A clinical primer for the glymphatic system

Abstract

The complex and dynamic system of fluid flow through the perivascular and interstitial spaces of the CNS has new-found implications for neurological diseases. CSF movement throughout the CNS parenchyma is more dynamic than could be explained via passive diffusion mechanisms alone. Indeed, a semistructured glial-lymphatic (glymphatic) system of astrocyte-supported extracellular perivascular channels serves to directionally channel extracellular fluid, clearing metabolites and peptides to optimize neurological function. Clinical studies of the glymphatic network have to date proven challenging, with most data gleaned from rodent models and post-mortem investigations. However, increasing evidence suggests that disordered glymphatic function contributes to the pathophysiology of CNS ageing, neurodegenerative disease and CNS injuries, as well as normal pressure hydrocephalus. Unlocking such pathophysiology could provide important avenues towards novel therapeutics. We here provide a multidisciplinary overview of glymphatics and critically review accumulating evidence regarding its structure, function and hypothesized relevance to neurological disease. We highlight emerging technologies of relevance to the longitudinal evaluation of glymphatic function in health and disease. Finally, we discuss the translational opportunities and challenges of studying glymphatic science.

glymphatic system, beta-amyloid, idiopathic normal pressure hydrocephalus, Alzheimer’s disease, aging

Introduction

The general lymphatic system is a well-described mechanism observed throughout the body, which subserves the core physiological functions of extravascular solute clearance and extracellular fluid circulation that is not removed by venous blood flow. Historically, lymphatics have been considered to be absent from the CNS, largely due to lack of conventional perivascular channels and associated lymph nodes that are characteristic of lymphatic angioarchitecture in non-CNS tissues. However, contemporary studies in rodent models, as well as emerging evidence in humans, have revealed a critical network of extravascular channels that provide a conduit for the circulation of CSF and interstitial fluid within and through the CNS parenchyma. These almost imperceptibly fine channels between vasculature and adjacent perivascular astrocytes help buffer and circulate metabolites, peptides and extracellular electrolytes across highly cellular regions with dynamic metabolic activity.^1-3

The so-called glia-lymphatic or glymphatic system is thought to exhibit relatively low function during periods of wakefulness and is switched on during slow-wave sleep.^4,5 Interestingly, recent evidence suggests that drainage—or efflux—directly into extracranial lymphatics appears to have a contrasting diurnal pattern, with higher rates during periods of wakefulness, thus is less effective for parenchymal waste removal.⁶ The glymphatic clearance system then facilitates flow into more conventional extracranial lymphatic networks within the surrounding dorsal dura, skull base and cranial nerves, ultimately coalescing in the large venous drainage system of the head and neck.^7–11 Glymphatic dysfunction has been implicated in the pathogenesis of numerous ageing processes and disease-states—including Alzheimer’s disease, idiopathic normal pressure hydrocephalus (iNPH), traumatic brain injury (TBI) and stroke, among others and will serve as the predominant focus of this review.

The existence of a glymphatic system has been theorized since the mid-1970s, since the initial discovery that ligating cervical lymphatic vessels precipitated marked cerebral oedema and inhibited clearance of parenterally delivered carbon molecules, providing early evidence of a lymphatic-type system for ‘waste’ removal from the CNS.^12,13 It was also noted that this previously undefined system was at least partially reliant on arterial pulsations.¹⁴ However, it was not until 2012 when better characterization of the system was achieved by Iliff and colleagues, utilizing in vivo two-photon imaging to observe fluorescent tracer actively circulating in interstitial perivascular spaces and exiting the CNS via perivenous channels.² Importantly, animals lacking the transmembrane water channel aquaporin 4 (AQP4)—normally expressed on perivascular astrocytic endfeet—demonstrated decreased CSF influx into CNS parenchyma and profound reduction in metabolite clearance. These data demonstrated an active role for astrocytic endfeet in bulk pericellular fluid circulation.^1,2,15,16

The glymphatic system model continues to evolve, but according to our contemporary understanding, it is broadly characterized by CSF transit through a two-phase process. In the first, CSF moves from the ventricular system into convexity subarachnoid spaces and ultimately into periarterial channels by bulk-flow—driven by arterial pulsations, inspiratory–expiratory pressure changes and CSF production.⁶ From the glymphatic channels, flow is then facilitated across astrocytic endfeet and via AQP4 into the brain interstitium, where the CSF flux admixes with the brain extracellular fluid, peptides and metabolites, which collectively outflow along perivenous space or cross the dura and clear via meningeal to cervical lymphatics that coalesce via routine pathways into the systemic circulation.^8,9,17

Does the glymphatic system exist in humans?

The glymphatic system, characterized initially in the rodent brain, is theorized to exist and function similarly in humans, with some minor anatomic differences pertinent to the final clearance pathways. One important translational stepping stone is characterizing the glymphatic system in a larger, highly gyrified brain with similar comparable cortical architecture to humans, such as a pig. Surprisingly, the perivascular influx of tracer in parenchymal interstitial spaces is up to four times larger in pig compared to mouse brain (Fig. 1).¹⁸ These findings not only demonstrate that larger, more complex gyrified brains have enhanced glymphatic system function, but begs the question whether more complex brains require a more comprehensive glymphatic system for normal physiology—paving the way for determining whether glymphatic system impairment is an aetiological contributor to human neurological ageing and disease.

Figure 1

Glymphatic transport in perivascular spaces is better developed in highly gyrified large mammals compared to rodents. 3D reconstruction utilizing light sheet microscopy of optically cleared mouse (A) and porcine (C) cortex following CSF tracer injection allowing visualization of perivascular flow. Scale bar = 2 mm. Representative cross-sectional images of mouse (B) and porcine (D) perivascular channels parallel to cortical surfaces. Comparative reconstructive imaging of cortical regions demonstrates pervasive, higher interstitial influx in a larger, more complex and gyrated brain of the pig compared to the mouse. Particularly high CSF tracer influx, as an indicator of glymphatic flow, was identified in the pig hippocampus. In review, light sheet microscopy of optically cleared mammalian brains revealed a highly organized pattern of CSF entry into the brain across multiple species, with greater and more dynamic flow in the pig, suggesting a more complex and important glymphatic system in the gyrencephalic brain, particular in cell-dense areas and high-functioning subcortical regions such as the hippocampus. Brief methodological review: both pigs and mice underwent cisterna magna injections, and both were optically cleared in the same fashion.¹⁸ A fluorescently conjugated tracer was injection at 100 ml/mi for 2–6 h in pigs and 1 ml/min for 30 min in mice. The brain tissue was imaged using an Ultramicroscope II light-sheet microscope.

In the first human study of its kind, Ringstad and colleagues utilized intrathecal gadobutrol—a low molecular weight MRI contrast agent used clinically to diagnosis spontaneous CSF leaks—to perform dynamic MR imaging in healthy subjects and patients with iNPH.^19–21 Findings revealed antegrade flow along leptomeningeal arteries into brain parenchyma prior to clearance with decreased rate of clearance in patients with iNPH, comparable to the findings previously observed in rodents. Interestingly, they also identified a relative gradient flow of CSF that was minimal or absent at the upper brain convexities in patients with iNPH, suggesting reduced CSF resorption at the level of the parasagittal arachnoid granulations connected to the dural lymphatics.²¹ Variations in distinct cortical regions were also observed, which may reflect local differences in cellular density, glial–neuronal complex ratios, distance from ventricular spaces, or AQP4 expression.^21,22

The function of the glymphatic system appears well coordinated, and likely driven by vascular pulsations from the heart cycle and the respiratory cycle, with eventual drainage both directly into the venous system and via the cervical lymph nodes.^2,23–27 Nine subjects without known neurological disease underwent ultra-fast MR encephalography, which allows the simultaneous temporal recording of electrographic and cardiopulmonary pulsations across the entire cortex. Investigators demonstrated that arterial pulsations, respiratory flow and low-frequency pulsatory flow collectively impacted CSF flow into interstitial spaces.²⁵ Conversely, Dreha-Kulaczewski et al.²⁸ reported respiratory inspiration to be the primary driver of solute efflux and CSF flow—largely from transmitted thoracic pressure changes that draw venous blood from cranial and spinal regions. These findings also suggest that body position and other related physiological factors may impact CSF flow and glymphatic system function. Indeed, CSF production rate and anatomic body orientation have been shown to also contribute to CSF dynamics—a particularly interesting finding when considering the interpretation of supine MRI findings to disease states impacting upright bipedal function.^29–31

With these observations in mind, several key questions emerge, including: (i) does CSF flow within the ventricular system and CSF dynamics in the perivascular and interstitial spaces share the same physiology? and (ii) What is the physiological importance of the glymphatic system? It is most likely that the ventricular system is not a simple, macroscopic generalization of the glymphatic system; rather, both systems may serve parallel physiological purposes, mediated by differential clusters of cellular and subcellular mechanisms. In keeping with historical methods of studying neurological function, much of the emerging information regarding glymphatic function in humans has been garnered in the course of clinical evaluation of disease states, such as iNPH, CSF leaks, Alzheimer’s disease and other neurodegenerative or senescent phenomena. Collectively, the findings pertinent to possible glymphatic involvement in these disease states suggest an association between cognitive impairment and impaired glymphatic function, with each disease process highlighting a range of possible mechanistic links.

What is the function of the glymphatic system?

Structurally, the glymphatic system is challenging to conceptualize—particularly in contrast to its peripheral counterpart, which consists of formal vessels and lymph nodes, rather than continually irrigated potential spaces between glia and neurovasculature that constitute the glymphatic system. Yet, the glymphatic system may play an integral role in maintaining CNS function.

Debate continues regarding the mechanistic functionality of glymphatics in both rodents and humans. Evidence from murine studies suggests glymphatic transport of bulk CSF flow from the subarachnoid and perivascular spaces across the glial basement membrane into the pericellular interstitial compartment, facilitated by the water channel AQP4 on astrocytic endfeet.² It is in this interstitial compartment where CSF flow encounters and transports metabolite waste and basal interstitial fluid, effluxing through perivenous channels into larger cerebral venous structures before ultimately returning the solutes back into systemic circulation.^2,9,17,32

In addition to local intraparenchymal clearance, there are recent discoveries about macrocellular CNS clearance of fluid and metabolites related to lymphatic pathways. The recirculation and efflux of CSF has numerous distinct pathways within the CNS; however, emerging evidence suggest meningeal lymphatic routes may play a key role in broader interstitial fluid and waste product clearance, as well as providing likely roles in immune surveillance.^7,9,33 These lymphatic vessels are located throughout the skull base and cerebral convexity dura, such as adjacent to the superior sagittal sinus, and have distinct structure differences from their peripheral counterparts—including having a less-ramified structure and predominantly filtering into venous sinus structures and deep cervical lymph chains.^7,9,34 Anterior CNS drainage occurs robustly along the olfactory/cribriform subfrontal and nasal mucosa pathways, which feed CSF and metabolites mainly into cervical lymph nodes (Fig. 2).^{12–14,35,36} Posterior and superior region lymphatics converge into the dorsal dural and skull base lymph channels, often running paired with cranial nerves or major dural sinuses, which then fuse and exit via venous sinuses and the internal jugular veins (Fig. 2). These meningeal channels predominantly function more as traditional lymphatic systems providing immune surveillance, in addition to metabolite clearance—particularly larger macro, and possible lipophobic, compounds. Emerging evidence suggests these glymphatic and meningeal lymphatic structures work in tandem, providing both distinct and shared functions; however, the role of immune surveillance, the precise dural/tissue location and the haemodynamic considerations remain poorly understand, with even less known about any possible associate with meningeal lymphatics in neurological disease, although drainage does decline with ageing in mice and the dural lymphatic vessels become atrophic.^27,37–39

Figure 2

CSF and interstitial fluid outflow. The outflow paravenous interstitial fluid and waste metabolites exiting the cerebral cortex drains into larger CSF spaces, such as the subarachnoid space and cranial nerve sheaths. Several major pathways may then facilitate further clearance, including anterior pathways along the olfactory/cribriform regions through to the nasal mucosa and into cervical lymphatics; or posteriorly through dorsal dural lymphatics or skull base cranial nerve foramina into the internal jugular venous outflow system.

Biomechanical and biophysical signals play significant roles in brain development and neuronal wiring, cognitive function and circuitry modulation and circulation of blood and interstitial fluid, with clear links to the glymphatic system and its components. Indeed, the mechanical sensitivity of excitatory neurotransmitter receptors is hypothesized to play a role in the normal physiology of fluid flow in the glymphatic system, and may be associated with dysfunction following traumatic injury.⁴⁰ Moreover, amyloid peptides have been shown to increase resting stress in F-actin and modulate mechanosensitive channels, as well as cause pericyte contraction, possibly creating a negative feedback loop once glymphatic clearance of amyloid is impaired.^41,42

Many neurological disorders are characterized by the pathological accumulation of abnormal proteins. Impairments in the function of the glymphatic system due to ageing, disruption of physiologica; processes—such as sleep, or from neurologic disease causes—may permit this accumulation (Fig. 3).⁴³ Disrupted homeostatic clearance of solutes such as amyloid-β, phosphorylated-tau and lactate can contribute to the pathophysiology of neurodegenerative disorders and to the sequelae of vascular and traumatic brain diseases (Table 1).^1,30,44–46 As has been so commonly done in the history of neuroscience research, we will review distinct disease processes as a mechanism for better understanding the glymphatic system and the role it plays in human neurological function.

Figure 3

Illustrative review of the glymphatic system during normal and abnormal function. (A) Under healthy conditions, CSF flows into the brain cortex through peri-arterial vascular channels bordered by astrocytic endfeet. AQP4 regulates fluid dispersion into the interstitial spaces, where it collects various peptides, metabolites and neurotransmitters before recycling through perivenous channels and ultimately into systemic circulation. (B) In diseased states, AQP4 often becomes dysregulated and a lack of interstitial flow leads to accumulation of peptides and metabolites.

Table 1

Review of glymphatic preclinical and clinical associations

	AQP4 effect	Molecular and cellular effects	Glymphatic impact in humans
Neurodegenerative
Alzheimer’s disease	Knockout mice exhibit diminished perivascular spaces. Loss of AQP4 polarization	Impairment in AQP4 increases amyloid-β accumulation, worsens cognitive function	Aberrant AQP4 polarization correlates with age-related cognitive decline and associated increased amyloid plaque burden. In mice, impaired meningeal lymphatic drainage leads to elevated amyloid-β levels and negatively impacts learning and memory
Parkinson’s disease	Aberrant AQP4 expression in the substantia nigra linked with pro-inflammatory role	AQP4 dysfunction associated with elevated levels of α-synuclein, oedema and cell loss within the substantia nigra	Reduced perivascular spaces, impaired flow—correlates with disease severity. Dural lymphatic flow dysfunction correlated with disease burden in typical vs atypical disease
iNPH	Brain biopsies in human age-matched controls demonstrated reduced perivascular AQP4 expression and impaired polarization	Increased amyloid-β plaques and neurofibrillary tangles, although to a lesser degree than Alzheimer’s disease	Severely delayed flow through sylvian/precentral sulci and impaired periarterial flow along cortical surfaces. Delayed clearance in entorhinal cortex, correlating with cognitive impairments
Acute injury/vascular
TBI	Acute and chronic AQP4 mislocalization, loss of polarization	Elevated amyloid-β levels, may improve over time; altered fluid dynamics and increased cerebral oedema	Known sleep and cognitive impairments, no formal glymphatic association. In rats, mild repetitive TBI leads to reduced glymphatic function in the hypothalamus, amygdala, hippocampus and olfactory regions, with associated cognitive and gait impairments
Ischaemic stroke	AQP4 mislocalization contributes to cerebral oedema	Increased amyloid plaque burden within the ischaemic region	Unknown. In mice leads to impaired function and associated cerebral oedema
Aneurysmal subarachnoid haemorrhage	Acute AQP4 upregulation with loss of polarization	Elevated perivascular fibrin	Unknown. Profoundly reduced function in mice within 24 h, not improved with craniotomy/pressure reduction, improved with fibrinolysis. Severe impairments in non-human primate model, particularly in the ipsilateral hemisphere
Craniotomy/neurosurgery	Dysregulated AQP4 levels and loss of polarization	Elevated astrocytic and microglial reactivity	Unknown. In mice, unilateral craniectomy reduces arterial pulsatility and reduces glymphatic circulation in the acute and chronic periods for both ipsilateral and contralateral hemispheres, with associated cognitive and motor impairments
Environmental/other
Ageing	Disrupted AQP4 expression, altered polarization	amyloid-β accumulation; stiffened arterial vessels	Aged brain demonstrates impairment function in both glymphatic circulation and meningeal lymphatic outflow
Sleep disorders	Knockout mice demonstrated an elevated inflammatory state, accumulation of amyloid and tau, and synaptic unit destruction	Sleep deprivation leads to increased amyloid-β and tau burden in mice. Clearance of amyloid is twofold higher in asleep vs awake mice	Sleep deprivation leads to increased amyloid-β burden in humans, including an increase after a single night of a disrupted sleep cycle (PET)
Space travel/microgravity	Altered expression during spaceflight in mice	Elevated amyloid levels, perivascular inflammation	Unknown direct effects, but humans experience vision and cognition impairments similar to NPH/Alzheimer’s disease. Similarly, mice develop elevated amyloid plaque burdens, perivascular inflammation and accelerated cognitive impairment
Alcohol	Increased AQP4 mislocalization with chronic use	Increased gliosis, speculated to have impaired amyloid-β clearance	Unknown, but elevated gliosis likely impairs function. In mice, low ethanol doses temporarily enhance glymphatic function; however, chronic heavy use impairs function

Glymphatics and sleep

The homeostatic functions of glymphatic clearance appear to fluctuate diurnally, with pronounced enhancement during periods of healthy sleep. Diurnal fluctuations in glymphatic efficiency and metabolite clearance offers a provocative mechanistic explanation for the impacts of acute and chronic sleep deprivation on CNS function.^5,47–49 Preliminary murine studies have demonstrated increased latency in the clearance of CSF tracers surrounding the brain parenchyma during wakefulness, with more effective clearance in sleep.⁵ Although contemporary data remain somewhat controversial, consensus increasingly recognizes a concentration gradient underlying glymphatic flow, with reproducible modulation occurring in concert with sleep cycles. More specifically, glymphatics are not explicitly ‘on’ or ‘off’ in states of wakefulness or sleep. Rather, the presence and magnitude of slow-wave activity during sleep strongly correlates with glymphatic flow—a pattern that is not observed during general anaesthesia, which suppresses most functions associated with sleep physiology.^4,50,51 Interestingly, alcohol has a nuanced impact on the glymphatic system function, with low doses of ethanol enhancing function while persistent heavy use leads to reactive gliosis and impaired function—possibly contributing to the cognitive impairments and dementia seen in chronic alcoholics.⁵² Other studies suggest a composite mechanism, with maximal CSF influx and metabolite clearance during slow-wave sleep, followed by marked increases in CSF and metabolite efflux from the CNS into the systemic circulation and cervical lymph system during wakefulness.⁶ Supportive data include PET studies, which have identified peaks in amyloid-β accumulation during the day, followed by brisk clearance overnight—a process that is dramatically impaired by sleep deprivation.^48,53,54 Indeed, even a single night of sleep deprivation in humans was found to raise thalamic and hippocampal amyloid-β levels by 5%—a change sufficient to induce neuritic dystrophy and impaired neuronal function in rodents.^48,55 Evidence in both mice and humans suggests that the relationship between sleep and amyloid-β clearance is AQP4-dependent.^48,53,54,56 Knockout AQP4 models that are sleep-deprived have an elevated inflammatory state, increased amyloid and tau burden and synaptic unit destruction.⁵⁷ Moreover, AQP4 localization to astrocytic endfeet on perivascular spaces is dependent on dystrophin-associated complex, which is under tight circadian regulation.⁶ Indeed, the awake brain reduces glymphatic influx into the interstitium, largely by reducing AQP4 expression at astrocytic endfeet.^5,6 It is also possible that increased oxygen demand caused by elevated activity during periods of wakefulness results in increased cerebral blood volume, thus reducing interstitial space for CSF influx. Interestingly, iNPH and Alzheimer’s disease are strongly associated with sleep-disordered breathing, largely obstructive sleep apnoea, which could reasonably contribute to glymphatic system dysfunction, likely due to apnoea-based sleep architecture fragmentation, if not more direct pathophysiology.^5,58–62

If the glymphatic system is predominantly active during slow-wave sleep, it invites a key question regarding CSF flow and production during wakefulness. In humans, CSF is produced roughly at 0.3–0.6 ml/min, with the mean gross ventricular volume of 140–160 ml turning over approximately four times daily in healthy adults.⁶³ Studies in humans suggest that CSF production peaks overnight, highlighting a potential driver of increased glymphatic influx.^64,65 Investigations using inert tracers, such as amyloid-β and ¹⁴C-sucrose, confirmed significant decreases in of CSF solute clearance rates during periods of waking in mice, while similar measurements of CSF influx assessed via tracer injections at the cisterna magna showed a parallel decrease of approximately 95%.^5,63,66 Importantly, studies evaluating the flow of larger tracer agents observed that those larger than 2000 kDa do not pass into the interstitial regions of the brain.² These findings suggest in turn that CSF flux does not require passage through the interstitium for outflow circulation. As such, glymphatic flow is not required for all CSF passage; however, solute clearance is markedly impaired without it. Still, much remains incompletely understood regarding the mechanistic relationships between glymphatic activity, sleep–wake cycles and short- or long-term patterns in CNS peptide concentrations in both healthy individuals and disease states.

Glymphatics in neurological disease

Alzheimer’s disease and neurodegenerative disease

Much of the foundational work regarding the physiology and pathophysiology of the glymphatic system has been conducted in preclinical models of Alzheimer’s disease. Acknowledging the existing limitations of Alzheimer’s disease rodent models, valuable insights have been gleaned regarding glymphatic function from such efforts.⁶⁷ Astrocytes play a substantial role in amyloid-β clearance, but become reactive and dysfunctional as Alzheimer’s disease progresses.^68–70 Murine models have demonstrated that amyloid-β clearance is dependent on AQP4 function through specialized perivascular channels, and dysregulation of AQP4 localization has been implicated in the pathogenesis of Alzheimer’s disease.^2,17,53,56 More specifically, AQP4-null mice exhibit dramatically diminished perivascular spaces, as well as substantially increased amyloid-β plaque accumulation, collectively precipitating cognitive dysfunction.^{2,46,56,71,72} Although the mechanistic importance of amyloid-β is under increased scrutiny given the failed clinical trials targeting amyloid-β levels, impaired amyloid-β and phosphorylated-tau clearance appear at least indirectly associated with the characteristic pathophysiological changes of Alzheimer’s disease.^46,73–76

A leading hypothesis that is highly congruent with data supporting the current model of glymphatic system function suggests that amyloid-β accumulation is a biomarker for an underlying dysfunction of the CNS metabolic clearance system.^21,32,74 In this formulation of Alzheimer’s disease pathophysiology, toxic metabolite accumulation at the cellular level is the primary driver of disease, a phenomenon that is more difficult to appreciate than the characteristic amyloid-β plaques. In post-mortem examination of Alzheimer’s disease brains, there was an association with severity of disease and impaired AQP4 polarization and increased amyloid plaque burden.⁷⁷ The ‘sundowning’ phenomenon, a collection of symptoms including confusion, anxiety and impaired executive abilities that occur towards the end of the day, which is observed in many neurodegenerative diseases, would support the hypothesis that reduced clearance of metabolic waste products may play a crucial role in their pathogenesis, given that the maximal pathological metabolite accumulation should occur at the end of a typical day, potentially triggering the transient features of cognitive decline and emotional lability that are partially relieved by sleep, during which metabolite clearance is more effective.^58,60 Furthermore, the broader outflow of metabolites through dural lymphatics likely becomes impaired with age as well, as these pathways become dysfunctional and demonstrate reduced drainage capacity.^10,33,34,37 In Alzheimer’s mammalian modelling, impairing meningeal lymphatic drainage leads to elevated CNS amyloid-β levels and negatively impacts learning and memory.^34,37 Gaining a better understanding of these physiological processes may provide novel interventions for Alzheimer’s disease, such as focused-ultrasound microbubbles that aid in amyloid-β clearance through glymphatic and lymphatic mechanisms.⁷⁸

Parkinson’s disease is another neurodegenerative that may also have associations with impairments in glymphatic and meningeal lymphatic function.^79–81 Parkinson’s patients frequently have coinciding sleep disorders, in addition to pathological accumulation and reduced clearance of alpha-synuclein.^79–81 Utilizing emerging imaging modalities, including diffusion tensor imaging along perivascular spaces, which is capable of visualizing perivascular fluid dynamics along periventricular white matter, Parkinson’s patients have demonstrated glymphatic impairment when compared those with non-Parkinsonian tremor.⁸¹ Furthermore, the associated perivascular fluid dysfunction was closely correlated with disease severity.⁸¹ Indeed, both global and discrete perivascular region structures were significantly altered in individuals with Parkinson’s disease compared to age-matched controls.⁸⁰ However, there may well be regional differences, as opposing findings have been reported in the basal ganglia regions of Parkinson’s patients—also indicating the nuances of glymphatic function and that disease severity may be only partially correlated with perivascular space volumes.⁸² Beyond perivascular-associated dysfunction, further downstream impairment of dural lymphatic flow has also been characterized in Parkinson’s disease using dynamic contrast-enhanced MRI—an emerging technology for imaging dural lymphatics.⁷⁹ Associated increases in alpha-synuclein accumulation paralleled impairment of meningeal lymphatic flow in typical Parkinson’s disease when compared to atypical Parkinson’s disease and age-matched control.⁷⁹ Region-specific perivascular space volumes and dural lymphatic dynamics may be useful radiographic markers of glymphatic dysfunction and associated Parkinson’s disease severity, but further characterization and study is required.

Idiopathic normal pressure hydrocephalus

The most common form of adult-onset hydrocephalus is iNPH, which is characterized clinically by progressive gait disturbance, urinary incontinence and neuropsychological impairment.^83,84 iNPH is a primary pathophysiological process, without a specific causative event or other inciting disease such as infection, trauma or haemorrhage. INPH lacks a definitive diagnostic test; rather, diagnosis is made via clinical criteria in the absence of another primary aetiology and in the presence of characteristic findings on imaging studies and response to CSF drainage.⁸⁴ Indeed, the diagnostic evaluation with the largest positive predictive value is resolution of symptoms following large-volume lumbar puncture (e.g. 30–50 ml) or extended lumbar drainage trial, in the setting of a normal-range opening pressure.⁸⁵ Although iNPH remains poorly understood, altered CSF circulation is a putative mechanism for this disease and other related processes.^76,85,86

INPH represents a major opportunity for translational study of glymphatics, particularly given that it is a relatively common disease and that the most common treatment—ventriculoperitoneal shunting—is far more likely to improve gait abnormalities than cognitive dysfunction, indicating that simple CSF diversion is an imperfect and incomplete treatment strategy.⁸⁷ Mounting evidence suggests that the observed excess in extracellular fluid volume and associated peptides and other metabolites may be attributable to glymphatic dysfunction.^21,75,88,89 More specifically, patients with iNPH demonstrate significantly delayed CSF tracer penetration and subsequent clearance from the entorhinal cortex as compared to age-matched controls.⁸⁹ Brain biopsies in age-matched controls demonstrated reduced AQP4 expression at endfeet and impaired AQP4 polarization.^90,91

Blunted CSF flow and delayed clearance would be consistent with impaired brain parenchymal compliance and suppressed neurovascular pulsations thought to underlie iNPH, which may contribute to the associated dementia—although it should be noted that some cases may be confounded by co-occurrence with Alzheimer’s disease or other neurodegenerative disease, as these patients have a higher rate of subsequent neurodegenerative diagnoses than the general age-matched population.^73,92,93 Interestingly, emerging evidence suggests that patients with iNPH who subsequently develop comorbid Alzheimer’s disease appear to derive substantial overall cognitive benefits from early shunting.^93–95 Shunt-responsive patients also appear to have lower pathological burdens of neuritic plaques or intraventricular amyloid and tau; however, it is unclear whether this difference reflects improved plaque clearance as a function of shunt placement, or if shunt responsiveness is simply a surrogate marker for a different pathophysiology, although it seems likely there is a degree of underlying aetiological overlap. Further unknown are the underlying mechanisms for why shunting directly improved neurological function in iNPH and may possibly do so in Alzheimer’s disease. Luikku et al.⁹³ propose performing cortical biopsies of patients that met specific clinical and radiographic disease criteria during initial shunt placement to provide earlier diagnosis for patients with concomitant iNPH and Alzheimer’s disease—longitudinal surveillance and comparison of these subgrouped patients will be insightful.

Ageing

Ageing, which is associated with peptide accumulation and frequent sleep impairment, has been hypothesized to be both directly and indirectly associated with glymphatic dysfunction. Age-related cognitive decline—which already shares clinical features with Alzheimer’s disease—is strongly associated with amyloid-β accumulation and other proposed markers of dysfunctional CNS metabolite clearance.⁹⁶ Notably, the glymphatic system also appears to undergo progressive dysfunction with age, which may represent an independent source of progressive CSF flow disruption in elderly patients.^{7,9,10,97–99} In a human trial evaluating glymphatic flow following intrathecal contrast-agent injection, it was identified that the aged brain demonstrates impairment in both glymphatic circulation and meningeal lymphatic vessel outflow—with glymphatic circulation seeming to be the upstream driver.¹⁰⁰ Ultimately, the question becomes: is glymphatic dysfunction a driver of cerebral ageing and age-related cognitive impairments? Further study is required to address this possibility, including a focused assessment of the relationship between changes in structural compliance in the brain parenchyma and cerebral vasculature that may predispose to impaired CSF flow and other neurodegenerative drivers such as vascular dementia and small vessel disease.^101,102

Glymphatic function appears to be, at least partially, mediated through arterial pulsations that create pressure differentials within the brain interstitial spaces and help drive CSF circulation out of and into perivascular channels of flow.^17,103 These cerebrovascular pulsations alter tissue stiffness, and modelling suggests that they may influence macromolecular transit through the interstitium.^17,103 These findings are in accordance with other novel observations regrading cerebral stiffness in humans with iNPH, Alzheimer’s disease and other neurodegenerative diseases assessed via magnetic resonance elastography (MRE). Indeed, disease processes including Alzheimer’s disease and NPH are characterized by marked changes in extracellular matrix composition and parenchymal stiffness, highlighting an unmet translational need to further develop MRE and other innovative, non-invasive neurodiagnostics.^104–106

An area of emerging research that may ultimately shed new light on the understanding of glymphatic flow is the relationship between hypertension and flux of CSF and metabolites. This is particularly relevant in the elderly population, where hypertension is highly prevalent and often coexistent with iNPH and neurodegenerative diseases - possibly contributing to the adverse neurocognitive changes associated with general ageing. In a rodent model of spontaneous hypertension, animals demonstrated elevated AQP4 expression and increased rate of tracer flow in perivascular regions.^107,108 These findings may reflect a transient compensatory response, akin to the elevated AQP4 levels observed during the healing phases of spinal cord injury or cerebral ischaemia.^109,110

Ageing is a complex phenomenon, with likely numerous pathophysiological mediators. For CNS interstitial fluid and metabolite/waste product clearance, there are multiple impairments along the processes associated with ageing—particularly within the brain parenchyma and dural lymphatics. It remains unclear whether any of these are drivers of the ageing phenomenon, or rather downstream interrelated effects and consequences. It is also possible that changes to the primary CNS cellular units—neurons, astrocytes, oligodendroglia, and/or microglia—are the initiators of ageing’s deleterious effects. Ultimately, striving for comprehensive evaluation of these systems and development of a more global perspective on the primary cellular and molecular initiators of ageing and various disease processes will be crucial to better understand these complex phenomena, and only then can we effectively work towards mechanistic interventions and therapies.

Traumatic brain injury

Severe head trauma is a likely disruptor of glymphatic system function, as TBI often leads to cerebral fluid abnormalities, including altered neurovascular autoregulation and parenchymal vasogenic oedema. Individuals that suffer TBI frequently experience disrupted sleep and cognitive impairment, among numerous other neurological sequela.⁴⁵ In the acute phase following injury, intracellular AQP4 localization becomes dysregulated, shifting away from astrocytic endfeet—a pathophysiological change that leads to adverse peptide and metabolite accumulation.^1,111–113 As the cellular response sets in and the chronic phase of injury recovery predominates, prolonged AQP4 mislocalization leads to sustained disruptions of the flow and clearance of perivascular fluids and solutes.^1,31,114,115 In rats subjected to mild repetitive trauma, extensive disruption in glymphatic circulation was identified throughout the hypothalamus, amygdala, hippocampus and olfactory regions, with associated dysfunction noted in cognition and gait.¹¹⁶ Interestingly, studies evaluating amyloid-β as a potential biomarker for TBI severity have noted acute-phase increases in CSF amyloid-β that are not reflected in systemic circulation, indicating a potential mechanistic link at the point of solute clearance from the CNS into the systemic circulation via glymphatics.^117–119 Still further support of this hypothesis is noted in the subsequent increases of systemic amyloid-β observed during the subacute and chronic phases of TBI, potentially indicating a partial restoration of glymphatic function.^117,120 In addition to its utility for clinically relevant biomarkers in TBI patients, the disrupted perivascular spaces may also be a possible therapeutic target. Indeed, endeavours to open larger CSF cisterns and interstitial fluid pathways, including dural lymphatics, may improve glymphatic function and metabolite/peptide clearance in TBI patients, although far more investigation is required.^1,31,121

Vascular insult

Like TBI, focal cerebral infarction causes AQP4 mislocalization away from astrocytic endfeet, contributing to rapid cerebral oedema.^122–125 In a rodent proximal middle cerebral artery occlusion model, glymphatic circulation decreased by >30% at 3 h post-occlusion, as assessed by MR-detected parenchymal levels of low-molecular weight compounds.¹²⁶ Following cerebral reperfusion, glymphatic function normalized at 24 h after.¹²⁶ There is also associated elevated amyloid plaque burden within the ischaemic region.¹²⁷ The importance of restoration of glymphatic function for post-stroke recovery deserves further investigation; however, there is potential that modulating AQP4 may impact cerebral oedema in humans and could be useful for tempering malignant cerebral swelling and spreading depolarization, or potentially reduce cerebral activity and therefore act to limit hypoxic injury.¹²⁸

In a mouse model of aneurysmal subarachnoid haemorrhage, glymphatic function becomes severely impaired within 24 h of aneurysm rupture, with MR evidence demonstrating >50% loss of parenchymal circulation, attributable to a build-up of perivascular fibrin.¹²⁶ Cortical structures seem to be more particularly impacted, presumably through occlusion of para-arterial influx by subarachnoid blood products, with associated AQP4 acute upregulation and loss of polarization.¹²⁹ Glymphatic function did not improve following bilateral decompressive craniotomies, suggesting that intracranial hypertension was not the primary driver of dysfunction,¹²⁶ although performing a craniotomy leads to AQP4 expression changes, as well as reduced arterial pulsatility and associated impaired glymphatic circulation in mice, therefore confounding greater inerpretations.^130,131 Instead, intraventricular administration of tissue-type plasminogen activator resulted in dramatic improvements in glymphatic perfusion and solute clearance, highlighting a possible additional mechanism of benefit for fibrinolysis in this population.¹²⁶ Furthermore, in a non-human primate model of subarachnoid haemorrhage, tracer MRI revealed severely impaired cerebral CSF circulation, particularly in the ipsilateral hemisphere.¹³² Meningeal lymphatics also appear to play a role in the clearance of extravasated erythrocytes from CSF into the cervical lymphatics.¹³³ The relationship between glymphatic dysfunction and the development of delayed cerebral ischaemia (e.g. clinically significant vasospasm) remains poorly understood; however, this represents a major area for potential intervention to improve outcomes in a vulnerable population.

Brain in space

The exposure to microgravity states is a fascinating area of neuroscience investigation, and prolonged duration may provoke alterations in intracranial fluid dynamics through various mechanisms. Changes include increased jugular venous pressure, accumulation of CSF around the optic nerves and greater ventricular CSF volumes. These changes may help explain alterations in vision and cognition observed in astronauts returning from space missions.^134–136

Altered gravitation forces and biophysical forces may have direct impacts on pericyte and interstitial fluid dynamics as well. Pericytes experience different magnitudes of perivascular hydrodynamic stresses as the transmural pressure difference decreases from arterioles to venules, and altered gradients in space may have profound impacts on fluid mechanisms and perivascular structural function.^137,138. Interestingly, there may be evidence of a glymphatic-like pathway within the optic nerve as well, as assessed by astronauts who underwent 6-month-long missions to the International Space Station and experienced T₂ hyperintensity of the optic nerve. These changes are thought to be related to the pressure dynamics exerted on perioptic CSF, thereby driving interstitial fluid into the perivascular spaces as a result of prolonged microgravity.^139,140

Sleep deficiency is also very prevalent among astronauts in space, which could further negatively influence glymphatic system function, particularly if endured for sustained periods of time.¹⁴¹ Furthermore, space travel increases amyloid plaque burden, leads to perivascular inflammation and accelerated cognitive impairment in mice, with likely implications for impairing glymphatic function.¹⁴² Space travel also leads to dysregulated AQP4 expression.¹⁴³ While glymphatic function has yet to be fully investigated in relation to microgravity, forthcoming research is on the horizon.

Challenges of investigating the glymphatic system

Animal and human studies have reached mixed conclusions regarding the nuances of glymphatic function, including the definitive role of AQP4. While important questions related to both the molecular physiology and the practical function of the glymphatic system remain incompletely understood, many of the observed discrepancies regarding the relationships of disease processes, circadian rhythms and ageing at the level of the glymphatic system may be more attributable to differences in experimental design than biology. More specifically, relatively minor differences in tracer agent diameter, size and location of calvarial removal and other experimental parameters have considerable local impacts on pressure dynamics, which may in turn influence measurements and outcomes.^50,144–146 Still further changes representing sources of possible systematic error include differential changes in global CSF volume or pressure associated with tracer infusions, the nature of the anaesthetic agents used in experimental protocols and their relative impacts on cerebral perfusion and simple changes in head or body position, which appear to have an appreciable impact on glymphatic function in rats.²⁹

While the use of tracer-based in vivo imaging and optically cleared preclinical studies have provided invaluable insight into glymphatic physiology, these techniques are not translatable into in vivo human studies. Less-invasive imaging modalities will need to be investigated to better characterize the role of glymphatics in normal physiology and pathophysiology. Indeed, emerging less-invasive techniques are forthcoming. Multiple echo time arterial spin labelling is capable of assessing AQP4-regulated transport of fluid along perivascular channels. MR-based diffusion tensor imaging is another possible resource, which promises to better assess the diffusivity of fluid through the perivascular regions and has even correlated changes in water flow with cognitive assessment.^147,148 Ultimately, these differences are characteristic of a nascent field-of-study, where understanding is still emerging and standardization is an afterthought. Notwithstanding, given the subtlety of the glymphatic system and its presumed vulnerability to minor changes in the experimental milieu, these preliminary inconsistencies emphasize the importance of experimental rigour. In the service of both greater physiological understanding and the eventual improvement of patient-centred outcomes, reproducibility, standardization and stringent objectivity in the interpretation of data should be emphasized in all glymphatic research, particularly in research laying the groundwork for eventual therapeutic interventions.

Emerging technologies

The goal of precision medicine is to target disease and disease pathophysiology while simultaneously minimizing off-target side effects—ideally paired with real-time biological or radiographic feedback. Many of these evolving interventions and diagnostic endeavours are moving away from ‘one size fits all’ medicine, with increased interest in individualized and tailored approaches. Paired therapeutic and diagnostic modalities—termed theranostics—is an evolving subject of nanomedicine and molecular imaging that seeks to integrate therapeutic and imaging functionalities on a single platform. Theranostics seeks to combine mechanistic diagnostic imaging with drug delivery and drug monitoring real-time.¹⁴⁹ There is growing interest that these theoretical possibilities, harnessing advances in electronic, optoelectronic and microfluidic interfaces, may greatly benefit the study and medical intervention of the glymphatic system.

As a case example, investigations into non-invasive brain stimulation to elicit brain-wide effects on patients with Alzheimer’s disease-associated pathology, such as altered cerebrovascular dynamics, impaired amyloid clearance and glymphatics functionality, may be able to enhance cognitive function.¹²⁵ Interestingly, it has been reported that inducing gamma oscillations with a non-invasive light flicker impacted pathology in the visual cortex of a mouse model of Alzheimer’s disease.¹²⁵ A follow-up study from the same group showed that auditory tone stimulation drove gamma frequency neural activity in different brain areas.¹⁵⁰ Combined auditory and visual stimulation induced a microglia-clustering response, resulting in reduced amyloid load.¹⁵¹ Furthermore, it may also be possible to modulate neurovascular physiology through non-invasive neurostimulation. Transcranial electrical stimulation may alter neurovascular responses from secondary responders to primary modulators of the neuronal response to electrical current.¹⁵² The potential application of transcranial electrical stimulation in modulating the glymphatic function warrants further investigation, but may be promising. Remote fields, from light, sound and/or magnetism, may also be combined for the purposes of monitoring and direct stimulation and provide unique closed-loop applications for the treatment of abnormal physiological processes within glymphatic subcomponents, including dural lymphatic structures, perivascular AQP4 localization, interstitial fluid dynamics, calvarial bone marrow–dural interaction and metabolic waste clearance.

Invivo imaging using optical coherence tomography (OCT), a tracer-free, non-invasive cross-sectional imaging technique, is a feasible and promising modality for monitoring interstitial and lymphatic drainage.^153,154 Several OCT techniques and functional modalities have been developed in the past decade allowing fine delineation of anatomical structures, including microangiography, Doppler and dynamic contrast OCT for visualizing blood and dural lymphatics.^155–157 Intravascular OCT is similar to intravascular ultrasound; however, rather than measuring sound waves, the OCT catheter samples reflections of the near-infrared light.¹⁵⁸ Cellular and molecular imaging utilizing contrast-enhanced and magnetomotive OCT has been useful for monitoring early-stage vascular atherosclerosis and plaque formation—as the resolution of these technologies continues to improve, it may ultimately be possible to directly detect subcellular cerebrovascular and perivascular plaques.¹⁵⁸

The interaction between the calvarial bone marrow and dural immune microenvironment has gained a lot of attention, as it may impact glymphatic system function.¹⁵⁹ A recent study showed that age-related loss of CCR7 expression can be attributed to altered communication between the immune environments of the meninges and the skull bone marrow, in addition to the dysfunction of the glymphatic flow.¹⁶⁰ Emerging remote-field technologies may provide a modality for targeting these distinct microenvironments.¹⁶¹ Optogenetics, and associated capabilities to modulate neuronal activity, angiogenesis, and cellular differentiation, may target perivascular complexes with lost physical and biological structure to improve pericyte function, enhance interstitial fluid dynamics and improve cellular and fluid-based clearance of metabolites and peptides.¹⁶² The development of single-beam optical traps known as ‘optical tweezers’, which possess high spatial precision, may even provide cellular and subcellular mechanisms for targeting discrete regions of interest, to more broadly impact cognitive or glymphatic function.¹⁶¹

However, innovation within convention imaging modalities is showing promise as well. Ultrasound, employing microvascular imaging technology, is capable of detecting CSF flow and cerebrovascular flow in microvessels.^163,164 Exciting techniques that selectively target the blood–brain barrier to increase permeability to contrast agents may allow real-time monitoring of the contrast agent through the glymphatics and dural lymphatics.¹⁶⁵ Beyond diagnostic purposes, ultrasound-directed release of microbubbles, the technology is being employed for treatment purposes as well.^166–168 Focused ultrasound, which is also capable of modulating gene expression and cellular signalling, can modulate genetic circuits and thereby neurological function.¹⁶⁸ Indeed, focused ultrasound has been demonstrated to improve targeted amyloid clearance through perivascular spaces.⁷⁸ Beyond imaging modalities, conventional therapeutic routes, including AQP4 agonist, may work to restore lost receptor polarization or improve residual function; however, these have to be comprehensively investigated to determine whether they may be viable options. Taken together, the continued development of new imaging modalities, and fresh uses for more conventional technologies, may help provide unique diagnostic and intervention avenues for cerebrovascular and glymphatic purposes.

Conclusion

Until recently, conventional wisdom in biomedicine held that the CNS had no lymphatic system—an assumption based on the absence of the characteristic lymphatic channels or lymph nodes seen throughout non-CNS tissues. Yet, numerous studies in rodent models and increasingly robust studies in humans have provided convincing evidence in support of the glymphatic system as a critical network of pericellular extravascular channels whose core function is to enable CSF flux, delivering electrolytes and proteins to highly cellular regions of CNS cortex, while simultaneously providing an active clearance system for metabolites and potentially pathogenic peptides. Dysfunction of this glymphatic system has been implicated in an increasing number of neurological diseases and injuries, including iNPH, Alzheimer’s disease, ischaemic stroke, subarachnoid haemorrhage, TBI and others, suggesting a major opportunity for advancement in disease pathogenesis, mechanistic therapuetic interventions, and homeostatic CNS function—with broad implications on public health. Furthermore, the glymphatic system may be involved in the progressive changes underlying brain ageing and chronic cognitive changes. Notwithstanding, additional investigations are critical, with an emphasis on increased refinement and standardization in the development of tools and metrics for the study of human glymphatic physiology. At present, the most important goal for the future of the field is definitive characterization of the pathological mechanisms underlying glymphatic dysfunction, including focused study of high-prevalence model diseases such as Alzheimer’s disease.

Acknowledgements

We would like to thank Moshik Gulst, a brilliant artist and cartoonist, for lending us his creative skills towards development of the glymphatic figure.

Funding

No direct funding was received towards this work.

Competing interests

The authors report no competing interests.

Abbreviations

 
• iNPH

  idiopathic normal pressure hydrocephalus

 
• TBI

  traumatic brain injury