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Mild traumatic brain injury (mTBI), often referred to as concussion, is a major cause of morbidity and mortality worldwide. Sleep disturbances are common after mTBI. Moreover, subjects who develop subjective sleep complaints after mTBI also report more severe somatic, mental health, and cognitive impairment and take longer to recover from mTBI sequelae. Despite many previous studies addressing the role of sleep in post-mTBI morbidity, the mechanisms linking sleep to recovery after mTBI remain poorly understood. The glymphatic system is a brain-wide network that supports fluid movement through the cerebral parenchyma and the clearance of interstitial solutes and wastes from the brain. Notably, the glymphatic system is active primarily during sleep. Clearance of cellular byproducts related to somatic, mental health, and neurodegenerative processes (e.g., amyloid-β and tau, among others) depends in part on intact glymphatic function, which becomes impaired after mTBI. In this viewpoint, we review the current knowledge regarding the association between sleep disturbances and post-mTBI symptoms. We also discuss the role of glymphatic dysfunction as a potential link between mTBI, sleep disruption, and post-traumatic morbidity. We outline a model where glymphatic dysfunction and sleep disruption caused by mTBI may have an additive effect on waste clearance, leading to cerebral dysfunction and impaired recovery. Finally, we review the novel techniques being developed to examine glymphatic function in humans and explore potential interventions to alter glymphatic exchange that may offer a novel therapeutic approach to those suffering from poor sleep and prolonged symptoms after mTBI.

OBJECTIVECranial neurosurgical procedures can cause changes in brain function. There are many potential explanations, but the effect of simply opening the skull has not been addressed, except for research into syndrome of the trephined. The glymphatic circulation, by which CSF and interstitial fluid circulate through periarterial spaces, brain parenchyma, and perivenous spaces, depends on arterial pulsations to provide the driving force for bulk flow; opening the cranial cavity could dampen this force. The authors hypothesized that a craniectomy, without any other pathological insult, is sufficient to alter brain function due to reduced arterial pulsatility and decreased glymphatic flow. Furthermore, they postulated that glymphatic impairment would produce activation of astrocytes and microglia; with the reestablishment of a closed cranial compartment, the glymphatic impairment, astrocytic/microglial activation, and neurobehavioral decline caused by opening the cranial compartment might be reversed.METHODSUsing two-photon in vivo microscopy, the pulsatility index of cortical vessels was quantified through a thinned murine skull and then again after craniectomy. Glymphatic influx was determined with ex vivo fluorescence microscopy of mice 0, 14, 28, and 56 days following craniectomy or cranioplasty; brain sections were immunohistochemically labeled for GFAP and CD68. Motor and cognitive performance was quantified with rotarod and novel object recognition tests at baseline and 14, 21, and 28 days following craniectomy or cranioplasty.RESULTSPenetrating arterial pulsatility decreased significantly and bilaterally following unilateral craniectomy, producing immediate and chronic impairment of glymphatic CSF influx in the ipsilateral and contralateral brain parenchyma. Craniectomy-related glymphatic dysfunction was associated with an astrocytic and microglial inflammatory response, as well as with the development of motor and cognitive deficits. Recovery of glymphatic flow preceded reduced gliosis and return of normal neurological function, and cranioplasty accelerated this recovery.CONCLUSIONSCraniectomy causes glymphatic dysfunction, gliosis, and changes in neurological function in this murine model of syndrome of the trephined.

Background Understanding molecular transport in the brain is critical to care and prevention of neurological disease and injury. A key question is whether transport occurs primarily by diffusion, or also by convection or dispersion. Dynamic contrast-enhanced (DCE-MRI) experiments have long reported solute transport in the brain that appears to be faster than diffusion alone, but this transport rate has not been quantified to a physically relevant value that can be compared to known diffusive rates of tracers. Methods In this work, DCE-MRI experimental data is analyzed using subject-specific finite-element models to quantify transport in different anatomical regions across the whole mouse brain. The set of regional effective diffusivities ($$D_{eff}$$ D eff ), a transport parameter combining all mechanisms of transport, that best represent the experimental data are determined and compared to apparent diffusivity ($$D_{app}$$ D app ), the known rate of diffusion through brain tissue, to draw conclusions about dominant transport mechanisms in each region. Results In the perivascular regions of major arteries, $$D_{eff}$$ D eff for gadoteridol (550 Da) was over 10,000 times greater than $$D_{app}$$ D app . In the brain tissue, constituting interstitial space and the perivascular space of smaller blood vessels, $$D_{eff}$$ D eff was 10–25 times greater than $$D_{app}$$ D app . Conclusions The analysis concludes that convection is present throughout the brain. Convection is dominant in the perivascular space of major surface and branching arteries (Pe > 1000) and significant to large molecules (> 1 kDa) in the combined interstitial space and perivascular space of smaller vessels (not resolved by DCE-MRI). Importantly, this work supports perivascular convection along penetrating blood vessels.

Background Slowed clearance of amyloid β (Aβ) is believed to underlie the development of Aβ plaques that characterize Alzheimer’s disease (AD). Aβ is cleared in part by the glymphatic system, a brain-wide network of perivascular pathways that supports the exchange of cerebrospinal and brain interstitial fluid. Glymphatic clearance, or perivascular CSF-interstitial fluid exchange, is dependent on the astroglial water channel aquaporin-4 (AQP4) as deletion of Aqp4 in mice slows perivascular exchange, impairs Aβ clearance, and promotes Aβ plaque formation. Methods To define the role of AQP4 in human AD, we evaluated AQP4 expression and localization in a human post mortem case series. We then used the α-syntrophin (Snta1) knockout mouse model which lacks perivascular AQP4 localization to evaluate the effect that loss of perivascular AQP4 localization has on glymphatic CSF tracer distribution. Lastly, we crossed this line into a mouse model of amyloidosis (Tg2576 mice) to evaluate the effect of AQP4 localization on amyloid β levels. Results In the post mortem case series, we observed that the perivascular localization of AQP4 is reduced in frontal cortical gray matter of subjects with AD compared to cognitively intact subjects. This decline in perivascular AQP4 localization was associated with increasing Aβ and neurofibrillary pathological burden, and with cognitive decline prior to dementia onset. In rodent studies, Snta1 gene deletion slowed CSF tracer influx and interstitial tracer efflux from the mouse brain and increased amyloid β levels. Conclusions These findings suggest that the loss of perivascular AQP4 localization may contribute to the development of AD pathology in human populations.

Aquaporin channels facilitate bidirectional water flow in all cells and tissues. AQP4 is highly expressed in astrocytes. In the CNS, it is enriched in astrocyte endfeet, at synapses, and at the glia limitans, where it mediates water exchange across the blood–spinal cord and blood–brain barriers (BSCB/BBB), and controls cell volume, extracellular space volume, and astrocyte migration. Perivascular enrichment of AQP4 at the BSCB/BBB suggests a role in glymphatic function. Recently, we have demonstrated that AQP4 localization is also dynamically regulated at the subcellular level, affecting membrane water permeability. Ageing, cerebrovascular disease, traumatic CNS injury, and sleep disruption are established and emerging risk factors in developing neurodegeneration, and in animal models of each, impairment of glymphatic function is associated with changes in perivascular AQP4 localization. CNS oedema is caused by passive water influx through AQP4 in response to osmotic imbalances. We have demonstrated that reducing dynamic relocalization of AQP4 to the BSCB/BBB reduces CNS oedema and accelerates functional recovery in rodent models. Given the difficulties in developing pore-blocking AQP4 inhibitors, targeting AQP4 subcellular localization opens up new treatment avenues for CNS oedema, neurovascular and neurodegenerative diseases, and provides a framework to address fundamental questions about water homeostasis in health and disease.

Humans are exposed to extreme environmental stressors during spaceflight and return with alterations in brain structure and shifts in intracranial fluids. To date, no studies have evaluated the effects of spaceflight on perivascular spaces (PVSs) within the brain, which are believed to facilitate fluid drainage and brain homeostasis. Here, we examined how the number and morphology of magnetic resonance imaging (MRI)-visible PVSs are affected by spaceflight, including prior spaceflight experience. Fifteen astronauts underwent six T

Impaired clearance of perivascular waste in the brain may play a critical role in morbidity after mild traumatic brain injury (mTBI). We aimed to determine the effect of mTBI on the burden of magnetic resonance imaging (MRI)-visible perivascular spaces (PVSs) in a cohort of U.S. military veterans and whether sleep modulates this effect. We also investigated the correlation between PVS burden and severity of persistent post-concussive symptoms. Fifty-six Iraq/Afghanistan veterans received 3 Tesla MRI as part of a prospective cohort study on military blast mTBI. White matter PVS burden (i.e., number and volume) was calculated using an established automated segmentation algorithm. Multi-variate regression was used to establish the association between mTBIs sustained in the military and PVS burden. Covariates included age, blood pressure, number of impact mTBIs outside the military, and blast exposures. Correlation coefficients were calculated between PVS burden and severity of persistent post-concussive symptoms. There was a significant positive relationship between the number of mTBIs sustained in the military and both PVS number and volume (p = 0.04). A significant interaction was found between mTBI and poor sleep on PVS volume (p = 0.04). A correlation was found between PVS number and volume, as well as severity of postconcussive symptoms (p = 0.03). Further analysis revealed a moderate correlation between PVS number and volume, as well as balance problems (p

Despite the recent description of meningeal lymphatic vessels draining solutes from the brain interstitium and cerebrospinal fluid (CSF), the physiological factors governing cranial lymphatic efflux remain largely unexplored. In agreement with recent findings, cervical lymphatic drainage of 70 kD and 2000 kD fluorescent tracers injected into the adult mouse cortex was significantly impaired in the anesthetized compared to waking animals (tracer distribution across 2.1 ± 4.5% and 23.7 ± 15.8% of deep cervical lymph nodes, respectively); however, free-breathing anesthetized mice were markedly hypercapnic and acidemic (paCO2 = 64 ± 8 mmHg; pH = 7.22 ± 0.05). Mechanical ventilation normalized arterial blood gases in anesthetized animals, and rescued lymphatic efflux of interstitial solutes in anesthetized mice. Experimental hypercapnia blocked cervical lymphatic efflux of intraparenchymal tracers. When tracers were injected into the subarachnoid CSF compartment, glymphatic influx into brain tissue was virtually abolished by hypercapnia, while lymphatic drainage was not appreciably altered. These findings demonstrate that cervical lymphatic drainage of interstitial solutes is, in part, regulated by upstream changes in glymphatic CSF-interstitial fluid exchange. Further, they suggest that maintaining physiological blood gas values in studies of glymphatic exchange and meningeal lymphatic drainage may be critical to defining the physiological regulation of these processes.

Large vessel disease and carotid stenosis are key mechanisms contributing to vascular cognitive impairment (VCI) and dementia. Our previous work, and that of others, using rodent models, demonstrated that bilateral common carotid stenosis (BCAS) leads to cognitive impairment via gradual deterioration of the glial-vascular unit and accumulation of amyloid-β (Aβ) protein. Since brain-wide drainage pathways (glymphatic) for waste clearance, including Aβ removal, have been implicated in the pathophysiology of VCI via glial mechanisms, we hypothesized that glymphatic function would be impaired in a BCAS model and exacerbated in the presence of Aβ. Male wild- type and Tg-SwDI (model of microvascular amyloid) mice were subjected to BCAS or sham surgery which led to a reduction in cerebral perfusion and impaired spatial learning and memory. After 3 months survival, glymphatic function was evaluated by cerebrospinal fluid (CSF) fluorescent tracer influx. We demonstrated that BCAS caused a marked regional reduction of CSF tracer influx in the dorsolateral cortex and CA1-DG molecular layer. In parallel to these changes increased reactive astrogliosis was observed post-BCAS. To further investigate the mechanisms that may lead to these changes, we measured the pulsation of cortical vessels. BCAS impaired vascular pulsation in pial arteries in WT and Tg-SwDI mice. Since our findings show that BCAS may influence VCI by impaired glymphatic drainage and reduced vascular pulsation we propose that these additional targets need to be considered when treating VCI.

Idiopathic intracranial hypertension is a brain disease incorporating cerebrospinal fluid disturbance, increased intracranial pressure and visual failure, but with unknown cause. This study examined a hypothesis that glymphatic function is impaired in idiopathic intracranial hypertension patients. The MRI contrast agent gadobutrol was utilized as a cerebrospinal fluid tracer following intrathecal administration. Consecutive standardized T1 MRI acquisitions over 48 h were done to assess tracer distribution within brain of 15 idiopathic intracranial hypertension patients and 15 reference individuals who were comparable in age and gender distribution. Using FreeSurfer software, we semi-quantified tracer level in multiple brain regions as T1 MRI signal change. The tracer enriched the entire brain of idiopathic intracranial hypertension and reference subjects. In idiopathic intracranial hypertension, tracer enrichment was increased and clearance of tracer delayed from a wide range of brain regions, including both grey and white matter. Differences were most evident in frontal and temporal regions. The pulsatile intracranial pressure was measured overnight and tracer propagation in brain compared between individuals with pathological and normal pulsatile intracranial pressure. In individuals with pathological pulsatile intracranial pressure, tracer enrichment was stronger and clearance from brain delayed, particularly in regions nearby large artery trunks at the brain surface. The present in vivo observations provide evidence for impaired glymphatic function in several brain regions of idiopathic intracranial hypertension patients. Glymphatic failure may imply altered clearance of metabolic byproducts, which may precede neurodegeneration. Further studies are needed to characterize glymphatic failure in idiopathic intracranial hypertension., Intrathecal contrast-enhanced MRI provided evidence for impaired glymphatic function in many brain regions, including both grey and white matter, of patients with idiopathic intracranial hypertension, a brain disease characterized by CSF disturbance and increased intracranial pressure of unknown cause., Graphical Abstract Graphical Abstract

The glymphatic system is a recently defined brain-wide network of perivascular spaces along which cerebrospinal fluid (CSF) and interstitial solutes exchange. Astrocyte endfeet encircling the perivascular space form a physical barrier in between these two compartments, and fluid and solutes that are not taken up by astrocytes move out of the perivascular space through the junctions in between astrocyte endfeet. However, little is known about the anatomical structure and the physiological roles of the astrocyte endfeet in regulating the local perivascular exchange. Here, visualizing astrocyte endfoot-endfoot junctions with immunofluorescent labeling against the protein megalencephalic leukoencephalopathy with subcortical cysts-1 (MLC1), we characterized endfoot dimensions along the mouse cerebrovascular tree. We observed marked heterogeneity in endfoot dimensions along vessels of different sizes, and of different types. Specifically, endfoot size was positively correlated with the vessel diameters, with large vessel segments surrounded by large endfeet and small vessel segments surrounded by small endfeet. This association was most pronounced along arterial, rather than venous segments. Computational modeling simulating vascular trees with uniform or varying endfeet dimensions demonstrates that varying endfoot dimensions maintain near constant perivascular-interstitial flux despite correspondingly declining perivascular pressures along the cerebrovascular tree through the cortical depth. These results describe a novel anatomical feature of perivascular astroglial endfeet and suggest that endfoot heterogeneity may be an evolutionary adaptation to maintain perivascular CSF-interstitial fluid exchange through deep brain structures.

The cerebral vasculature serves as the crossroads of the CNS, supporting exchange of nutrients, metabolic wastes, solutes and cells between the compartments of the brain, including the blood, brain interstitium, and cerebrospinal fluid (CSF). The blood-brain barrier (BBB) regulates the entry and efflux of molecules into brain tissue. The cells of the neurovascular unit regulate cerebral blood flow, matching local metabolic demand to blood supply. The blood-CSF barrier at the choroid plexus secretes CSF, which supports the brain and provides a sink for interstitial solutes not cleared across the BBB. Recent studies have characterized the glymphatic system, a brain-wide network of perivascular spaces that supports CSF and interstitial fluid exchange and the clearance of interstitial solutes to the CSF. The critical role that these structures play in maintaining brain homeostasis is illustrated by the established and emerging roles that their dysfunctions play in the development of neurodegenerative diseases, such as Alzheimer's disease (AD). Loss of BBB and blood-CSF barrier function is reported both in rodent models of AD, and in human AD subjects. Cerebrovascular dysfunction and ischemic injury are well established contributors to both vascular dementia and to a large proportion of cases of sporadic AD. In animal models, the slowed glymphatic clearance of interstitial proteins, such as amyloid β or tau, are proposed to contribute to the development of neurodegenerative diseases, including AD. In total, these findings suggest that cellular and molecular changes occurring within and around the cerebral vasculature are among the key drivers of neurodegenerative disease pathogenesis.

The cerebral vasculature serves as the crossroads of the CNS, supporting exchange of nutrients, metabolic wastes, solutes and cells between the compartments of the brain, including the blood, brain interstitium, and cerebrospinal fluid (CSF). The blood-brain barrier (BBB) regulates the entry and efflux of molecules into brain tissue. The cells of the neurovascular unit regulate cerebral blood flow, matching local metabolic demand to blood supply. The blood-CSF barrier at the choroid plexus secretes CSF, which supports the brain and provides a sink for interstitial solutes not cleared across the BBB. Recent studies have characterized the glymphatic system, a brain-wide network of perivascular spaces that supports CSF and interstitial fluid exchange and the clearance of interstitial solutes to the CSF. The critical role that these structures play in maintaining brain homeostasis is illustrated by the established and emerging roles that their dysfunctions play in the development of neurodegenerative diseases, such as Alzheimer's disease (AD). Loss of BBB and blood-CSF barrier function is reported both in rodent models of AD, and in human AD subjects. Cerebrovascular dysfunction and ischemic injury are well established contributors to both vascular dementia and to a large proportion of cases of sporadic AD. In animal models, the slowed glymphatic clearance of interstitial proteins, such as amyloid β or tau, are proposed to contribute to the development of neurodegenerative diseases, including AD. In total, these findings suggest that cellular and molecular changes occurring within and around the cerebral vasculature are among the key drivers of neurodegenerative disease pathogenesis.

Waste clearance from the brain parenchyma occurs along perivascular pathways. Enlargement of the perivascular space (ePVS) is associated with pathologic features of Alzheimer's disease (AD), although the mechanisms and implications of this dilation are unclear. Fluid exchange along the cerebral vasculature is dependent on the perivascular astrocytic water channel aquaporin-4 (AQP4) and loss of perivascular AQP4 localization is found in AD. We directly measured ePVS in postmortem samples of pathologically characterized tissue from participants who were cognitively intact or had AD or mixed dementia (vascular lesions with AD). We found that both AD and mixed dementia groups had significantly increased ePVS compared to cognitively intact subjects. In addition, we found increased global AQP4 expression of the AD group over both control and mixed dementia groups and a qualitative reduction in perivascular localization of AQP4 in the AD group. Among these cases, increasing ePVS burden was associated with the presence of tau and amyloid-β pathology. These findings are consistent with the existing evidence of ePVS in AD and provide novel information regarding differences in AD and vascular dementia and the potential role of astroglial pathology in ePVS.

Introduction The glymphatic system is a brain-wide perivascular network that facilitates clearance of proteins, including amyloid β, from the brain interstitium through the perivascular exchange of cerebrospinal fluid and interstitial fluid. The astrocytic water channel aquaporin-4 (AQP4) is required for glymphatic system function, and impairment of glymphatic function in the aging brain is associated with altered AQP4 expression and localization. In human cortical tissue, alterations in AQP4 expression and localization are associated with Alzheimer's disease (AD) status and pathology. Although this suggests a potential role for AQP4 in the development or progression of AD, the relationship between of naturally occurring variants in the human AQP4 gene and cognitive function has not yet been evaluated. Methods Using data from several longitudinal aging cohorts, we investigated the association between five AQP4 single-nucleotide polymorphisms (SNPs) and the rate of cognitive decline in participants with a diagnosis of AD. Results None of the five SNPs were associated with different rates of AD diagnosis, age of dementia onset in trial subjects. No association between AQP4 SNPs with histological measures of AD pathology, including Braak stage or neuritic plaque density was observed. However, AQP4 SNPs were associated with altered rates of cognitive decline after AD diagnosis, with two SNPS (rs9951307 and rs3875089) associated with slower cognitive decline and two (rs3763040 and rs3763043) associated with more rapid cognitive decline after AD diagnosis. Discussion These results provide the first evidence that variations in the AQP4 gene, whose gene product AQP4 is vital for glymphatic pathway function, may modulate the progression of cognitive decline in AD.

Traumatic brain injury (TBI) is a major public health concern in the USA and worldwide. Sleep disruption and headaches are two of the most common problems reported by patients after TBI. In this manuscript, we review the current knowledge regarding the relation between post-traumatic sleep disruption and headaches. We also describe the role of the glymphatic system as a potential link between TBI, sleep, and headaches. Recent studies show a reciprocal relation between post-traumatic sleep disruption and headaches: patients with sleep disruption after TBI report more headaches, and post-traumatic headaches are a risk factor for developing disrupted sleep. Despite this clinical association, the exact mechanisms linking post-traumatic sleep disruption and headaches are not well understood. The glymphatic pathway, a newly described brain–wide network of perivascular spaces that supports the clearance of interstitial solutes and wastes from the brain, is active primarily during sleep, and becomes dysfunctional after TBI. We propose a model where changes in glymphatic function caused by TBI and post-traumatic sleep disruption may impair the clearance of neuropeptides involved in the pathogenesis of post-traumatic headaches, such as CGRP. The relation between TBI, post-traumatic sleep disruption, and post-traumatic headaches, although well documented in the literature, remains poorly understood. Dysfunction of the glymphatic system caused by TBI offers a novel and exiting explanation to this clinically observed phenomenon. The proposed model, although theoretical, could provide important mechanistic insights to the TBI-sleep-headache association.

The deposition of misfolded proteins, including amyloid beta plaques and neurofibrillary tangles is the histopathological hallmark of Alzheimer’s disease (AD). The glymphatic system, a brain-wide network of perivascular pathways that supports interstitial solute clearance, is dependent upon expression of the perivascular astroglial water channel aquaporin-4 (AQP4). Impairment of glymphatic function in the aging rodent brain is associated with reduced perivascular AQP4 localization, and in human subjects, reduced perivascular AQP4 localization is associated with AD diagnosis and pathology. Using human transcriptomic data, we demonstrate that expression of perivascular astroglial gene products dystroglycan (DAG1), dystrobrevin (DTNA) and alpha-syntrophin (SNTA1), are associated with dementia status and phosphorylated tau (P-tau) levels in temporal cortex. Gene correlation analysis reveals altered expression of a cluster of potential astrocytic endfoot components in human subjects with dementia, with increased expression associated with temporal cortical P-tau levels. The association between perivascular astroglial gene products, including DTNA and megalencephalic leukoencephalopathy with subcortical cysts 1 (MLC1) with AD status was confirmed in a second human transcriptomic dataset and in human autopsy tissue by Western blot. This suggests changes in the astroglial endfoot domain may underlie vulnerability to protein aggregation in AD.

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.

The glymphatic system is a brain-wide clearance pathway; its impairment contributes to the accumulation of amyloid-β. Influx of cerebrospinal fluid (CSF) depends upon the expression and perivascular localization of the astroglial water channel aquaporin-4 (AQP4). Prompted by a recent failure to find an effect of Aqp4 knock-out (KO) on CSF and interstitial fluid (ISF) tracer transport, five groups re-examined the importance of AQP4 in glymphatic transport. We concur that CSF influx is higher in wild-type mice than in four different Aqp4 KO lines and in one line that lacks perivascular AQP4 (Snta1 KO). Meta-analysis of all studies demonstrated a significant decrease in tracer transport in KO mice and rats compared to controls. Meta-regression indicated that anesthesia, age, and tracer delivery explain the opposing results. We also report that intrastriatal injections suppress glymphatic function. This validates the role of AQP4 and shows that glymphatic studies must avoid the use of invasive procedures.

Astrocytes play a critical role in regulating the interface between the cerebral vasculature and the central nervous system. Contributing to this is the astrocytic endfoot domain, a specialized structure that ensheathes the entirety of the vasculature and mediates signaling between endothelial cells, pericytes and neurons. The astrocytic endfoot has been implicated as a critical element of the glymphatic pathway and changes in protein expression profiles in this cellular domain are linked to Alzheimer’s disease pathology. Despite this, basic physiological properties of this structure remain poorly understood including the developmental timing of its formation, and the protein components that localize there to mediate its functions. Here we use human transcriptome data from male and female subjects across several developmental stages and brain regions to characterize the gene expression profile of the dystrophin associated complex (DAC), a known structural component of the astrocytic endfoot that supports perivascular localization of the astroglial water channel aquaporin-4 (AQP4). Transcriptomic profiling is also used to define genes exhibiting parallel expression profiles to DAC elements, generating a pool of candidate genes that encode gene products that may contribute to the physiological function of the perivascular astrocytic endfoot domain. We found that several genes encoding transporter proteins are transcriptionally associated with DAC genes.

Amyloid-β (Aβ) plaques are a key histopathological hallmark of Alzheimer's disease (AD), and soluble Aβ species are believed to play an important role in the clinical development of this disease. Emerging biomarker data demonstrate that Aβ plaque deposition begins decades before the onset of clinical symptoms, suggesting that understanding the biological determinants of the earliest steps in the development of AD pathology may provide key opportunities for AD treatment and prevention. Although a clinical association between sleep disruption and AD has long been appreciated, emerging clinical studies and insights from the basic neurosciences have shed important new light on how sleep and Aβ homeostasis may be connected in the setting of AD. Aβ, like many interstitial solutes, is cleared in part through the exchange of brain interstitial fluid and cerebrospinal fluid along a brain-wide network of perivascular pathways recently termed the glymphatic system. Glymphatic function is primarily a feature of the sleeping brain, rather than the waking brain, and is slowed in the aging and posttraumatic brain. These changes may underlie the diurnal fluctuations in interstitial and cerebrospinal fluid Aβ levels observed in both the rodent and the human. These and other emerging studies suggest that age-related sleep disruption may be one key factor that renders the aging brain vulnerable to Aβ deposition and the development of AD. If this is true, sleep may represent a key modifiable risk factor or therapeutic target in the preclinical phases of AD.

This chapter reviews the basis of brain interstitial fluid homeostasis and discusses the roles of the glymphatic system in the clearance of fluid and solutes. The anatomical organization of the glymphatic system and its drainage into cervical and meningeal lymph vessels, as well as the role of astrocytes, the perivascular space, and the sleep–wake cycle in the regulation of interstitial space and bulk flow, are described. The existence of the glymphatic system opens up new ways to understand the mechanisms underlying brain fluid dynamics and acute edema formation following stroke. Influx of cerebrospinal fluid along the perivascular spaces surrounding cerebral arteries and failure of ischemic cells to clear excess interstitial fluid, as well as perivasculitis, likely contribute to cytotoxic and vasogenic brain edema. As such, diagnostic imaging or manipulation of the glymphatic pathway may unlock novel therapeutic opportunities for cerebrovascular disease.

Objective: In the brain, protein waste removal is partly performed by paravascular pathways that facilitate convective exchange of water and soluble contents between cerebrospinal fluid (CSF) and interstitial fluid (ISF). Several lines of evidence suggest that bulk flow drainage via the glymphatic system is driven by cerebrovascular pulsation, and is dependent on astroglial water channels that line paravascular CSF pathways. The objective of this study was to evaluate whether the efficiency of CSF–ISF exchange and interstitial solute clearance is impaired in the aging brain. Methods: CSF–ISF exchange was evaluated by in vivo and ex vivo fluorescence microscopy and interstitial solute clearance was evaluated by radiotracer clearance assays in young (2–3 months), middle-aged (10–12 months), and old (18–20 months) wild-type mice. The relationship between age-related changes in the expression of the astrocytic water channel aquaporin-4 (AQP4) and changes in glymphatic pathway function was evaluated by immunofluorescence. Results: Advancing age was associated with a dramatic decline in the efficiency of exchange between the subarachnoid CSF and the brain parenchyma. Relative to the young, clearance of intraparenchymally injected amyloid-b was impaired by 40% in the old mice. A 27% reduction in the vessel wall pulsatility of intracortical arterioles and widespread loss of perivascular AQP4 polarization along the penetrating arteries accompanied the decline in CSF–ISF exchange. Interpretation: We propose that impaired glymphatic clearance contributes to cognitive decline among the elderly and may represent a novel therapeutic target for the treatment of neurodegenerative diseases associated with accumulation of misfolded protein aggregates. ANN NEUROL 2014;76:845–861

Importance Cognitive impairment and dementia, including Alzheimer disease (AD), are common within the aging population, yet the factors that render the aging brain vulnerable to these processes are unknown. Perivascular localization of aquaporin-4 (AQP4) facilitates the clearance of interstitial solutes, including amyloid-β, through the brainwide network of perivascular pathways termed the glymphatic system , which may be compromised in the aging brain. Objectives To determine whether alterations in AQP4 expression or loss of perivascular AQP4 localization are features of the aging human brain and to define their association with AD pathology. Design, Setting, and Participants Expression of AQP4 was analyzed in postmortem frontal cortex of cognitively healthy and histopathologically confirmed individuals with AD by Western blot or immunofluorescence for AQP4, amyloid-β 1-42, and glial fibrillary acidic protein. Postmortem tissue and clinical data were provided by the Oregon Health and Science University Layton Aging and Alzheimer Disease Center and Oregon Brain Bank. Postmortem tissue from 79 individuals was evaluated, including cognitively intact “young” individuals aged younger than 60 years (range, 33-57 years), cognitively intact “aged” individuals aged older than 60 years (range, 61-96 years) with no known neurological disease, and individuals older than 60 years (range, 61-105 years) of age with a clinical history of AD confirmed by histopathological evaluation. Forty-eight patient samples (10 young, 20 aged, and 18 with AD) underwent histological analysis. Sixty patient samples underwent Western blot analysis (15 young, 24 aged, and 21 with AD). Main Outcomes and Measures Expression of AQP4 protein, AQP4 immunoreactivity, and perivascular AQP4 localization in the frontal cortex were evaluated. Results Expression of AQP4 was associated with advancing age among all individuals ( R2 = 0.17; P = .003). Perivascular AQP4 localization was significantly associated with AD status independent of age (OR, 11.7 per 10% increase in localization; z = −2.89; P = .004) and was preserved among eldest individuals older than 85 years of age who remained cognitively intact. When controlling for age, loss of perivascular AQP4 localization was associated with increased amyloid-β burden ( R2 = 0.15; P = .003) and increasing Braak stage ( R2 = 0.14; P = .006). Conclusions and Relevance In this study, altered AQP4 expression was associated with aging brains. Loss of perivascular AQP4 localization may be a factor that renders the aging brain vulnerable to the misaggregation of proteins, such as amyloid-β, in neurodegenerative conditions such as AD.

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.

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 usein vivotwo-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 usingin vivotwo-photon andex vivofluorescence 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.

Taking Out the Trash The purpose of sleep remains mysterious. Using state-of-the-art in vivo two-photon imaging to directly compare two arousal states in the same mouse, Xie et al. (p. 373 ; see the Perspective by Herculano-Houzel ) found that metabolic waste products of neural activity were cleared out of the sleeping brain at a faster rate than during the awake state. This finding suggests a mechanistic explanation for how sleep serves a restorative function, in addition to its well-described effects on memory consolidation.

The brain is unique among virtually all somatic organs in its lack of a conventional lymphatic vasculature.1–3 In the periphery, the lymphatic circulation facilitates the clearance of extracellular proteins and excess fluid from the interstitium, a role critical to tissue homeostasis and function.4,5 Yet within the brain, despite its complex architecture and high metabolic activity and neural cells’ sensitivity to changes in the extracellular environment, no specialized organ-wide anatomic structure has yet been identified that facilitates the efficient lymphatic clearance of extracellular solutes and fluid from the brain parenchyma. For small molecules and hydrophobic compounds, efflux across the blood–brain barrier is relatively unrestricted. Molecules that are substrates for specific blood–brain barrier transporters are also readily cleared from the brain.6,7 Other compounds must be cleared from the brain interstitium to the cerebrospinal fluid (CSF) compartment, where they are ultimately eliminated to the blood stream via arachnoid granulations or to peripheral lymphatics along cranial nerves.1,8,9 However, the distances between much of the brain tissue and the CSF compartments are too great for efficient clearance by simple diffusion, particularly for large molecules (such as peptides and proteins) with low diffusion coefficients.6 Rather, the clearance of these interstitial solutes from the brain is attributed to bulk flow, by which convective currents of interstitial fluid (ISF) sweep solutes along at a high rate that is largely independent of molecular size.1,2,6,7 In a controversial series of studies, Grady et al10,11 suggested that brain ISF may exchange with CSF along paravascular routes surrounding cerebral blood vessels. Because these findings seemed to be subsequently refuted by Cserr et al,12,13 such retrograde movement of CSF into the brain parenchyma is now thought to …

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 features 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 propose 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.

Because it lacks a lymphatic circulation, the brain must clear extracellular proteins by an alternative mechanism. The cerebrospinal fluid (CSF) functions as a sink for brain extracellular solutes, but it is not clear how solutes from the brain interstitium move from the parenchyma to the CSF. We demonstrate that a substantial portion of subarachnoid CSF cycles through the brain interstitial space. On the basis of in vivo two-photon imaging of small fluorescent tracers, we showed that CSF enters the parenchyma along paravascular spaces that surround penetrating arteries and that brain interstitial fluid is cleared along paravenous drainage pathways. Animals lacking the water channel aquaporin-4 (AQP4) in astrocytes exhibit slowed CSF influx through this system and a ~70% reduction in interstitial solute clearance, suggesting that the bulk fluid flow between these anatomical influx and efflux routes is supported by astrocytic water transport. Fluorescent-tagged amyloid β, a peptide thought to be pathogenic in Alzheimer's disease, was transported along this route, and deletion of the Aqp4 gene suppressed the clearance of soluble amyloid β, suggesting that this pathway may remove amyloid β from the central nervous system. Clearance through paravenous flow may also regulate extracellular levels of proteins involved with neurodegenerative conditions, its impairment perhaps contributing to the mis-accumulation of soluble proteins.

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.