The glymphatic system is not a lymphatic structure comprising fully matured LECs (Iliff et al., 2012). However, owing to its similar functionality as peripheral lymphatic vessels in promoting the clearance and drainage of metabolic waste from the ISF of the brain, it is referred to as the glymphatic system. Its primary constituents include astrocytes and their end-feet protein, AQP4 (Iliff et al., 2012), along with perivascular spaces known as Virchow-Robin spaces (VRS) (Rennels et al., 1985). Astrocytes mainly play a supportive role by adhering to the penetrating vessels of the brain. Meanwhile, AQP4 is expressed on their surface, enabling the delivery of nutrient-rich CSF into the brain parenchyma for nourishing neurons, glial cells, and other neurocytes (Plog and Nedergaard, 2018). The perivascular spaces encompass both arteriolar and venular perivascular spaces that serve as conduits for the inflow of CSF and outflow of ISF (Rasmussen et al., 2018). Penetrating intracerebral arteries traverse these spaces to reach deep into blind-ended regions of brain parenchyma. Facilitated by the action of AQP4, CSF can follow these spaces and infiltrate extensively within the brain parenchyma. This pathway also provides an opportunity for intrathecally injected drugs in CSF to access deep regions of brain parenchyma (Lohela et al., 2022).

The development of MLVs is characterized by multiple facets. After birth, MLVs develop and extend in synchrony with the growth direction of nerves and blood vessels (Antila et al., 2017). By utilizing MRI techniques, non-invasive visualization and quantification of MLVs, along with studying the flow dynamics within the human MLVs, have been successfully accomplished (Kim et al., 2023). Preliminary results indicate that the flow velocity in dorsal MLVs ranges from 2.2 to 2.7 mm per second in humans. Moreover, a gender discrepancy in MLV volumes is apparent between males and females, with male MLV volumes significantly exceeding those of females (Jacob et al., 2022). In both mice and humans, the drainage capacity of MLVs diminishes with age (Da Mesquita et al., 2018; Albayram et al., 2022). Remarkably, the MLVs exhibited plasticity and regenerative capacity. An increase in Vascular Endothelial Growth Factor C (VEGF-C), a positive factor promoting lymphatic vessel development, induces the sprouting, development, and maintenance of MLVs. Conversely, silencing or blocking VEGF-C signaling leads to noticeable degradation of MLVs, whereas the knockout of Vascular Endothelial Growth Factor D (VEGF-D) does not significantly impact them (Antila et al., 2017). Basal MLVs, adjacent to the subarachnoid space (SAS), possess lymphatic valves akin to peripheral lymphatic vessels and serve as “hotspot” sites for clearing large molecules within CSF (Ahn et al., 2019).

While there is no direct anatomical connection between the glymphatic system and the meningeal lymphatic system, they functionally interact to collectively form a unique network responsible for waste clearance and immune regulation in the brain (Louveau et al., 2015b). In terms of waste clearance, these systems collaboratively establish a CSF drainage pathway: CSF-ISF-CSF-MLV-dcLNs (Li G. et al., 2022). Iliff et al. (2012) demonstrated that the majority of the CSF in the SAS infiltrates into the brain parenchyma. As shown in Figure 2, CSF flows into the brain parenchyma through perivascular spaces surrounding pia mater arteries and penetrating arterioles, where it undergoes mixing as well as nutrients and metabolic waste exchange with ISF . ISF, loaded with interstitial solutes, is expelled from the brain parenchyma via venous perivascular routes (intracerebral and caudal rhinal veins) and eliminated into the CSF (Iliff et al., 2012).

The dural lymphatic vessels then uptake absorb ISF and CSF from the SAS, subsequently transporting them to the dcLNs (Aspelund et al., 2015; Louveau et al., 2015b) (Figure 3). The principal site of absorption is identified as the MLVs located at the base of the skull (Ahn et al., 2019). This discovery offers a novel perspective on the clearance of large molecular solutes in neurodegenerative disorders. Furthermore, CSF is drained through multiple pathways. Traditionally, it is posited that arachnoid granulations facilitate the absorption of CSF from the SAS via a valve-like mechanism, allowing CSF to enter and ultimately be absorbed by the venous sinuses (Welch and Friedman, 1960; Wolpow and Schaumburg, 1972) (Figure 2). Additionally, the arachnoid cuff exit points, formed by discontinuities as the bridging veins traverse the arachnoid barrier, create openings that permit the exchange of CSF and molecules between the SAS and the dura mater, facilitating CSF drainage (Smyth et al., 2024). Recently, the nasopharyngeal lymphatic plexus (NPLP) has been demonstrated to serve as a major hub for the outflow of CSF to the dcLNs (Yoon et al., 2024). Research indicates that the NPLP is connected with upstream lymphatic vessels, including those near the pituitary gland and the cavernous sinus, as well as through the cribriform plate, where CSF is drained to the dcLNs via the deep cervical lymphatic vessels. These CSF drainage structures collaborate with the glymphatic system and the meningeal lymphatic system to jointly sustain the balance of cerebral fluids and the clearance of metabolic waste.

On the other hand, both the glymphatic system and the meningeal lymphatic system play crucial roles in immune cell drainage and immunosurveillance, functioning synergistically (Li G. et al., 2022). Prior to the discovery of the brain lymphatic system, research on neuroimmunity primarily focused on microglia. However, with the identification of this system, it has revitalized studies in neuroimmunology (Sun et al., 2018). Research indicates that antigens generated within the CNS can travel through the glymphatic pathway to reach the SAS before draining into MLVs or passing through the cribriform plate into nasal lymphatics and eventually reaching cervical lymph nodes (cLNs). This process stimulates adaptive immune responses (Louveau et al., 2015a; Louveau et al., 2015b). Mechanistically, neuroinflammation can induce the formation of MLVs adjacent to the cribriform plate. These newly formed lymphatic vessels containing CD4⁺ T cells and CD11c+ cells capable of capturing CNS antigens and presenting them to T cells (Hsu et al., 2022). Additionally, a substantial number of immune cells are located around the meningeal sinuses which serve as an ecological niche known as a “regional immune center.” These cells collaborate with MLVs to perform neuroimmunosurveillance (Rustenhoven et al., 2021; Smyth et al., 2024). Studies have demonstrated that CNS antigens present in the CSF reside around dural sinuses, where they are presented by antigen presenting cells associated with dura mater for patrolling T cells from venous sinuses. These activated T cells migrate through interstitial spaces of MLV endothelial cells into MLVs and are subsequently drained towards cLNs, thereby inducing the generation of T cell effector functions and promoting tissue retention (Rustenhoven et al., 2021). Therefore, MLVs provide a feasible physical conduit for the interaction between CNS antigens and peripheral T cells.

Several studies have highlighted dysfunction in CNS lymphatic drainage across a range of brain diseases (Table 1). For instance, in models of subarachnoid hemorrhage (SAH), the glymphatic system is obstructed by fibrin clot blockages in perivascular spaces, thereby impeding CSF inflow into the brain parenchyma (Pu et al., 2019). In cytotoxic brain edema, hypoxia-induced energy depletion results in glymphatic system dysfunction, promoting CSF inflow into the brain parenchyma while inhibiting ISF outflow (Thrane et al., 2014). In the meningeal lymphatic system of idiopathic Parkinson’s disease (PD) patients, a reduction in MLV flow was observed in the superior sagittal sinus and sigmoid sinus regions, along with delayed CSF drainage in deep lymph nodes (Ding et al., 2021), and impaired clearance of α-synuclein (α-syn) by CNS lymphatics (Zou et al., 2019). These findings provide evidence of MLV dysfunction in PD patients. Moreover, both the meningeal lymphatic drainage capability and MLV coverage were compromised in an Alzheimer’s disease (AD) mouse model, accompanied by cognitive impairment (Da Mesquita et al., 2018). Dysfunctions in both the glymphatic system and MLVs frequently co-occur in CNS diseases. Glymphatic system dysfunction often results in inadequate removal of metabolic waste from CSF through downstream MLVs, while disruption of MLVs hinders glymphatic clearance of ISF, leading to concurrent dysfunction between these systems (Da Mesquita et al., 2018; Li W. et al., 2022). For instance, traumatic brain injury (TBI) can impair AQP4 polarity and reduce glymphatic function by approximately 60% (Iliff et al., 2014). Concurrently, TBI can cause persistent MLV dysfunction for up to 1 month post-injury, indicating an interconnected pathology (Bolte et al., 2020). Besides, evidence for a simultaneous dysfunction of both glymphatic system and meningeal lymphatics in the context of chronic migraine conditions has been consolidated through non-invasive MRI imaging techniques (Wu et al., 2024). Analyses highlight an attenuated diffusion of water molecules surrounding the vasculature in individuals grappling with chronic migraines, in stark contrast to a control population manifesting healthy parameters. Investigations further reveal a decelerated fluid flow rate within the MLVs, culminating in a compromised effectiveness in CSF clearance, thereby convincingly illustrating a simultaneous dysfunction of both lymphatic systems (Table 1).

Furthermore, in the presence of preexisting CNS diseases, impaired CNS lymphatic drainage often acts as an exacerbating factor. For instance, in an AD mouse model, inhibition of glymphatic system function via AQP4 knockout led to decelerated CSF inflow into the brain parenchyma, resulting in heightened accumulation of Amyloid-β (Aβ) and more pronounced cognitive deficits (Iliff et al., 2012). In a PD mouse model, dysfunction of glymphatic system drainage caused by ligation of the MLVs exacerbated α-syn accumulation, increased neuroinflammation, and worsened motor function impairment (Zou et al., 2019). In the context of stroke, mice with impaired MLVs often present with a larger infarct volume compared to wild-type mice (Yanev et al., 2020) (Table 1). When considering brain tumors, such as glioma and metastatic melanoma, obstruction of dorsal skull MLVs in mouse models diminishes the therapeutic effects of anti-Programmed Cell Death Protein 1/Cytotoxic T-Lymphocyte-Associated Protein 4 checkpoint combined therapy on these tumors (Hu et al., 2020). In an AD mouse model, ablation of MLVs intensifies AD pathology and cognitive performance decline by promoting Aβ accumulation, activating microglia proliferation, causing behavioral deficits and deterioration, while also reducing the effectiveness of anti-Aβ immunotherapy (Da Mesquita et al., 2018; Wang et al., 2019). Conversely, the latest research suggests that the blockade of MLVs function did not lead to an increase in the accumulation of pathological proteins in AD mouse models, nor did it exacerbate cognitive impairments (Antila et al., 2024). Such discrepancies may be attributed to variations in animal models, methodologies, and compensatory mechanisms of CSF dynamics. The complexity of CNS disease pathophysiology involves numerous factors and pathways, with various studies potentially revealing different aspects of this network. Therefore, further investigation of MLVs in the CNS diseases to clarify their role in disease pathology is needed.

The evidence presented indicates that dysfunction in the glymphatic system and meningeal lymphatic system is observed across a spectrum of neurological diseases, where compromised CNS lymphatic drainage frequently exacerbates disease severity, thus establishing a detrimental cycle. Therefore, the regulation of CNS lymphatic functions assumes paramount importance for the effective treatment of CNS diseases.

Emerging studies suggest that gamma multisensory stimulation exerts regulatory effects on the glymphatic system and ameliorates the pathological manifestations of AD. Researchers have induced corresponding neural activities in certain brain areas through multisensory gamma stimulation, which enhances clearance functionalities within the glymphatic system, leading to a reduction in amyloid protein accumulation in AD mouse models (Murdock et al., 2024). This outcome is attributed to gamma stimulation, which promotes polarization of the water channel protein AQP4 in astroglial cells and expands MLVs. Moreover, it promotes glymphatic clearance by mediating pulsations in cerebral arteries through Vasointestinal Peptide (VIP) neuron-dependent activity (Nedergaard and Goldman, 2020). Repetitive transcranial magnetic stimulation (rTMS) has exhibited similar effects. Notably, early high-frequency rTMS treatment in AD mouse models effectively reduces long-term memory loss and mitigates AD-related pathological development, including Aβ deposition and glial cell activation, by enhancing the excretory efficiency of the glymphatic system (Lin et al., 2021). Consequently, gamma multisensory stimulation and rTMS, along with other physical therapies alike, represent promising therapeutic strategies for improving glymphatic system function, thereby ameliorating the prognosis of neurodegenerative diseases.

In summary, modulation of glymphatic system functionality holds significant value for the treatment of CNS diseases. The enhancement of this system’s capabilities through physiological regulation, pharmacological intervention, and physical therapy can facilitate more efficient clearance of detrimental substances within the brain parenchyma. This process may mitigate or prevent the onset and progression of neurodegenerative disorders.

With the advancement of precision medicine, there is an increasing demand for non-invasive and non-traumatic treatment modalities (Salehpour et al., 2022). PMT, an emerging approach for neuroprotection, employs visible and near-infrared light to stimulate the biochemical activities of mitochondrial components, thereby modulating cellular processes (Wong-Riley et al., 2005). Consequently, researchers have utilized non-invasive near-infrared light treatments to mediate cerebral diseases by regulating the lymphatic system (Salehpour et al., 2022). For instance, by directing near-infrared irradiation upon MLVs based on their superficial distribution characteristics, investigators observed improved cognitive function and reduced Aβ deposition in AD mice. This was attributed mechanistically to PMT’s regulation of mitochondrial metabolism and cell conjunction in meningeal LECs (Zinchenko et al., 2019; Wang M. et al., 2024). Additionally, near-infrared light irradiation of MLVs at doses of 5 and 10 J/cm² demonstrated increased permeability of the lymphatic endothelium, promoting relaxation of lymphatic vessels and enhancing clearance and drainage of macromolecules within the brain (Semyachkina-Glushkovskaya et al., 2020). Furthermore, accelerating the elimination of RBCs is of utmost importance in alleviating complications arising from intraventricular hemorrhage in preterm infants. Following low-level infrared light stimulation of MLVs, it was discovered that the removal RBCs within the ventricles was accelerated, leading to their drainage to dcLNs through MLVs. This process effectively reduces the toxic side effects associated with intraventricular RBC retention and enhances neural function recovery after hemorrhage (Li et al., 2023). While PMT targeting MLVs holds significant implications for the treatment of brain diseases, further exploration is warranted due to current limitations such as cranial scattering effects, laser wavelength optimization, irradiation duration adjustment, and potential side reactions.

In the exploration of immunotherapeutic strategies for brain diseases, a growing body of research has substantiated the integral role played by MLVs in this field (Schiller et al., 2021). Subsequent sections will elucidate the immunomodulatory functions of MLVs in various cerebral diseases and how these pathways can be targeted to develop innovative therapeutic strategies.

Initially focusing on MLV-mediated immunotherapy in cerebral neoplasms, accumulating evidence suggests that VEGF-C signaling plays a crucial role in enhancing immune surveillance of brain tumors (Song et al., 2020). By delivering VEGF-C mRNA through an adenovirus vector, researchers have successfully enhanced specific expression of VEGF-C within the brain, thereby eliciting adaptive immune responses in deep lymph nodes. This cascade facilitates the migration of CD8⁺ T cells towards the microenvironment of glioblastomas via MLVs, ultimately reshaping the immune microenvironment with the ultimate goal being tumor destruction (Song et al., 2020). Moreover, the efficacy of inhibiting glioblastoma growth can be significantly boosted by combining immune checkpoint inhibitors with VEGF-C mRNA treatment (Song et al., 2020), offering new strategic avenues for GBM therapy. Further investigations have indicated that the conjunction of VEGF-C mRNA therapy with radiotherapy - by expanding MLVs - promotes dendritic cell migration and activates CD8⁺ T cells, thereby amplifying radiotherapy-induced antitumor immune responses (Zhou et al., 2022). However, whether MLVs can enhance the release of tumor-specific antigens induced by radiotherapy necessitates additional scrutiny.

In contrast to malignancies, treatment strategies for autoimmune disorders need the prevention of immune cell activation and brain invasion (Hsu et al., 2019). Take experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, for instance; disease pathogenesis is accompanied by the development of lymph vessels at the cribriform plate. These vessels facilitate the transportation of inflammatory signals from the CNS to cLNs, triggering adaptive immune responses (Hsu et al., 2019). By utilizing MAZ51, a VEGFR3 tyrosine kinase inhibitor that hinders VEGFR3-dependent lymphangiogenesis and eliminates MLVs, researchers have observed a reduction in the severity of EAE and suppression of inflammatory reactions by brain-reactive T cells (Hsu et al., 2019; Li G. et al., 2022). Nevertheless, the long-term implications of MLV blockade on overall health continue to prompt further investigations. In addition, researchers have initiated a clinical trial (NCT05414487) utilizing Ofatumumab as an immunomodulatory drug for treating multiple sclerosis patients to explore its impact on MLVs and dynamic changes in immune cells. Consequently, scientists have dedicated significant efforts towards elucidating strategies for autoimmune disease treatment involving MLVs, yet additional research is still required.

In neurodegenerative diseases, such as AD, the immunological role mediated by MLVs remains crucial. Elderly mice often demonstrate functional impairment of MLVs, leading to the accumulation of misfolded proteins and metabolic waste within the brain parenchyma. This accumulation triggers neurodegenerative diseases (Da Mesquita et al., 2018). Rectifying this age-related dysfunction in older mice represents a promising therapeutic target for managing neurodegenerative diseases. Through single-cell RNA sequencing, researchers have identified prominent alterations in meningeal immunity among elderly mice. These alterations are characterized by a notable increase in Interferon γ (IFN-γ) expression primarily attributed to activated CD4⁺ T cells and CD8⁺ cytotoxic T cells, which ultimately affects CSF drainage through MLVs (Rustenhoven et al., 2023). Therefore, it has been demonstrated that the drainage function of MLVs can be significantly improved by peripherally administering anti-IFNγ neutralizing antibodies, verifying the reversibility of age-related MLV impairment (Rustenhoven et al., 2023).

In conclusion, MLVs are observed to play a multifaceted and intricate role in the therapeutic management of cerebral diseases. By fostering an in-depth comprehension of the functionality and regulatory mechanisms of MLVs under varying disease states, there lies potential for developing more precise and efficacious strategies to improve the quality of life for individuals diagnosed with brain diseases. Notably, VEGF-C has been recognized for its significant potential clinical translation among these therapeutic approaches. Copious research reports (Salvador et al., 2024), suggest that VEGF-C, as a well-established lymphangiogenic factor, stimulates growth and development of the lymphatic system and promotes MLVs’ drainage, consequently improving CNS disease pathology and clinical manifestations. However, transitioning these findings to clinical practice may likely necessitate additional scrutiny to ascertain the optimal therapeutic strategies, dosage, and safety.

The existence of the BBB serves as a significant hindrance to the treatment of CNS diseases (Terstappen et al., 2021). MLVs, serving as conduits connecting the peripheral lymphatic system with the CNS (Aspelund et al., 2015), offer a potential pathway for delivering drugs to the intracranial space. Drug delivery through this route may hold the prospect of precision therapy for CNS diseases. In the treatment of glioblastoma, the injection of Poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with Indocyanine Green (ICG) near the cLNs, it was found that the nanoparticles entered the MLVs, which then resulted in a 44-fold higher uptake of the nanoparticle drugs in the brain than conventional intravenous injection. The excitation of ICG by near-infrared light for photodynamic treatment of tumors demonstrated that the residual nanoparticles significantly inhibited glioblastoma growth (Zhao et al., 2020). Additionally, immune cell membranes have also been utilized to encapsulate drugs for delivery via MLVs to treat CNS diseases. For instance, curcumin encapsulated within natural killer cell membranes has been utilized in PD, and it was discovered that the encapsulated drug colocalized with MLVs marked by Lyve-1, with a marked enrichment observed within the brain (Liu et al., 2023). Additionally, research has demonstrated that Fibroblast Growth Factor-21 (FGF21), a neuroprotective factor (Chen et al., 2019), when encapsulated in BV2 cell membranes, similarly exhibits the ability to traverse the MLVs and target areas affected by AD. This approach has been shown to reduce the accumulation of tau protein and ameliorate neuroinflammation (Wang H. C. et al., 2024). Therefore, drug delivery mediated by the MLVs offers a novel approach for the treatment of CNS diseases; however, its feasibility and the underlying mechanisms necessitate further experimental validation.

In addition to transporting drugs from peripheral to intracranial locations, the conveyance of drugs from the CSF to the deep brain parenchyma also has significant implications. Studies have shown that systemic injections of hypertonic saline can enhance the binding of anti-Aβ antibodies to Aβ in AD mouse models, owing to a reduction in brain tissue volume caused by elevated plasma osmotic pressure. Interestingly, this effect enlarges the perivascular space and enhances glymphatic system function while leaving brain arterial volume unaffected (Plog et al., 2018). Similar effects of hypertonic saline have been substantiated in spinal drug treatments as well (Blomqvist et al., 2022). Furthermore, researchers have initiated investigations aiming to utilize the glymphatic system for directing nanoparticles from CSF into deep brain parenchymal regions. It has been established that following systemic injection of hypertonic saline and intrathecal administration of radioactively labeled gold nanoparticles, both single-photon emission computed tomography and MRI detected extensive distribution of nanoparticles within deep brain tissue regions, with rapid renal metabolic clearance occurring within 24 h (Lilius et al., 2023). Leveraging the perivascular space in the glymphatic system as a pathway for delivering immunopharmacological agents or nanodrugs into deep brain parenchymal regions presents a promising approach. These insights lay a solid foundation for new strategies involving intrathecal drug delivery assisted by the glymphatic system.