Glymphatic Clearance Dynamics in Traumatic Brain Injury: Mechanisms, Imaging Biomarkers, and Application Prospects

Abstract

The pathological increase in brain catabolites after traumatic brain injury strongly correlates with a higher risk of neurodegenerative disease. This review examines the pathogenic role of glymphatic clearance dysfunction in that process. The glymphatic network enables cerebrospinal and interstitial fluid exchange and paracellular flow. These processes are mediated by astrocytic aquaporin-4. Glymphatic function is regulated by arterial pulsatility, sleep-wake cycles, and intramural periarterial drainage, with meningeal lymphatic vessels acting as the final drainage site. Mechanical trauma causes aquaporin-4 depolarization and mislocalization; it also triggers neuroinflammatory activation and blood-brain barrier disruption. These processes ultimately impair glymphatic function and neurotoxic proteins become more localized and overproduced. Previous studies have linked clearance defects to secondary neuron injury. Current evidence in humans has come mostly from pilot studies. Recent advances in neuroimaging provide new assessment tools. Dynamic contrast-enhanced magnetic resonance imaging (MRI) reveals delayed tracer clearance. Diffusion tensor imaging along perivascular spaces shows abnormalities in key parameters. These imaging findings preliminarily associate with fluctuations in cerebrospinal fluid catabolites. Therapeutic research suggests several reparative strategies. Physical exercise improves aquaporin-4 polarization integrity. Cannabidiol administration in experimental models increases meningeal lymphatic drainage and reduces tau pathology. Angiotensin II type 1 receptor antagonists may indirectly improve clearance by stabilizing the blood-brain barrier. Lymphatic pathways have been used as therapeutic targets for cannabidiol. Biological evidence also supports their role in traumatic brain injury progression. Further investigation is needed to validate whether these represent independent contributing processes. Multimodal imaging, novel biomarker assays, and chronobiological modulation strategies are improving visualization. Microfluidic modeling could clarify the glymphatic-biomarker relationship; it may also advance precision medicine approaches for traumatic brain injury.

Graphical abstract

Keywords

traumatic brain injury / glymphatic system / aquaporin-4 / intramural periarterial drainage

1. Introduction

Traumatic Brain Injury (TBI) affects over 50 million individuals annually and represents a significant worldwide health burden [1, 2, 3, 4]. Long-term neuropathological sequelae of TBI, such as chronic traumatic encephalopathy and Alzheimer’s disease (AD), impose substantial socioeconomic costs [2, 3, 5]. As evidenced by clinical investigations, changes in cerebrospinal fluid (CSF) biomarker concentrations are directly associated with cognition deficits in months to years following moderate–severe TBI [4, 6, 7, 8, 9, 10, 11]. The exact mechanisms governing fluctuation in CSF biomarker levels remains incompletely understood. The majority of existing theories propose this fluctuation is caused by irregular production resulting from neuronal injuries, emerging evidence further reveal that deficits of neuronal clearance pathways may equally contribute to change in CSF biomarker levels [12, 13, 14].

As a main pathway for cerebral waste clearance, the glymphatic system maintains homeostasis through a tightly orchestrated, hierarchical mechanism. The glymphatic circulation initiates with arterial pulsations driving CSF influx along periarterial spaces into the brain parenchyma. Polarized aquaporin-4 (AQP4) channels distributed densely in the astrocyte endfeet mediate rapid CSF-interstitial fluid (ISF) exchange [8, 13, 14, 15, 16, 17, 18]. Following this exchange, metabolic waste products, such as \beta-amyloid (A\beta) and phosphorylated tau proteins (p-tau) are cleared from the brain via two different pathways. Primary drainage via perivenous spaces, with partial elimination through meningeal lymphatic vessels to deep cervical lymph nodes [15, 16, 17, 19]. The complementary clearance pathway is the intramural periarterial drainage (IPAD) along vascular basement membranes. Efficiency of the glymphatic system is regulated by multiple physiological factors, including sleep-wake cycles, neurovascular coupling, and circadian regulation of AQP4 membrane trafficking [12, 20]. Glymphatic dysfunction has been identified in AD and stroke models as a driver of A\beta plaque deposition and tau tangle formation, the extent to which glymphatic dysfunction influences the generation of peripheral fluid biomarkers after TBI has not been adequately investigated [21, 22, 23].

This review introduces a novel glymphatic-biomarker axis model that unites the molecular, imaging, and clinical aspects to reveal the phenomenon of post-TBI clearance dysregulation, and reveals the molecular pathogenesis including different connected pathways and mechanisms. Mechanical shear forces in TBI disrupt astrocytic endfoot architecture, inducing AQP4 depolarization and mislocalization. Coexisting blood-brain barrier (BBB) damage will also promote extravasation of plasma proteins, forming unsoluble A\beta-protein aggregates to obstruct interstitial flow. Secondary neuroinflammation like NACHT, LRR and PYD domains-containing protein 3 (NLRP3)-caspase-1 mediated pyroptosis further degrades IPAD efficiency by impairing basement membrane integrity, creating a multi-hit clearance deficit. The clinical assessment of glymphatic function encompasses advanced neuroimaging techniques, including dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and diffusion tensor imaging along the perivascular space (DTI-ALPS), which provide noninvasive evaluation methods. These complementary approaches allow for comprehensive characterization of glymphatic activity. DCE-MRI measures CSF-ISF exchange efficiency through gadolinium tracer kinetics, and DTI-ALPS evaluates interstitial flow dynamics by quantifying perivascular water diffusivity patterns. Both methods are associated with the extent of cerebrospinal fluid biomarkers for neurodegeneration, particularly A\beta and phosphorylated tau protein levels, suggesting their potential utility in clinical diagnostics and disease monitoring. Preclinical evidence supports several potential therapeutic interventions for modulating glymphatic dysfunction that have yielded promising findings. Aerobic exercise has been shown to enhance A\beta clearance through two distinct mechanisms: augmentation of arterial pulsatility and restoration of AQP4 polarization. Cannabidiol (CBD) can promote waste efflux via activation of meningeal lymphatic endothelial receptors. Angiotensin II Type 1 (AT1) receptor antagonists could protect the integrity of BBB by preserving tight junction proteins, thereby indirectly improve glymphatic flow. These interventions collectively address multiple aspects of glymphatic impairment, offering potential avenues for therapeutic development in neurodegenerative disorders.

2. Physiological Architecture and Dynamic Characteristics of the Glymphatic System

2.1 Definition and Structural Composition

The glymphatic system, a recently characterized intracranial clearance pathway, derives its nomenclature from the functional integration of glial cells and lymphatic-like drainage mechanisms [8, 12, 13, 20, 24, 25, 26]. This system drives uni-directional flow of CSF into the brain parenchyma via periarterial spaces, where astrocytic endfoot AQP4 channels mediate exchange with ISF [8, 13, 20, 24, 25, 26, 27]. Substantial AQP4-mediated paracellular flow within the interstitial fluid (ISF) contributes to the active clearance of metabolic waste products toward the venous drainage system [28, 29]. These wastes are transported along perivenous pathways into meningeal lymphatics and deeper cervical nodes through meningeal lymphatics [12, 16, 17, 18, 19, 30, 31]. The polarized distribution of AQP4 on astrocytic vascular endfeet serves as the structural foundation of glymphatic dynamics [9, 10, 32, 33, 34, 35, 36, 37]. This spatial arrangement, which is anchored on the vascular interface, engages in tripartite interactions with neurons and cerebral vasculature. It creates an exclusive brain-fluid microenvironment [12, 38]. Beyond the classical glymphatic route, the IPAD pathway contributes significantly to interstitial solute clearance [16, 32, 36, 39, 40]. This alternative mechanism relies on the structural integrity of the vascular basement membrane, extracellular matrix composition, and the homeostasis of scaffolding proteins such as \alpha-dystrobrevin [9, 41, 42]. Recent investigations have identified surfactant protein-G (SP-G) as a novel participant in CSF rheology and waste clearance [9]. SP-G exhibits co-localization with AQP4, glial fibrillary acidic protein, and platelet endothelial cell adhesion molecule-1 (PECAM-1) at perivascular and choroid plexus epithelial sites, suggesting its potential role in modulating glymphatic efficiency [9]. These findings allow us to add more specific molecular targets for the modulation of glymphatic functions.

2.2 Glymphatic Transport Dynamics

The kinetics of CSF-ISF exchange within the brain are driven mainly by physiologic forces. Mechanical factors including arterial pulsations from cardiac cycles, respiratory movements, and vasomotion collectively push CSF influx along periarterial spaces into the parenchyma, while perivenous spaces serve as efflux pathways for metabolic waste products [12, 18, 30, 31, 35, 43]. Water transport occurs selectively through the interstitial space via highly specific AQP4 water channels, which are densely expressed in astrocytic end-feet enveloping the central nervous system vasculature [28]. The efficiency of this system exhibits marked state-dependent modulation. During sleep, the cerebral interstitial space undergoes transient expansion exceeding 30%, resulting in enhanced CSF influx and accelerated clearance of neurotoxic metabolites. Wakefulness is correlated with lower activity of the systemic circuit and reduced removal of the waste product [12]. Circadian rhythms show significant regulatory influence on both AQP4 polarization patterns and glymphatic flux [33, 44, 45]. Core clock genes and circadian signaling pathways modulate the subcellular localization and expression of AQP4 in astrocytic endfeet through complex molecular mechanisms, resulting in temporally stratified clearance efficiency that correlates with diurnal variations in cognitive performance observed in aged murine models [38, 46, 47, 48, 49]. The cerebral surfactant protein SP-G demonstrates abundant expression in CSF outflow-associated structures. Its marked elevation in CSF from normal pressure hydrocephalus and central nervous system (CNS) infection cases suggests potential involvement in both cerebrospinal fluid rheology and immunoprotection, with positive correlation to total CSF protein levels reflecting dynamic alterations in CSF drainage under pathological conditions [9]. This leads to multifactorial processes that include not only bidirectional transport across the CSF-ISF exchange but also BBB function. Multiple related systems together function to preserve overall brain homeostasis through such multipoint flux dynamics [39].

2.3 Role of AQP4 and IPAD in Clearance Dynamics

Aquaporins (AQPs) constitute a family of integral membrane proteins initially characterized as passive channels dedicated to facilitating water movement across biological membranes, thereby playing an essential role in maintaining osmotic balance [50, 51]. Over the last ten years, growing evidence has broadened the understanding of their functional diversity beyond mere water permeability [52, 53, 54]. In addition to water, certain AQPs, known as aquaglyceroporins, enable transmembrane passage of small neutral solutes including glycerol and various metabolites, linking them to metabolic and inflammatory pathways as well as diverse physiological processes such as renal water reabsorption, cerebral fluid dynamics, adipocyte-liver triglyceride flux, and ocular lens stability [53, 55]. AQPs are also implicated in cellular volume regulation. Recent findings suggest their involvement extends beyond passive pore function to include potential roles in signal transduction, though the underlying mechanisms remain poorly elucidated. The concurrent expression of multiple AQP isoforms in many cell types raises the possibility of physical or functional interactions among different family members [50, 51].

Despite being promising drug targets for conditions like cerebral edema, metabolic dysregulation, and impaired glymphatic clearance, no pharmacological agent targeting AQPs has achieved clinical approval to date [56, 57, 58, 59]. The development of such therapies is hindered by the challenge of simultaneously addressing comorbid processes like edema and dysfunctional waste clearance with a single agent. As a result, the cooperative mechanisms between AQPs and other therapeutic targets remain an underinvestigated area deserving further exploration [54].

AQP4 is the primary aquaporin water channel expressed in the CNS, where it is highly enriched in astrocytic end-feet and serves as a key regulator of water homeostasis and glymphatic clearance. Its polarized localization on astrocytic endfoot membranes facilitates rapid water exchange between CSF and ISF, with its spatial distribution critically determining the directionality and efficiency of fluid transport [11, 38, 47, 48]. MR imaging utilizing H₂¹⁷O as a tracer revealed a substantial bulk flow of ISF, facilitating significantly accelerated clearance of waste molecules from the parenchyma compared to purely diffusion-driven transport [28]. Experimental evidence demonstrates that AQP4-knockout mice exhibit markedly reduced CSF-ISF exchange efficiency, accompanied by impaired clearance of A\beta and p-tau, ultimately exacerbating neuropathological alterations [60]. Administration of an AQP4 inhibitor resulted in significantly delayed tracer imaging within the brain [28]. AQP4 dysfunction is manifested not only by alterations in protein expression levels but also by aberrant subcellular localization [61]. Dysregulation of glymphatic clearance has been linked to disrupted perivascular polarization of AQP4. In rodent models, the dynamic redistribution of AQP4 to both the blood-spinal cord and blood-brain barriers attenuates CNS edema and promotes functional recovery [29, 62].

The IPAD pathway represents an alternative clearance mechanism that depends on the structural integrity of the vascular basement membrane and extracellular matrix [16, 63, 64]. Genetic ablation of \alpha-dystrobrevin, a key scaffolding protein, not only disrupts AQP4 polarization but also induces vascular basement membrane remodeling. These changes lead to compromised IPAD efficiency, ISF accumulation, and protein deposition, thereby promoting cerebrovascular amyloid pathology [41]. Schematic patterns of glymphatic system is shown in Fig. 1.

Recent advances in microphysiological systems have enabled the development of “glymphatics-on-a-chip” platforms [47, 65]. These in vitro models have validated the mechanistic link between neuroinflammation (e.g., lipopolysaccharide or A\beta oligomer exposure), extracellular matrix degradation, and impaired fluid transport under conditions of AQP4 inhibition. These technological advancements provide the necessary means to manipulate the AQP4 polarization and the reconstruction of the extracellular matrix in subsequent, forward-facing therapies [47]. These studies highlight the indispensable functional roles of both AQP4 and IPAD in regulating the cerebral clearance rate and they offer a theoretical groundwork on glymphatic dysfunction after TBI [47, 65].

3. Manifestations of Glymphatic Dysfunction in Neurological Disorders

3.1 Glymphatic Dysfunction and Protein Aggregation in AD

A hallmark neuropathological feature of AD is cerebellar A\beta plaque deposit and neurofibrillary tangles constituted of p-tau which is the main mediator for neuronal degeneration. Currently growing evidence indicates that glymphatic impairment plays an important part in AD pathogenesis [15, 16, 26]. The tauopathies exhibit reduced CSF-ISF exchange efficiency and abnormal AQP4 polarity, and such functional disorders might induce faster aggregation of tau protein, as well as more rapidly transmit among neurons, thus aggravate neurodegenerative disease process and cognitive dysfunction [60, 66].

Pharmacological studies using the selective AQP4 inhibitor TGN-020 have established the central importance of AQP4-mediated glymphatic flow in tau clearance [31, 60, 67]. TGN-020 has been proposed as a potential AQP4 inhibitor candidate. Huber et al. [68] demonstrated, through osmotic swelling assays in Xenopus laevis oocytes, that this compound inhibits AQP4-mediated water transport in a dose-dependent manner. Experiments conducted in primary human and rat astrocytes have indicated a lack of AQP4 inhibitory activity, suggesting that its functional efficacy requires further investigation [69, 70]. Experimental inhibition not only obstructs CSF-ISF exchange but also significantly reduces tau clearance rates, identifying this pathway as a potential therapeutic target [60]. The analysis of experiments that tracked the fluorescent label of tau injected into mouse brain in vivo provided a clear sign of glymphatic outflow path, and through the quantification of the data it was indicated that the outflow was reliant upon the function of AQP4, showing the exact steps needed to allow for the movement of tau and its removal [71].

Neuroinflammatory mediators such as Chitinase-3-like protein 1 (CHI3L1, commonly referred to as YKL-40), elevated in AD and other neuroinflammatory disorders, are secreted by activated astrocytes and may indirectly impair glymphatic waste clearance through Chemoattractant Receptor-Homologous Molecule expressed on T Helper 2 Cells (CRTH2) receptor-mediated suppression of neural stem cell \beta-catenin signaling, ultimately disrupting neurogenesis [11, 15, 34, 72]. These findings establish a positive correlation between glymphatic dysfunction and A\beta/p-tau accumulation in AD, suggesting that therapies aimed at increasing AQP4 polarization and restoring CSF-ISF circulation may help to attenuate neurodegeneration [73].

3.2 Dysregulation of the Glymphatic System in Proteinopathies (SAH, PD, and Related Disorders)

Evidences implicate glymphatic dysfunction plays an important role in multiple protein aggregation disorders like subarachnoid hemorrhage (SAH) and Parkinson’s disease (PD). In contrast to the initial view focusing only on alterations in A\beta and tau proteostasis via CSF analysis, numerous CSF protein biomarkers associated with pathways affecting glymphatic function have also been found in patient groups with decreased cerebrospinal fluid drainage, such as congenital hydrocephalus. Some cases have elevated levels of soluble amyloid precursor protein alpha (sAPP\alpha), soluble amyloid precursor protein beta (sAPP\beta), A\beta42 in CSF and lack apparent changes in the level of AQP4, suggesting a disequilibrium between CSF production and clearance is the fundamental pathophysiologic mechanism [6, 16, 40]. In PD and related movement disorders, the accumulation of neurotoxic proteins such as \alpha-synuclein may be exacerbated by compromised glymphatic clearance. Current reviews highlight that the synergistic effects of AQP4 depolarization, neuroinflammation, and sleep fragmentation collectively promote cerebral protein accumulation, representing a potential final common pathway in neurodegenerative processes [16, 74]. The functional roles of novel surfactant proteins (e.g., SP-G) in neuroinflammation and CSF rheology remain to be fully elucidated. Similar to conventional surfactant proteins (SP-A/B/C/D), these molecules may modulate CSF viscosity and flow dynamics at the molecular level, thereby influencing protein clearance kinetics [9]. The precise mechanisms of glymphatic function alteration in proteinopathic disorder cases still need more studies in detail [75].

3.3 Cerebrovascular Disorders and Glymphatic Dysfunction (Stroke, Cerebral Hemorrhage, and Hydrocephalus)

Cerebrovascular pathologies, including ischemia-reperfusion injury, post-stroke neuroinflammation, subdural hematoma, and intracerebral hemorrhage with consequent CSF pathway obstruction, consistently demonstrate glymphatic impairment accompanied by pathological accumulation of metabolic byproducts such as A\beta and p-tau [76]. Experimental investigations using middle cerebral artery occlusion/reperfusion (MCAO/R) models in rats reveal that pyroptosis-mediated neuroinflammatory responses not only compromise BBB integrity but also induce AQP4 mislocalization, resulting in cerebral edema and intraparenchymal aggregation of A\beta1–42 oligomers that exacerbate neuronal dysfunction [77]. Complementary multimodal studies combining dynamic contrast-enhanced MRI with immunofluorescence in rat thalamic secondary injury zones demonstrate synchronous peaking of contrast agent retention and amyloid precursor protein (APP) deposition at two weeks’ post-stroke, coinciding with significant reduction in AQP4 polarization ratios. These findings establish that temporal deterioration of glymphatic function can be effectively monitored through advanced neuroimaging modalities [19]. Clinical analyses further identify sAPP\alpha in congenital hydrocephalus patients’ CSF as exhibiting exceptional diagnostic sensitivity (Area Under the Curve (AUC) = 0.99), confirming strong correlations between CSF protein composition alterations, ventricular dilation severity, and CSF drainage dysregulation. This biomarker profile provides valuable clinical chemistry parameters for evaluating glymphatic-CSF outflow capacity [6, 78]. Edema formation following injury-induced hypoxia is AQP4-dependent and correlated with both an upregulation of total AQP4 expression and its subcellular translocation to the blood-spinal cord barrier (BSCB) [79]. Pharmacological inhibition of AQP4 translocation to the BSCB was shown to attenuate CNS edema and enhance functional recovery in injured rats. This role of AQP4 has been further corroborated by another study, which utilized a photothrombotic stroke model to show that targeting AQP4 effectively mitigates cerebral edema in the early acute phase of stroke [80]. Their study also revealed an associated alteration in brain energy metabolism, evidenced by elevated glycogen levels.

These observations indicate that the coexistent abnormality of glymphatic and BBB transport systems is the essential mechanism responsible for the buildup of pathological proteins in cerebrovascular disorders, while both neurological imaging and biochemistry play an essential role in diagnosis and prognosis.

4. Alterations in Glymphatic Function Following TBI

4.1 Alterations in Glymphatic Flux and AQP4 Polarization Following TBI

Mechanical shear forces and pressure waves induced by TBI trigger immediate astrocyte activation and loss of AQP4 polarization. Astrocytes play a complex and dual role in the progression of TBI, with their functions differing significantly between acute and chronic phases. During the acute phase, reactive astrogliosis primarily exerts protective effects. Activated astrocytes form a glial scar, referred to in the literature as the perilesional glial barrier, which sequesters the core injury area and confines the spread of damage and infiltration of inflammatory cells, thereby preserving surrounding healthy tissue. Astrocytes also contribute to the formation and regulation of cerebral edema through specific ion channels like AQP4 [79]. Although this activity may initially exacerbate cytotoxic edema, astrocyte activation and barrier formation are crucial for controlling subsequent secondary injury. During the chronic phase, the function of astrocytes is more contradictory: they maintain the activation and stabilization of the glial scar through long-term activity, thereby forming an obstacle to axonal regeneration and the recovery of neural circuits; yet, at the same time, it also has an impact on recovery in some active ways, including secretion of various neurotrophic factors. To promote neurogenesis and synaptogenesis, sustain angiogenesis, and aid in axonal remodelling, GDNF and VEGF stimulate additional factors, such as astrocytes. Through their effects on inflammation, cellular homeostasis, and the establishment of an appropriate environment for brain plasticity and repair, astrocytes ensure sufficient levels of GDNF and VEGF that facilitate these processes [79, 81, 82]. Experimental studies utilizing murine TBI models demonstrate significant reductions in parenchymal CSF-ISF exchange efficiency post-injury, accompanied by decreased polarized distribution of AQP4 at astrocytic endfeet membranes [44, 83]. These pathological changes facilitate rapid cerebral accumulation of metabolic byproducts (including A\beta and tau species), subsequently exacerbating neuroinflammatory responses and cellular apoptosis [18]. Notably, TBI models employing AT1 receptor knockout mice exhibit preserved expression of BBB tight junction proteins (Occludin and ZO-1), restoration of AQP4 polarization, and concomitant reductions in both A\beta40 and A\beta42 levels [13, 18, 84, 85]. These observations suggest that the adrenergic-angiotensin system modulates glymphatic functionality and exerts neuroprotective effects on post-TBI waste clearance [85]. The progressive actions of pyroptotic inflammation within secondary TBI pathogenesis merit special attention. Induced via NLRP3-caspase pathway engagement, this inflammatory response results in the simultaneous polarized depolarization of AQP4 and increased BBB permeability, leading to further obstructions for metabolic waste efflux [77].

It is clear that the outcomes can reveal possible impacts of post-TBI on various causes responsible for the glymphatic function disorders in general as well as present a basis for a targeted intervention to improve neurological outcomes after TBI. Besides, it has been demonstrated that the TBI affected clearance may involve neurovascular regulation and inflammatory pathways. Both seem essential elements of a treatment.

4.2 Mechanistic Interplay Between the BBB and Glymphatic System in TBI Pathogenesis

The BBB and the GL arranged for their separate but coordinated functions to accomplish the maintenance of neural homeostasis; however, a TBI would concurrently impair the BBB and GL despite their inherently divergent physiology. The TBI damages not only the tight junctional protein of the vascular endothelium (claudin-5 and occludin) in the BBB to increase its permeability, but also alters cerebral hemodynamics to decrease the glymphatic inflow driven by cardiac pulsation [39]. Extravasated blood components including albumin and hemoglobin infiltrate the interstitial compartment, triggering activation of neuroinflammatory cascades in astrocytes and microglia, and formation of persistent A\beta-protein complexes that obstruct interstitial channels and impair CSF-ISF exchange networks [86]. Genetic ablation studies using AT1 receptor knockout mice reveal a neuroprotective paradigm wherein BBB stabilization facilitates AQP4 repolarization at perivascular endfeet, partially restoring glymphatic flow and enhancing A\beta clearance—highlighting the therapeutic potential of combined BBB-glymphatic system protection [85].

The relationship between the BBB, iron deposition, and cognitive function is increasingly recognized as a critical axis in both neurodevelopmental and neurodegenerative processes. The BBB serves as a dynamic interface that regulates the exchange of substances between the blood and the brain, including iron, an essential element for neuronal metabolism, myelination, and neurotransmitter synthesis. Dysfunction of the BBB, through increased permeability or impaired water exchange, can lead to aberrant iron accumulation in susceptible brain regions such as the globus pallidus, substantia nigra, and thalamus [87, 88].

Quantitative susceptibility mapping (QSM) studies reveal that iron deposition follows a region-specific trajectory, with rapid accumulation during early development and a plateau in later life. These iron dynamics are closely coupled to BBB function, as measured by techniques such as diffusion-prepared pseudo-continuous arterial spin labeling (DP-pCASL), which quantifies the water exchange rate across the BBB. This rate serves as a sensitive indicator of BBB efficiency, with lower values associated with higher iron accumulation and potentially impaired clearance mechanisms [89].

In pathological contexts such as AD and cerebral small vessel disease, BBB breakdown facilitates the extravasation of neurotoxic substances promoting oxidative stress, neuroinflammation, and neuronal damage. The APOE \epsilon4 allele, a major genetic risk factor for AD, exacerbates BBB dysfunction and iron deposition, further accelerating cognitive decline. Conversely, in early development, regulated iron transport via the BBB is essential for normal cognitive maturation, and disruptions in this process may contribute to neurodevelopmental impairments [90].

These pathophysiological insights demonstrate that optimal post-TBI metabolic clearance requires coordinated therapeutic strategies targeting both BBB integrity restoration and glymphatic pathway reconstruction, emphasizing the need for dual-system intervention approaches in clinical management.

4.3 Post-TBI Protein Accumulation and Neurodegenerative Risk

The pathological accumulation of metabolic byproducts following TBI represents a critical determinant of long-term neurodegenerative consequences [91]. Impaired fluid dynamics coupled with elevated A\beta and p-tau levels in both cerebrospinal fluid and interstitial compartments not only acutely compromise neuronal synaptic function but also induce sustained neuroinflammatory responses and glial activation, thereby potentiating the risk of developing AD-like pathology or chronic traumatic encephalopathy [18, 92]. Beyond classical amyloid and tau pathology, astrocyte-derived secretory factors such as CHI3L1 demonstrate significant post-TBI elevation. These molecules may impair neural repair mechanisms by suppressing neural stem cell proliferation and differentiation, further compromising neuroregenerative capacity [15]. Concurrent activation of oxidative stress and pyroptotic pathways promotes the release of inflammatory mediators (caspases and IL-1\beta), which synergistically exacerbate the neurotoxic effects of A\beta and tau aggregates, establishing a self-perpetuating pathological cycle [77, 93]. Discoveries made using models with simulated microgravity show that endogenous formaldehyde accumulated via plasma protein cross-linking leads to A\beta interstitial space obstruction; hence, environmental co-factors could contribute to continued post-TBI interstitial congestion, synergistically contributing to the pathological process [86, 92]. The alterations in glymphatic function following TBI and the therapeutic interventions is shown in Fig. 2.

4.4 Advancements in Murine and Clinical Research

Preclinical investigations with murine TBI models indicated that CBD administration could enhance motor and cognitive recovery, accelerate cerebral tracer clearance, restore AQP4 polarization, and reduce p-tau/A\beta burden, suggesting therapeutic potential through glymphatic-lymphatic system modulation. All these beneficial effects disappeared when deep cervical lymphatics were ligated, providing definitive evidence for the essential role of the meningeal lymphatic network in CBD-mediated clearance enhancement [94].

Translational clinical studies employing DTI-ALPS have pioneered noninvasive glymphatic assessment in TBI patients. Initial correlational analyses reveal an inverse relationship between diminished ALPS indices and elevated CSF A\beta/tau concentrations, supporting the utility of this imaging biomarker for evaluating glymphatic clearance capacity [18]. Complementary DCE-MRI techniques quantitatively characterize glymphatic kinetics through contrast agent retention/clearance profiles, with methodology validation established in stroke models [19].

Even though existing clinical reports of accumulating evidence are currently few in number, such proof-of-concept studies lay an important groundwork for future multicenter clinical trials. Combining our preclinical mechanistic insights with the growing body of clinical biomarkers will continue to form the basis upon which we may provide disease-targeted interventions.

5. Aberrant Fluid Biomarkers in TBI and Their Association With Glymphatic Impairment

5.1 Clearance Kinetics of \beta-Amyloid and Phosphorylated Tau Proteins

The A\beta peptides and p-tau represent considerable fluid biomarkers of TBI-induced injury and subsequent axonal insult and deterioration that heavily depend on glymphatic pathways for cerebral clearance. Exercise interventions increase A\beta clearance rates with upregulated AQP4 and enhanced polarization efficiency while decreasing neuroinflammatory responses and restoring cognition deficits, irrespective of age-related change. However, no changes were noted for BBB permeability, suggesting specificity in the glymphatic mediations of A\beta clearance [37].

Limbrick et al. [6] reported in their clinical investigation elevated concentrations of CSF sAPP\alpha, sAPP\beta, A\beta42, p-tau in congenital hydrocephalus and postulated the increase in biomarkers as a consequence of CSF stasis and dysfunction of flow-out-of-cisterns. CSF stasis and inhibited flow lead to massive overproduction of CSF biomarkers, consistent with a previous report on TBI biomarkers [6]. Cutting-edge molecular biological approaches employing in vivo microinjection coupled with mass spectrometric detection have further validated tau transport through AQP4-dependent glymphatic routes into the CSF, enabling quantitative assessment of tau clearance kinetics via tracer methodology [71].

All these findings present a deeper understanding on how the glymphatic system is controlling the levels of certain proteins after TBI by regulating post-TBI changes in A\beta or p-tau concentration, while simultaneously linking all evidence. The integrated collection of interventional, clinical, and molecular evidence supports a general unifying framework that describes how proteinopathies resulting from neural injury change over time.

5.2 AQP4 Expression, Polarization and Localization Dynamics

The water channel protein AQP4 serves as the principal molecular effector of glymphatic function, with its quantitative expression and polarized distribution directly governing CSF-ISF exchange efficiency. Aerobic exercise training has been shown to upregulate AQP4 expression while reorganizing its polarized orientation, thereby enhancing A\beta clearance capacity and attenuating astrocytic/microglial activation [37]. TBI and stroke models have both demonstrated decreases in polarized AQP4 localization at the endfoot membrane along with a corresponding shift towards diffuse cellular localization. This depolazised state correlates highly with neuro-inflammation and the pyroptotic pathway activation, which also contributes to basement membrane remodelling. All of this results in impaired glymphatic flux and an increased accumulation of metabolic waste products [19, 77]. Clinical investigations have explored the diagnostic potential of AQP4-containing exosomes in CSF and plasma, with emerging evidence suggesting correlations between exosomal AQP4 levels and TBI severity/outcome measures. These findings support the development of AQP4 depolarization as a novel biochemical marker for TBI assessment. Recent mechanistic studies have identified the p38MAPK/NF-\kappaB signaling pathway as a regulator of AQP4 expression and its co-localization with tight junction proteins (Occludin, ZO-1) in intestinal epithelial cells. This discovery provides new insights into the tissue-specific regulatory mechanisms of AQP4 dynamics [95]. It might be the future development of therapies altering glymphatic drainage that extend to multiple organs. Dysregulation of glymphatic clearance has been linked to disrupted perivascular polarization of AQP4. In rodent models, the dynamic redistribution of AQP4 to both the blood-spinal cord and BBB attenuates CNS edema and promotes functional recovery [29, 62]. Therapeutics targeting AQP4 subcellular localization can halt CNS edema in rats [79].

5.3 Alterations in Lysosomal-Associated Markers LAMP-1/2

The research into both lysosome-associated membrane proteins LAMP-1 and LAMP-2 in the field of neurodegenerative diseases and lysosomal storage disorders is very extensive. Little attention has been given to the assessment of glymphatic biomarkers following TBI [42, 74]. Given the critical dependence of astrocytes and neurons on endosomal-lysosomal pathways for clearing both exogenous and endogenous protein aggregates, quantitative changes in LAMP-1/2 expression may serve as indicators of compromised intracellular degradative capacity. Such impairment could significantly influence the glymphatic system’s ability to intercept and transport metabolic waste products to clearance pathways [42].

Future investigations should employ integrated methodologies combining ELISA-based quantification of CSF LAMP-1/2 levels with advanced neuroimaging techniques to elucidate their biological significance in post-TBI clearance dysfunction. This multimodal approach may reveal novel mechanistic insights into the dysregulation of protein homeostasis following neural trauma [17]. SP-G is postulated to reflect alterations in the extracellular matrix, while LAMP-1/2 is indicative of lysosomal function/autophagy activity. These emerging molecular biomarkers have the potential to complement and enrich the AQP4-centric framework from orthogonal perspectives. They may provide critical insights into distinct pathological processes, such as neuroinflammation and impaired protein degradation. Looking forward, it may be feasible to construct a multimodal biomarker panel that integrates AQP4 (water transport), neuroimaging (system-level function), and these molecular indicators (microscopic pathology), thereby enabling more precise disease subtyping and therapeutic target selection. The key molecules of glymphatic system and their functional consequences following TBI are summarized in Table 1.

6. Predictive Utility of DCE-MRI and DTI-ALPS for TBI Fluid Biomarker Concentrations

6.1 Quantitative Assessment of Glymphatic Function Using DCE-MRI Kinetics

Dynamic contrast-enhanced MRI enables real-time visualization and quantification of glymphatic fluid dynamics through sequential monitoring of contrast agent (Gd-DTPA) influx, retention, and clearance patterns in cerebral tissue [96]. As reviewed by Verheggen et al. [39], this technique provides critical metrics including CSF-ISF exchange rates and contrast retention duration, reflecting both arterial pulsation-driven flow and AQP4 functional status [97].

The experimental application on rodent models of ischemic stroke verifies that the DCE-MRI parameters undergo synchronous changes in association with the APP deposition in secondary thalamic infarcts, confirming the feasibility of utilizing this method to analyze the abnormal glymphatic function due to post-traumatic or vascular injuries [19, 97]. Some preliminary studies applied DCE-MRI on the TBI population and found that compared to un-injured groups, both the lesioned and peri-lesional areas showed prolonged CSF clearance time which correlated with higher CSF A\beta and p-tau concentrations. Thus, DCE-MRI is highly suitable for identifying an altered glymphatic function after neural trauma [98].

6.2 Correlation Between DTI-ALPS Indices and Fluid Biomarkers

The DTI-ALPS approach is an objective method to quantify the glymphatic system by depicting the water molecule diffusion pattern inside the peri-vessels and demonstrate the reduction of ALPS index after TBI presents strong inverse correlations with cognitive impairment and increased A\beta/tau species levels in CSF; meanwhile, the proposed method demonstrates excellent reproducibility, non-invasiveness and full-brain coverage [18]. Some preliminary validations have been performed on small TBI cohort that the association between the ALPS parameters and fluid biomarkers are consistent and robust, providing theoretical foundation for this non-invasive neuroimaging surrogate for assessing glymphatic function in different condition and pathological states [99].

6.3 Integrated Multimodal Neuroimaging Analysis

In order to cope with the inherent shortcomings of single-modality imaging techniques, researchers in today’s era increasingly rely on multimodal neuroimaging. For example, there is a typical scheme that jointly employs kinetic parameters acquired from DCE-MRI and DTI-ALPS indices to evaluate the rates of contrast agent clearance and the flow of aqueous diffusion simultaneously [17, 97]. This integrated approach, when correlated with multi-omics data including CSF/plasma concentrations of A\beta, p-tau, and AQP4-containing exosomes, yields a comprehensive assessment of glymphatic system functionality [17]. The primary challenges in translating DCE-MRI and DTI-ALPS into clinical practice include the need to standardize data acquisition and analysis protocols across different centers and scanner platforms, to improve technical reproducibility and standardization, and to establish normative values and diagnostic thresholds in large-scale, diverse TBI cohorts. It is essential to evaluate the specificity and sensitivity of these imaging techniques in distinguishing TBI from other neurological disorders, and to longitudinally correlate imaging findings with clinical outcomes, such as cognitive recovery. Considerations of accessibility and cost-effectiveness are critical to ensure that these advanced MRI sequences can be widely adopted and sustainably implemented in routine clinical settings. Future investigations should focus on validating the diagnostic and prognostic utility of such combined imaging protocols through large-scale multicenter studies. This effort will establish a robust dual-parameter biomarker system integrating neuroimaging and biochemical profiles to advance personalized therapeutic strategies for TBI management [32, 35, 99].

7. Therapeutic Interventions and Future Directions

7.1 Therapeutic Interventions Targeting Glymphatic Function: Exercise, Pharmacological and Molecular Approaches

Evidences show that glymphatic-targeted interventions improve the efficiency of waste clearance in TBI as well as other neuropathologies. Aerobic exercise changes both the cardiac-induced arterial pulsatility and AQP4 expression/repolarization to boost the CSF-ISF exchange and diminish the deposition and inflammation of A\beta, which confer cognitive protections on aging mouse models [37]. Given its exceptional safety profile, low cost, and broad health benefits, we consider it the most feasible strategy worthy of prioritization for large-scale clinical trials, particularly in the context of rehabilitation management. Therapeutic strategies targeting AQP4 relocalization demonstrate considerable potential. For instance, administration of trifluoperazine (TFP) to inhibit AQP4 relocalization has been shown to significantly ameliorate CNS edema in rat models [79, 100, 101]. The neuroprotective phytocannabinoid CBD exhibits multimodal therapeutic actions in TBI, including augmentation of meningeal lymphatic and glymphatic flow velocities, restoration of AQP4 polarization, and reduction of p-tau/A\beta burden. These mechanisms collectively improve motor and memory deficits, with complete abolition of therapeutic effects following cervical lymphatic blockade—confirming the essential role of the brain-lymphatic axis [94]. Molecular targeting strategies show particular promise, as demonstrated by AQP4 inhibitors regulating tau clearance in AD models, suggesting potential applications for modulating dysfunctional AQP4 in chronic TBI [60]. Despite encouraging preclinical results, clinical evidence remains preliminary. Complicated regulatory landscapes across regions, alongside unresolved issues related to dosing and standardization, suggest that CBD should be investigated at a later stage, following further resolution of these constraints. AT1 receptor antagonists preserve BBB integrity and restore glymphatic flow post-TBI, identifying novel pharmacological targets for post-traumatic waste clearance [85]. As approved antihypertensive agents with well-established safety profiles and substantial preclinical and emerging epidemiological evidence, these compounds present a clear translational pathway. Their strong supporting evidence justifies priority consideration for drug repurposing clinical trials. Interventions targeting the CHI3L1/CRTH2/\beta-catenin signaling pathway may dually modulate astrocytic function and neurogenesis, offering therapeutic potential for chronic neuroinflammatory conditions with impaired clearance [15]. These findings collectively establish a framework for developing precision therapies targeting glymphatic dysfunction across neurological disorders. The therapeutic strategies targeting the glymphatic system are summarized in Table 2.

7.2 Biomimetic Microfluidic Models for In Vitro Investigation of Glymphatic Dynamics

The development of microfluidic chip technology (glymphatics-on-a-chip) represents a significant advancement in elucidating the molecular regulatory mechanisms governing glymphatic system dynamics. These innovative platforms employ co-culture systems incorporating human astrocytes and cerebral microvascular endothelial cells to accurately simulate CSF-ISF flow dynamics [102]. Experimental validation using this model has demonstrated the dual effects of LPS, A\beta oligomers, and AQP4 inhibitors on both fluid transport pathways and A\beta clearance efficiency. Flow resistance increased in cells following LPS treatment even after cell lysis, indicating that both matrix degradation and cell-dependent clearance mechanisms might coexist simultaneously [47]. When integrated with experimental TBI paradigms, such microfluidic systems enable precise modulation of injury severity, inflammatory status, and AQP4 polarization patterns in vitro. This technological approach establishes a high-throughput, cost-effective platform for subsequent pharmacological screening and pathological mechanism investigation, offering substantial advantages over conventional in vivo models.

7.3 Chronobiological Rhythms and Sleep Modulation of Glymphatic Function

Research on the circadian regulation of glymphatic activity suggests that good sleep quality and circadian rhythms stabilization may be alternative ways to improve post-TBI metabolic clearance without pharmaceutical intervention. Chronic sleep-deprivation animal models reveal much higher nocturnal AQP4 polarization and glymphatic clearance capacity than those during daytime [103]. Sustained sleep limitation annihilates this circadian difference with correspondingly high brain A\beta1–42 and p-tau accumulation leading to accelerated cognitive deterioration, which underpins the need to preserve normal sleep wake cycles to ensure physiologically normal clearance kinetics [46].

7.4 Future Directions

Advancing investigations should focus on multidimensional data integration (incorporating neuroimaging, CSF/plasma biomarkers, and genomic profiles) to establish international consensus standards for the glymphatic-biomarker axis, facilitating early TBI diagnosis and precision therapeutics. Beyond AQP4, emerging molecular targets including surfactant protein SP-G, CHI3L1, and LAMP-1/2 may expand both diagnostic and therapeutic landscapes [9, 42, 102, 104]. Multifactorial studies incorporating microgravity simulation and environmental stressors will elucidate complex endogenous/exogenous influences on glymphatic function. Ultimately, interdisciplinary collaboration will prove pivotal in transforming fundamental discoveries into innovative clinical management strategies for neural trauma and related disorders.

8. Conclusion

We present glymphatic dysfunction as an important pathologic feature observed after TBI in post-TBI biomarkers for development of TBI precision therapies, and propose the foundations of chronotherapeutic targeting to restore glymphatic function clearance. Continued investigation regarding multimodal biomarker validation is needed along with investigation of chronotherapeutic interventions aimed at maximally enhancing glymphatic function.

References

[1] Raja HAA, Chaurasia B. Prognostication in traumatic brain injury. Neurosurgical Review. 2024; 47: 314. https://doi.org/10.1007/s10143-024-02574-y.

[2] Dams-O’Connor K, Juengst SB, Bogner J, Chiaravalloti ND, Corrigan JD, Giacino JT, et al. Traumatic brain injury as a chronic disease: insights from the United States Traumatic Brain Injury Model Systems Research Program. The Lancet. Neurology. 2023; 22: 517–528. https://doi.org/10.1016/S1474-4422(23)00065-0.

[3] Capizzi A, Woo J, Verduzco-Gutierrez M. Traumatic Brain Injury: An Overview of Epidemiology, Pathophysiology, and Medical Management. The Medical Clinics of North America. 2020; 104: 213–238. https://doi.org/10.1016/j.mcna.2019.11.001.

[4] Maas AIR, Menon DK, Manley GT, Abrams M, Åkerlund C, Andelic N, et al. Traumatic brain injury: progress and challenges in prevention, clinical care, and research. The Lancet. Neurology. 2022; 21: 1004–1060. https://doi.org/10.1016/S1474-4422(22)00309-X.

[5] Guan B, Anderson DB, Chen L, Feng S, Zhou H. Global, regional and national burden of traumatic brain injury and spinal cord injury, 1990-2019: a systematic analysis for the Global Burden of Disease Study 2019. BMJ Open. 2023; 13: e075049. https://doi.org/10.1136/bmjopen-2023-075049.

[6] Limbrick DD, Jr, Baksh B, Morgan CD, Habiyaremye G, McAllister JP, II, Inder TE, et al. Cerebrospinal fluid biomarkers of infantile congenital hydrocephalus. PLoS ONE. 2017; 12: e0172353. https://doi.org/10.1371/journal.pone.0172353.

[7] Fang Y, Liu Y, Chen L, Wang J, Zhang J, Zhang H, et al. Cerebrospinal fluid markers of neuroinflammation and coagulation in severe cerebral edema and chronic hydrocephalus after subarachnoid hemorrhage: a prospective study. Journal of Neuroinflammation. 2024; 21: 237. https://doi.org/10.1186/s12974-024-03236-y.

[8] Ferrara M, Bertozzi G, Volonnino G, Di Fazio N, Frati P, Cipolloni L, et al. Glymphatic System a Window on TBI Pathophysiology: A Systematic Review. International Journal of Molecular Sciences. 2022; 23: 9138. https://doi.org/10.3390/ijms23169138.

[9] Krause M, Peukert N, Härtig W, Emmer A, Mahr CV, Richter C, et al. Localization, Occurrence, and CSF Changes of SP-G, a New Surface Active Protein with Assumable Immunoregulatory Functions in the CNS. Molecular Neurobiology. 2019; 56: 2433–2439. https://doi.org/10.1007/s12035-018-1247-x.

[10] Zhao ZA, Li P, Ye SY, Ning YL, Wang H, Peng Y, et al. Perivascular AQP4 dysregulation in the hippocampal CA1 area after traumatic brain injury is alleviated by adenosine A_2⁢A receptor inactivation. Scientific Reports. 2017; 7: 2254. https://doi.org/10.1038/s41598-017-02505-6.

[11] Connolly K, Lehoux M, O’Rourke R, Assetta B, Erdemir GA, Elias JA, et al. Potential role of chitinase-3-like protein 1 (CHI3L1/YKL-40) in neurodegeneration and Alzheimer’s disease. Alzheimer’s & Dementia. 2023; 19: 9–24. https://doi.org/10.1002/alz.12612.

[12] Rasmussen MK, Mestre H, Nedergaard M. Fluid transport in the brain. Physiological Reviews. 2022; 102: 1025–1151. https://doi.org/10.1152/physrev.00031.2020.

[13] Sullan MJ, Asken BM, Jaffee MS, DeKosky ST, Bauer RM. Glymphatic system disruption as a mediator of brain trauma and chronic traumatic encephalopathy. Neuroscience and Biobehavioral Reviews. 2018; 84: 316–324. https://doi.org/10.1016/j.neubiorev.2017.08.016.

[14] Beschorner N, Nedergaard M. Glymphatic system dysfunction in neurodegenerative diseases. Current Opinion in Neurology. 2024; 37: 182–188. https://doi.org/10.1097/WCO.0000000000001252.

[15] Huang SY, Zhang YR, Guo Y, Du J, Ren P, Wu BS, et al. Glymphatic system dysfunction predicts amyloid deposition, neurodegeneration, and clinical progression in Alzheimer’s disease. Alzheimer’s & Dementia. 2024; 20: 3251–3269. https://doi.org/10.1002/alz.13789.

[16] Yue Y, Zhang X, Lv W, Lai HY, Shen T. Interplay between the glymphatic system and neurotoxic proteins in Parkinson’s disease and related disorders: current knowledge and future directions. Neural Regeneration Research. 2024; 19: 1973–1980. https://doi.org/10.4103/1673-5374.390970.

[17] Naganawa S, Taoka T, Ito R, Kawamura M. The Glymphatic System in Humans: Investigations With Magnetic Resonance Imaging. Investigative Radiology. 2024; 59: 1–12. https://doi.org/10.1097/RLI.0000000000000969.

[18] Peters ME, Lyketsos CG. The glymphatic system’s role in traumatic brain injury-related neurodegeneration. Molecular Psychiatry. 2023; 28: 2707–2715. https://doi.org/10.1038/s41380-023-02070-7.

[19] Li C, Lin L, Sun C, Hao X, Yin L, Zhang X, et al. Glymphatic system in the thalamus, secondary degeneration area was severely impaired at 2nd week after transient occlusion of the middle cerebral artery in rats. Frontiers in Neuroscience. 2022; 16: 997743. https://doi.org/10.3389/fnins.2022.997743.

[20] Ghanizada H, Nedergaard M. The glymphatic system. Handbook of Clinical Neurology. 2025; 209: 161–170. https://doi.org/10.1016/B978-0-443-19104-6.00006-1.

[21] Zhu HH, Li SS, Wang YC, Song B, Gao Y, Xu YM, et al. Clearance dysfunction of trans-barrier transport and lymphatic drainage in cerebral small vessel disease: Review and prospect. Neurobiology of Disease. 2023; 189: 106347. https://doi.org/10.1016/j.nbd.2023.106347.

[22] Graham NS, Sharp DJ. Dementia after traumatic brain injury. BMJ. 2023; 383: 2065. https://doi.org/10.1136/bmj.p2065.

[23] Reuben DB, Kremen S, Maust DT. Dementia Prevention and Treatment: A Narrative Review. JAMA Internal Medicine. 2024; 184: 563–572. https://doi.org/10.1001/jamainternmed.2023.8522.

[24] Shlobin NA, Staple BL, Sclafani M, Harter DH. The Glymphatic System and Subarachnoid Lymphatic-Like Membrane: Recent Developments in Cerebrospinal Fluid Research. World Neurosurgery. 2024; 190: 147–156. https://doi.org/10.1016/j.wneu.2024.07.062.

[25] Holstein-Rønsbo S, Gan Y, Giannetto MJ, Rasmussen MK, Sigurdsson B, Beinlich FRM, et al. Glymphatic influx and clearance are accelerated by neurovascular coupling. Nature Neuroscience. 2023; 26: 1042–1053. https://doi.org/10.1038/s41593-023-01327-2.

[26] Lopes DM, Wells JA, Ma D, Wallis L, Park D, Llewellyn SK, et al. Glymphatic inhibition exacerbates tau propagation in an Alzheimer’s disease model. Alzheimer’s Research & Therapy. 2024; 16: 71. https://doi.org/10.1186/s13195-024-01439-2.

[27] Ye F, Keep RF, Hua Y, Garton HJL, Xi G. Glymphatic System and Post-hemorrhagic Hydrocephalus. Brain Hemorrhages. 2023; 4: 44–46. https://doi.org/10.1016/j.hest.2022.06.001.

[28] Alshuhri MS, Gallagher L, Work LM, Holmes WM. Direct imaging of glymphatic transport using H217O MRI. JCI Insight. 2021; 6: e141159. https://doi.org/10.1172/jci.insight.141159.

[29] Salman MM, Kitchen P, Halsey A, Wang MX, Törnroth-Horsefield S, Conner AC, et al. Emerging roles for dynamic aquaporin-4 subcellular relocalization in CNS water homeostasis. Brain. 2022; 145: 64–75. https://doi.org/10.1093/brain/awab311.

[30] Szlufik S, Kopeć K, Szleszkowski S, Koziorowski D. Glymphatic System Pathology and Neuroinflammation as Two Risk Factors of Neurodegeneration. Cells. 2024; 13: 286. https://doi.org/10.3390/cells13030286.

[31] Barichello de Quevedo JL, Leffa DT, Pascoal TA. Glymphatic system waste clearance and Alzheimer’s disease. Brazilian Journal of Psychiatry. 2023; 45: 385–386. https://doi.org/10.47626/1516-4446-2023-0049.

[32] Eide PK, Undseth RM, Pripp A, Lashkarivand A, Nedregaard B, Sletteberg R, et al. Impact of Subarachnoid Hemorrhage on Human Glymphatic Function: A Time-Evolution Magnetic Resonance Imaging Study. Stroke. 2025; 56: 678–691. https://doi.org/10.1161/STROKEAHA.124.047739.

[33] Xiong A, Li J, Xiong R, Xia Y, Jiang X, Cao F, et al. Inhibition of HIF-1α-AQP4 axis ameliorates brain edema and neurological functional deficits in a rat controlled cortical injury (CCI) model. Scientific Reports. 2022; 12: 2701. https://doi.org/10.1038/s41598-022-06773-9.

[34] Chachaj A, Gąsiorowski K, Szuba A, Sieradzki A, Leszek J. The Lymphatic System In The Brain Clearance Mechanisms - New Therapeutic Perspectives For Alzheimer’s Disease. Current Neuropharmacology. 2023; 21: 380–391. https://doi.org/10.2174/1570159X20666220411091332.

[35] Hussain R, Tithof J, Wang W, Cheetham-West A, Song W, Peng W, et al. Potentiating glymphatic drainage minimizes post-traumatic cerebral oedema. Nature. 2023; 623: 992–1000. https://doi.org/10.1038/s41586-023-06737-7.

[36] Sun YR, Lv QK, Liu JY, Wang F, Liu CF. New perspectives on the glymphatic system and the relationship between glymphatic system and neurodegenerative diseases. Neurobiology of Disease. 2025; 205: 106791. https://doi.org/10.1016/j.nbd.2025.106791.

[37] He XF, Liu DX, Zhang Q, Liang FY, Dai GY, Zeng JS, et al. Voluntary Exercise Promotes Glymphatic Clearance of Amyloid Beta and Reduces the Activation of Astrocytes and Microglia in Aged Mice. Frontiers in Molecular Neuroscience. 2017; 10: 144. https://doi.org/10.3389/fnmol.2017.00144.

[38] Li B, Wei M, Wan X, Chen Z, Liu M, Fan Z, et al. Neuroprotective effects of lentivirus-mediated aquaporin-4 gene silencing in rat model of traumatic brain injury. Neurological Research. 2022; 44: 692–699. https://doi.org/10.1080/01616412.2022.2039509.

[39] Verheggen ICM, Van Boxtel MPJ, Verhey FRJ, Jansen JFA, Backes WH. Interaction between blood-brain barrier and glymphatic system in solute clearance. Neuroscience and Biobehavioral Reviews. 2018; 90: 26–33. https://doi.org/10.1016/j.neubiorev.2018.03.028.

[40] Zhang Y, Zhang C, He XZ, Li ZH, Meng JC, Mao RT, et al. Interaction Between the Glymphatic System and α-Synuclein in Parkinson’s Disease. Molecular Neurobiology. 2023; 60: 2209–2222. https://doi.org/10.1007/s12035-023-03212-2.

[41] Sharp MM, Cassidy J, Thornton T, Lyles J, Keable A, Gatherer M, et al. The α-dystrobrevins play a key role in maintaining the structure and function of the extracellular matrix-significance for protein elimination failure arteriopathies. Acta Neuropathologica Communications. 2021; 9: 171. https://doi.org/10.1186/s40478-021-01274-8.

[42] Zhang J, Zeng W, Han Y, Lee WR, Liou J, Jiang Y. Lysosomal LAMP proteins regulate lysosomal pH by direct inhibition of the TMEM175 channel. Molecular Cell. 2023; 83: 2524–2539.e7. https://doi.org/10.1016/j.molcel.2023.06.004.

[43] Cai Y, Zhang Y, Leng S, Ma Y, Jiang Q, Wen Q, et al. The relationship between inflammation, impaired glymphatic system, and neurodegenerative disorders: A vicious cycle. Neurobiology of Disease. 2024; 192: 106426. https://doi.org/10.1016/j.nbd.2024.106426.

[44] Lu H, Zhan Y, Ai L, Chen H, Chen J. AQP4-siRNA alleviates traumatic brain edema by altering post-traumatic AQP4 polarity reversal in TBI rats. Journal of Clinical Neuroscience. 2020; 81: 113–119. https://doi.org/10.1016/j.jocn.2020.09.015.

[45] Dadgostar E, Rahimi S, Nikmanzar S, Nazemi S, Naderi Taheri M, Alibolandi Z, et al. Aquaporin 4 in Traumatic Brain Injury: From Molecular Pathways to Therapeutic Target. Neurochemical Research. 2022; 47: 860–871. https://doi.org/10.1007/s11064-021-03512-w.

[46] Zhai S, Yin MM, Sun HQ, Jiang XQ, Liu Y, Marshall C, et al. The day-night differences in cognitive and anxiety-like behaviors of mice after chronic sleep restriction. FASEB Journal. 2023; 37: e23034. https://doi.org/10.1096/fj.202202040RR.

[47] Soden PA, Henderson AR, Lee E. A Microfluidic Model of AQP4 Polarization Dynamics and Fluid Transport in the Healthy and Inflamed Human Brain: The First Step Towards Glymphatics-on-a-Chip. Advanced Biology. 2022; 6: e2200027. https://doi.org/10.1002/adbi.202200027.

[48] Wang M, Yu X, Li B, Gao C, Chen Y, Zhang X, et al. miR-211-5p targeting MMP9 regulates the expressions of AQP4 in traumatic brain injury. Acta Neurologica Belgica. 2023; 123: 1321–1329. https://doi.org/10.1007/s13760-023-02205-1.

[49] Ortiz C, Pearson A, McCartan R, Roche S, Carothers N, Browning M, et al. Overexpression of pathogenic tau in astrocytes causes a reduction in AQP4 and GLT1, an immunosuppressed phenotype and unique transcriptional responses to repetitive mild TBI without appreciable changes in tauopathy. Journal of Neuroinflammation. 2024; 21: 130. https://doi.org/10.1186/s12974-024-03117-4.

[50] Ishibashi K, Hara S, Kondo S. Aquaporin water channels in mammals. Clinical and Experimental Nephrology. 2009; 13: 107–117. https://doi.org/10.1007/s10157-008-0118-6.

[51] Kourghi M, Pei JV, De Ieso ML, Nourmohammadi S, Chow PH, Yool AJ. Fundamental structural and functional properties of Aquaporin ion channels found across the kingdoms of life. Clinical and Experimental Pharmacology & Physiology. 2018; 45: 401–409. https://doi.org/10.1111/1440-1681.12900.

[52] Kitchen P, Day RE, Salman MM, Conner MT, Bill RM, Conner AC. Beyond water homeostasis: Diverse functional roles of mammalian aquaporins. Biochimica et Biophysica Acta. 2015; 1850: 2410–2421. https://doi.org/10.1016/j.bbagen.2015.08.023.

[53] Kitchen P, Salman MM, Pickel SU, Jennings J, Törnroth-Horsefield S, Conner MT, et al. Water channel pore size determines exclusion properties but not solute selectivity. Scientific Reports. 2019; 9: 20369. https://doi.org/10.1038/s41598-019-56814-z.

[54] Verkman AS, Anderson MO, Papadopoulos MC. Aquaporins: important but elusive drug targets. Nature Reviews. Drug Discovery. 2014; 13: 259–277. https://doi.org/10.1038/nrd4226.

[55] Hara-Chikuma M, Verkman AS. Physiological roles of glycerol-transporting aquaporins: the aquaglyceroporins. Cellular and Molecular Life Sciences. 2006; 63: 1386–1392. https://doi.org/10.1007/s00018-006-6028-4.

[56] Wang S, Solenov EI, Yang B. Aquaporin Inhibitors. Advances in Experimental Medicine and Biology. 2023; 1398: 317–330. https://doi.org/10.1007/978-981-19-7415-1_22.

[57] Tradtrantip L, Jin BJ, Yao X, Anderson MO, Verkman AS. Aquaporin-Targeted Therapeutics: State-of-the-Field. Advances in Experimental Medicine and Biology. 2017; 969: 239–250. https://doi.org/10.1007/978-94-024-1057-0_16.

[58] Bill RM. Drugging aquaporins. Biochimica et Biophysica Acta. Biomembranes. 2024; 1866: 184164. https://doi.org/10.1016/j.bbamem.2023.184164.

[59] Abir-Awan M, Kitchen P, Salman MM, Conner MT, Conner AC, Bill RM. Inhibitors of Mammalian Aquaporin Water Channels. International Journal of Molecular Sciences. 2019; 20: 1589. https://doi.org/10.3390/ijms20071589.

[60] Harrison IF, Ismail O, Machhada A, Colgan N, Ohene Y, Nahavandi P, et al. Impaired glymphatic function and clearance of tau in an Alzheimer’s disease model. Brain. 2020; 143: 2576–2593. https://doi.org/10.1093/brain/awaa179.

[61] Salman MM, Kitchen P, Woodroofe MN, Brown JE, Bill RM, Conner AC, et al. Hypothermia increases aquaporin 4 (AQP4) plasma membrane abundance in human primary cortical astrocytes via a calcium/transient receptor potential vanilloid 4 (TRPV4)- and calmodulin-mediated mechanism. The European Journal of Neuroscience. 2017; 46: 2542–2547. https://doi.org/10.1111/ejn.13723.

[62] Ciappelloni S, Bouchet D, Dubourdieu N, Boué-Grabot E, Kellermayer B, Manso C, et al. Aquaporin-4 Surface Trafficking Regulates Astrocytic Process Motility and Synaptic Activity in Health and Autoimmune Disease. Cell Reports. 2019; 27: 3860–3872.e4. https://doi.org/10.1016/j.celrep.2019.05.097.

[63] Coloma M, Schaffer JD, Huang P, Chiarot PR. Boundary waves in a microfluidic device as a model for intramural periarterial drainage. Biomicrofluidics. 2019; 13: 024103. https://doi.org/10.1063/1.5080446.

[64] Aldea R, Weller RO, Wilcock DM, Carare RO, Richardson G. Cerebrovascular Smooth Muscle Cells as the Drivers of Intramural Periarterial Drainage of the Brain. Frontiers in Aging Neuroscience. 2019; 11: 1. https://doi.org/10.3389/fnagi.2019.00001.

[65] Spitz S, Ko E, Ertl P, Kamm RD. How Organ-on-a-Chip Technology Can Assist in Studying the Role of the Glymphatic System in Neurodegenerative Diseases. International Journal of Molecular Sciences. 2023; 24: 2171. https://doi.org/10.3390/ijms24032171.

[66] Xiong Y, Yu Q, Zhi H, Peng H, Xie M, Li R, et al. Advances in the study of the glymphatic system and aging. CNS Neuroscience & Therapeutics. 2024; 30: e14803. https://doi.org/10.1111/cns.14803.

[67] Yamada K, Iwatsubo T. Involvement of the glymphatic/meningeal lymphatic system in Alzheimer’s disease: insights into proteostasis and future directions. Cellular and Molecular Life Sciences. 2024; 81: 192. https://doi.org/10.1007/s00018-024-05225-z.

[68] Huber VJ, Tsujita M, Nakada T. Identification of aquaporin 4 inhibitors using in vitro and in silico methods. Bioorganic & Medicinal Chemistry. 2009; 17: 411–417. https://doi.org/10.1016/j.bmc.2007.12.040.

[69] Verkman AS, Smith AJ, Phuan PW, Tradtrantip L, Anderson MO. The aquaporin-4 water channel as a potential drug target in neurological disorders. Expert Opinion on Therapeutic Targets. 2017; 21: 1161–1170. https://doi.org/10.1080/14728222.2017.1398236.

[70] Choi YH, Laaker C, Hsu M, Cismaru P, Sandor M, Fabry Z. Molecular Mechanisms of Neuroimmune Crosstalk in the Pathogenesis of Stroke. International Journal of Molecular Sciences. 2021; 22: 9486. https://doi.org/10.3390/ijms22179486.

[71] Ishida K, Yamada K. Detection of Glymphatic Outflow of Tau from Brain to Cerebrospinal Fluid in Mice. Methods in Molecular Biology. 2024; 2754: 351–359. https://doi.org/10.1007/978-1-0716-3629-9_19.

[72] Formolo DA, Yu J, Lin K, Tsang HWH, Ou H, Kranz GS, et al. Leveraging the glymphatic and meningeal lymphatic systems as therapeutic strategies in Alzheimer’s disease: an updated overview of nonpharmacological therapies. Molecular Neurodegeneration. 2023; 18: 26. https://doi.org/10.1186/s13024-023-00618-3.

[73] Kelly L, Sharp MM, Thomas I, Brown C, Schrag M, Antunes LV, et al. Targeting lysyl-oxidase (LOX) may facilitate intramural periarterial drainage for the treatment of Alzheimer’s disease. Cerebral Circulation - Cognition and Behavior. 2023; 5: 100171. https://doi.org/10.1016/j.cccb.2023.100171.

[74] Ghavami S, Shojaei S, Yeganeh B, Ande SR, Jangamreddy JR, Mehrpour M, et al. Autophagy and apoptosis dysfunction in neurodegenerative disorders. Progress in Neurobiology. 2014; 112: 24–49. https://doi.org/10.1016/j.pneurobio.2013.10.004.

[75] Massoud AT, Noltensmeyer DA, Juranek J, Cox CS, Jr, Velasquez FC, Zhu B, et al. Insights into the Role of the Glymphatic System in the Pathogenesis of Post-hemorrhagic Hydrocephalus. Molecular Neurobiology. 2025; 62: 6537–6543. https://doi.org/10.1007/s12035-025-04692-0.

[76] Jiang D, Liu L, Kong Y, Chen Z, Rosa-Neto P, Chen K, et al. Regional Glymphatic Abnormality in Behavioral Variant Frontotemporal Dementia. Annals of Neurology. 2023; 94: 442–456. https://doi.org/10.1002/ana.26710.

[77] Lyu Z, Chan Y, Li Q, Zhang Q, Liu K, Xiang J, et al. Destructive Effects of Pyroptosis on Homeostasis of Neuron Survival Associated with the Dysfunctional BBB-Glymphatic System and Amyloid-Beta Accumulation after Cerebral Ischemia/Reperfusion in Rats. Neural Plasticity. 2021; 2021: 4504363. https://doi.org/10.1155/2021/4504363.

[78] Eide PK. Adult Hydrocephalus and the Glymphatic System. Neurosurgery Clinics of North America. 2025; 36: 127–140. https://doi.org/10.1016/j.nec.2024.11.002.

[79] Kitchen P, Salman MM, Halsey AM, Clarke-Bland C, MacDonald JA, Ishida H, et al. Targeting Aquaporin-4 Subcellular Localization to Treat Central Nervous System Edema. Cell. 2020; 181: 784–799.e19. https://doi.org/10.1016/j.cell.2020.03.037.

[80] Sylvain NJ, Salman MM, Pushie MJ, Hou H, Meher V, Herlo R, et al. The effects of trifluoperazine on brain edema, aquaporin-4 expression and metabolic markers during the acute phase of stroke using photothrombotic mouse model. Biochimica et Biophysica Acta. Biomembranes. 2021; 1863: 183573. https://doi.org/10.1016/j.bbamem.2021.183573.

[81] Linnerbauer M, Rothhammer V. Protective Functions of Reactive Astrocytes Following Central Nervous System Insult. Frontiers in Immunology. 2020; 11: 573256. https://doi.org/10.3389/fimmu.2020.573256.

[82] Alhadidi QM, Bahader GA, Arvola O, Kitchen P, Shah ZA, Salman MM. Astrocytes in functional recovery following central nervous system injuries. The Journal of Physiology. 2024; 602: 3069–3096. https://doi.org/10.1113/JP284197.

[83] Czyżewski W, Litak J, Sobstyl J, Mandat T, Torres K, Staśkiewicz G. Aquaporins: Gatekeepers of Fluid Dynamics in Traumatic Brain Injury. International Journal of Molecular Sciences. 2024; 25: 6553. https://doi.org/10.3390/ijms25126553.

[84] Lv C, Han S, Sha Z, Liu M, Dong S, Zhang C, et al. Cerebral glucagon-like peptide-1 receptor activation alleviates traumatic brain injury by glymphatic system regulation in mice. CNS Neuroscience & Therapeutics. 2023; 29: 3876–3888. https://doi.org/10.1111/cns.14308.

[85] Yang L, Chen Z, Wan X, Liu M, Wu J, Chen Y, et al. Angiotensin II type 1 receptor deficiency protects against the impairment of blood-brain barrier in a mouse model of traumatic brain injury. The International Journal of Neuroscience. 2023; 133: 604–611. https://doi.org/10.1080/00207454.2021.1946056.

[86] Mei T, Chen Y, Gao Y, Zhao H, Lyu X, Lin J, et al. Formaldehyde initiates memory and motor impairments under weightlessness condition. NPJ Microgravity. 2024; 10: 100. https://doi.org/10.1038/s41526-024-00441-0.

[87] Uchida Y, Kan H, Sakurai K, Oishi K, Matsukawa N. Quantitative susceptibility mapping as an imaging biomarker for Alzheimer’s disease: The expectations and limitations. Frontiers in Neuroscience. 2022; 16: 938092. https://doi.org/10.3389/fnins.2022.938092.

[88] Uchida Y, Kan H, Sakurai K, Oishi K, Matsukawa N. Contributions of blood-brain barrier imaging to neurovascular unit pathophysiology of Alzheimer’s disease and related dementias. Frontiers in Aging Neuroscience. 2023; 15: 1111448. https://doi.org/10.3389/fnagi.2023.1111448.

[89] Uchida Y, Kan H, Furukawa G, Onda K, Sakurai K, Takada K, et al. Relationship between brain iron dynamics and blood-brain barrier function during childhood: a quantitative magnetic resonance imaging study. Fluids and Barriers of the CNS. 2023; 20: 60. https://doi.org/10.1186/s12987-023-00464-x.

[90] Uchida Y, Kan H, Sakurai K, Horimoto Y, Hayashi E, Iida A, et al. APOE ɛ4 dose associates with increased brain iron and β-amyloid via blood-brain barrier dysfunction. Journal of Neurology, Neurosurgery, and Psychiatry. 2022; 93: 772–778. https://doi.org/10.1136/jnnp-2021-328519.

[91] Wilson L, Stewart W, Dams-O’Connor K, Diaz-Arrastia R, Horton L, Menon DK, et al. The chronic and evolving neurological consequences of traumatic brain injury. The Lancet. Neurology. 2017; 16: 813–825. https://doi.org/10.1016/S1474-4422(17)30279-X.

[92] Maigler KC, Buhr TJ, Park CS, Miller SA, Kozlowski DA, Marr RA. Assessment of the Effects of Altered Amyloid-Beta Clearance on Behavior following Repeat Closed-Head Brain Injury in Amyloid-Beta Precursor Protein Humanized Mice. Journal of Neurotrauma. 2021; 38: 665–676. https://doi.org/10.1089/neu.2020.6989.

[93] Abutarboush R, Reed E, Chen Y, Gu M, Watson C, Kawoos U, et al. Exposure to Low-Intensity Blast Increases Clearance of Brain Amyloid Beta. Journal of Neurotrauma. 2024; 41: 685–704. https://doi.org/10.1089/neu.2023.0284.

[94] Dong S, Zhao H, Nie M, Sha Z, Feng J, Liu M, et al. Cannabidiol Alleviates Neurological Deficits After Traumatic Brain Injury by Improving Intracranial Lymphatic Drainage. Journal of Neurotrauma. 2024; 41: e2009–e2025. https://doi.org/10.1089/neu.2023.0539.

[95] Xu R, Bi Y, He X, Zhang Y, Zhao X. Kidney-tonifying blood-activating decoction delays ventricular remodeling in rats with chronic heart failure by regulating gut microbiota and metabolites and p38 mitogen-activated protein kinase/p65 nuclear factor kappa-B/aquaporin-4 signaling pathway. Journal of Ethnopharmacology. 2024; 330: 118110. https://doi.org/10.1016/j.jep.2024.118110.

[96] Botta D, Hutuca I, Ghoul EE, Sveikata L, Assal F, Lövblad KO, et al. Emerging non-invasive MRI techniques for glymphatic system assessment in neurodegenerative disease. Journal of Neuroradiology. 2025; 52: 101322. https://doi.org/10.1016/j.neurad.2025.101322.

[97] Prasuhn J, Xu J, Hua J, van Zijl P, Knutsson L. Exploring neurodegenerative disorders using advanced magnetic resonance imaging of the glymphatic system. Frontiers in Psychiatry. 2024; 15: 1368489. https://doi.org/10.3389/fpsyt.2024.1368489.

[98] Bai Y, Yuan M, Mi H, Zhang F, Liu X, Lu C, et al. Hypothermia reduces glymphatic transportation in traumatic edematous brain assessed by intrathecal dynamic contrast-enhanced MRI. Frontiers in Neurology. 2022; 13: 957055. https://doi.org/10.3389/fneur.2022.957055.

[99] Kamagata K, Saito Y, Andica C, Uchida W, Takabayashi K, Yoshida S, et al. Noninvasive Magnetic Resonance Imaging Measures of Glymphatic System Activity. Journal of Magnetic Resonance Imaging. 2024; 59: 1476–1493. https://doi.org/10.1002/jmri.28977.

[100] Markou A, Kitchen P, Aldabbagh A, Repici M, Salman MM, Bill RM, et al. Mechanisms of aquaporin-4 vesicular trafficking in mammalian cells. Journal of Neurochemistry. 2024; 168: 100–114. https://doi.org/10.1111/jnc.16029.

[101] Mohaupt P, Vialaret J, Hirtz C, Lehmann S. Readthrough isoform of aquaporin-4 (AQP4) as a therapeutic target for Alzheimer’s disease and other proteinopathies. Alzheimer’s Research & Therapy. 2023; 15: 170. https://doi.org/10.1186/s13195-023-01318-2.

[102] Huang H, Lin L, Wu T, Wu C, Zhou L, Li G, et al. Phosphorylation of AQP4 by LRRK2 R1441G impairs glymphatic clearance of IFNγ and aggravates dopaminergic neurodegeneration. NPJ Parkinson’s Disease. 2024; 10: 31. https://doi.org/10.1038/s41531-024-00643-z.

[103] Astara K, Tsimpolis A, Kalafatakis K, Vavougios GD, Xiromerisiou G, Dardiotis E, et al. Sleep disorders and Alzheimer’s disease pathophysiology: The role of the Glymphatic System. A scoping review. Mechanisms of Ageing and Development. 2024; 217: 111899. https://doi.org/10.1016/j.mad.2023.111899.

[104] Jatczak-Pawlik I, Jurewicz A, Domowicz M, Ewiak-Paszyńska A, Stasiołek M. CHI3L1 in Multiple Sclerosis-From Bench to Clinic. Cells. 2024; 13: 2086. https://doi.org/10.3390/cells13242086.