic pathway function could be evaluated after lumbar intrathecal infusion. Results: Following lumbar intrathecal infusions, small molecular weight TR-d3 entered the brain along perivascular pathways and exchanged broadly with the brain ISF, consistent with the initial characterization of the glymphatic pathway in mice. Large molecular weight FITC-d500 remained confined to the perivascular spaces. Lumbar intrathecal infusions exhibited a reduced and delayed peak parenchymal fluorescence intensity compared to intracisternal infusions. Conclusion: Lumbar intrathecal contrast delivery is a clinically useful approach that could be used in conjunction with dynamic contrast enhanced MRI nuclear imaging to assess glymphatic pathway function in humans. [ABSTRACT FROM AUTHOR]

Traumatic spinal cord injury is characterized by an immediate. irreversible loss of tissue at the lesion site, as well as a secondary expansion of tissue damage overtime. Although secondary injury should, in principle, be preventable, no effective treatment options currently exist for patients with acute spinal cord injury (Sd). Excessive release of ATP by the traumatized tissue, followed by activation of high-affinity P2X7 receptors, has previously been implicated in secondary injury, but no clinically relevant strategy by which to antagonize P2X7 receptors has yet, to the best of our knowledge, been reported. Here we have tested the neuroprotective effects of a systemically administered P2X7R antagonist, Brilliant blue G (BBG), in a weight-drop model of thoracic Sd in rats. Administration of BBG 15 mm after injury reduced spinal cord anatomic damage and improved motor recovery without evident toxicity. Moreover, BBG treatment directly reduced local activation of astrocytes and microglia, as well as neutrophil infiltration. These observations suggest that BBG not only protected spinal cord neurons from purinergic excitotoxicity, but also reduced local inflammatory responses. Importantly, BBG is a derivative of a commonly used blue food color (FD&C blue No. 1), which crosses the blood-brain barrier. Systemic administration of BBG may thus comprise a readily feasible approach by which to treat traumatic SCI in humans. [ABSTRACT FROM AUTHOR]

The glymphatic system is a fluid-transport system that accesses all regions of the brain. It facilitates the exchange of cerebrospinal fluid and interstitial fluid and clears waste from the metabolically active brain. Astrocytic endfeet and their dense expression of the aquaporin-4 water channels promote fluid exchange between the perivascular spaces and the neuropil. Cerebrospinal and interstitial fluids are together transported back to the vascular compartment by meningeal and cervical lymphatic vessels. Multiple lines of work show that neurological diseases in general impair glymphatic fluid transport. Insofar as the glymphatic system plays a pseudo-lymphatic role in the central nervous system, it is poised to play a role in neuroinflammation. In this review, we discuss how the association of the glymphatic system with the meningeal lymphatic vessel calls for a renewal of established concepts on the CNS as an immune-privileged site. We also discuss potential approaches to target the glymphatic system to combat neuroinflammation. [ABSTRACT FROM AUTHOR]

Perivascular spaces include a variety of passageways around arterioles, capillaries and venules in the brain, along which a range of substances can move. Although perivascular spaces were first identified over 150 years ago, they have come to prominence recently owing to advances in knowledge of their roles in clearance of interstitial fluid and waste from the brain, particularly during sleep, and in the pathogenesis of small vessel disease, Alzheimer disease and other neurodegenerative and inflammatory disorders. Experimental advances have facilitated in vivo studies of perivascular space function in intact rodent models during wakefulness and sleep, and MRI in humans has enabled perivascular space morphology to be related to cognitive function, vascular risk factors, vascular and neurodegenerative brain lesions, sleep patterns and cerebral haemodynamics. Many questions about perivascular spaces remain, but what is now clear is that normal perivascular space function is important for maintaining brain health. Here, we review perivascular space anatomy, physiology and pathology, particularly as seen with MRI in humans, and consider translation from models to humans to highlight knowns, unknowns, controversies and clinical relevance. [ABSTRACT FROM AUTHOR]

The glymphatic system is a fluid transport network of cerebrospinal fluid (CSF) entering the brain along arterial perivascular spaces, exchanging with interstitial fluid (ISF), ultimately establishing directional clearance of interstitial solutes. CSF transport is facilitated by the expression of aquaporin-4 (AQP4) water channels on the perivascular endfeet of astrocytes. Mice with genetic deletion of AQP4 (AQP4 KO) exhibit abnormalities in the brain structure and molecular water transport. Yet, no studies have systematically examined how these abnormalities in structure and water transport correlate with glymphatic function. Here, we used high-resolution 3D magnetic resonance (MR) non-contrast cisternography, diffusion-weighted MR imaging (MR-DWI) along with intravoxel-incoherent motion (IVIM) DWI, while evaluating glymphatic function using a standard dynamic contrast-enhanced MR imaging to better understand how water transport and glymphatic function is disrupted after genetic deletion of AQP4. AQP4 KO mice had larger interstitial spaces and total brain volumes resulting in higher water content and reduced CSF space volumes, despite similar CSF production rates and vascular density compared to wildtype mice. The larger interstitial fluid volume likely resulted in increased slow but not fast MR diffusion measures and coincided with reduced glymphatic influx. This markedly altered brain fluid transport in AQP4 KO mice may result from a reduction in glymphatic clearance, leading to enlargement and stagnation of fluid in the interstitial space. Overall, diffusion MR is a useful tool to evaluate glymphatic function and may serve as valuable translational biomarker to study glymphatics in human disease. [ABSTRACT FROM AUTHOR]

The choroid plexus is generally agreed to be a key site of CSF production[4], although the relative contributions of choroid plexus in the lateral ventricle versus that in the fourth ventricle are uncertain and may differ between species. We agree that the questions of how much cerebrospinal fluid (CSF) is produced and from which structures remain unanswered. [Extracted from the article]

Recent discoveries of the glymphatic system and of meningeal lymphatic vessels have generated a lot of excitement, along with some degree of skepticism. Here, we summarize the state of the field and point out the gaps of knowledge that should be filled through further research. We discuss the glymphatic system as a system that allows CNS perfusion by the cerebrospinal fluid (CSF) and interstitial fluid (ISF). We also describe the recently characterized meningeal lymphatic vessels and their role in drainage of the brain ISF, CSF, CNS-derived molecules, and immune cells from the CNS and meninges to the peripheral (CNS-draining) lymph nodes. We speculate on the relationship between the two systems and their malfunction that may underlie some neurological diseases. Although much remains to be investigated, these new discoveries have changed our understanding of mechanisms underlying CNS immune privilege and CNS drainage. Future studies should explore the communications between the glymphatic system and meningeal lymphatics in CNS disorders and develop new therapeutic modalities targeting these systems. [ABSTRACT FROM AUTHOR]

The glymphatic system is a recently defined brain-wide paravascular pathway for cerebrospinal fluid (CSF) and interstitial fluid (ISF) exchange that facilitates efficient clearance of solutes and waste from the brain. CSF enters the brain along para-arterial channels to exchange with ISF, which is in turn cleared from the brain along para-venous pathways. Because soluble amyloid β clearance depends on glymphatic pathway function, we proposed that failure of this clearance system contributes to amyloid plaque deposition and Alzheimer's disease progression. Here we provide proof of concept that glymphatic pathway function can be measured using a clinically relevant imaging technique. Dynamic contrast-enhanced MRI was used to visualize CSF-ISF exchange across the rat brain following intrathecal paramagnetic contrast agent administration. Key fea-tures of glymphatic pathway function were confirmed, including visualization of para-arterial CSF influx and molecular size-dependent CSF-ISF exchange. Whole-brain imaging allowed the identification of two key influx nodes at the pituitary and pineal gland recesses, while dynamic MRI permitted the definition of simple kinetic parameters to characterize glymphatic CSF-ISF exchange and solute clearance from the brain. We pro-pose that this MRI approach may provide the basis for a wholly new strategy to evaluate Alzheimer's disease susceptibility and progression in the live human brain. [ABSTRACT FROM AUTHOR]