Function Of Meningeal Lymphatics Research Articles

Age-related changes in meningeal lymphatic function are closely associated with vascular endothelial growth factor-C expression

Meningeal lymphatic vessels (MLVs) have crucial roles in removing metabolic waste and toxic proteins from the brain and transporting them to the periphery. Aged mice show impaired meningeal lymphatic function. Nevertheless, as the disease progresses, and significant pathological changes manifest in the brain, treating the condition becomes increasingly challenging. Therefore, investigating the alterations in the structure and function of MLVs in the early stages of aging is critical for preventing age-related central nervous system degenerative diseases. We detected the structure and function of MLVs in young, middle-aged, and aged mice. Middle-aged mice, compared with young and aged mice, showed enhanced meningeal lymphatic function along with MLV expansion and performed better in the Y maze test. Moreover, age-related changes in meningeal lymphatic function were closely associated with vascular endothelial growth factor-C (VEGF-C) expression in the brain cortex. Our data suggested that the cerebral cortex may serve as a target for VEGF-C supplementation to ameliorate meningeal lymphatic dysfunction, thus providing a new strategy for preventing age-related central nervous system diseases.

Enhanced meningeal lymphatic drainage ameliorates lipopolysaccharide-induced brain injury in aged mice

BackgroundSepsis-associated encephalopathy (SAE) is an acute cerebral dysfunction caused by sepsis. Neuroinflammation induced by sepsis is considered a potential mechanism of SAE; however, very little is known about the role of the meningeal lymphatic system in SAE.MethodsSepsis was established in male C57BL/6J mice by intraperitoneal injection of 5 mg/kg lipopolysaccharide, and the function of meningeal lymphatic drainage was assessed. Adeno-associated virus 1-vascular endothelial growth factor C (AAV1-VEGF-C) was injected into the cisterna magna to induce meningeal lymphangiogenesis. Ligation of deep cervical lymph nodes (dCLNs) was performed to induce pre-existing meningeal lymphatic dysfunction. Cognitive function was evaluated by a fear conditioning test, and inflammatory factors were detected by enzyme-linked immunosorbent assay.ResultsThe aged mice with SAE showed a significant decrease in the drainage of OVA-647 into the dCLNs and the coverage of the Lyve-1 in the meningeal lymphatic, indicating that sepsis impaired meningeal lymphatic drainage and morphology. The meningeal lymphatic function of aged mice was more vulnerable to sepsis in comparison to young mice. Sepsis also decreased the protein levels of caspase-3 and PSD95, which was accompanied by reductions in the activity of hippocampal neurons. Microglia were significantly activated in the hippocampus of SAE mice, which was accompanied by an increase in neuroinflammation, as indicated by increases in interleukin-1 beta, interleukin-6 and Iba1 expression. Cognitive function was impaired in aged mice with SAE. However, the injection of AAV1-VEGF-C significantly increased coverage in the lymphatic system and tracer dye uptake in dCLNs, suggesting that AAV1-VEGF-C promotes meningeal lymphangiogenesis and drainage. Furthermore, AAV1-VEGF-C reduced microglial activation and neuroinflammation and improved cognitive dysfunction. Improvement of meningeal lymphatics also reduced sepsis-induced expression of disease-associated genes in aged mice. Pre-existing lymphatic dysfunction by ligating bilateral dCLNs aggravated sepsis-induced neuroinflammation and cognitive impairment.ConclusionThe meningeal lymphatic drainage is damaged in sepsis, and pre-existing defects in this drainage system exacerbate SAE-induced neuroinflammation and cognitive dysfunction. Promoting meningeal lymphatic drainage improves SAE. Manipulation of meningeal lymphangiogenesis could be a new strategy for the treatment of SAE.

Vascular Endothelial Growth Factor-C Treatment Enhances Cerebrospinal Fluid Outflow during Toxoplasma gondii Brain Infection but Does Not Improve Cerebral Edema

Cerebral edema frequently develops in the setting of brain infection and can contribute to elevated intracranial pressure, a medical emergency. How excess fluid is cleared from the brain is not well understood. Previous studies have shown that interstitial fluid is transported out of the brain along perivascular channels that collect into the cerebrospinal fluid (CSF)-filled subarachnoid space. CSF is then removed from the central nervous system through venous and lymphatic routes. The current study tested the hypothesis that increasing lymphatic drainage of CSF would promote clearance of cerebral edema fluid during infection with the neurotropic parasite Toxoplasma gondii. Fluorescent microscopy and magnetic resonance imaging was used to show that C57BL/6 mice develop vasogenic edema 4 to 5 weeks after infection with T. gondii. Tracer experiments were used to evaluate how brain infection affects meningeal lymphatic function, which demonstrated a decreased rate in CSF outflow in T. gondii-infected mice. Next, mice were treated with a vascular endothelial growth factor (VEGF)-C-expressing viral vector, which induced meningeal lymphangiogenesis and improved CSF outflow in chronically infected mice. No difference in cerebral edema was observed between mice that received VEGF-C and those that rececived sham treatment. Therefore, although VEGF-C treatment can improve lymphatic outflow in mice infected with T. gondii, this effect does not lead to increased clearance of edema fluid from the brains of these mice.

Regulation of meningeal lymphatic function by APOE

AbstractBackgroundThe meningeal layers form a unique interface between the central nervous and peripheral immune systems. The lymphatic vasculature that is present in the meningeal dura and drains cerebrospinal fluid (CSF) becomes dysregulated during aging and in models of neurodegenerative disorders such as Alzheimer’s disease (AD). Apolipoprotein E (APOE), and the isoform APOE4 in particular, increases the risk for late‐onset sporadic AD. However, it is still poorly understood whether changes in meningeal lymphatic morphology and drainage are mediating the deleterious effects of APOE4 expression. In the present study, we investigate the effects of human APOE3 or APOE4 on meningeal lymphatic function and immunity.MethodWe evaluated the morphology of lymphatic vessels in dural whole mounts of age‐matched wild type, Apoe‐KO, APOE3, and APOE4 mice at 2‐3, and 11‐12 months. This assessment was performed in both male and female mice. The drainage function of meningeal lymphatics is being assessed in the same mice by in vivo imaging of CSF‐derived fluorescent tracers into the deep cervical lymph nodes. We have also performed RNAscope and protein measurements using murine meningeal tissue to investigate the main cellular source(s) of Apoe, and single‐cell RNA sequencing to explore the cell‐specific transcriptomic signatures linked to the changes in meningeal lymphatic function.ResultLymphatic vessel length is increased in 11‐12‐month‐old APOE4 male mice, when compared to their APOE3 counterparts. Interestingly, the same was not detected in female mice. Dural myeloid cells, expressing Itgam (encoding for CD11b), are the main source of Apoe mRNA transcripts, and one of the main sources of apoE protein in the brain dura mater. APOE4 expression and age markedly alter the transcriptional profile of meningeal myeloid cells, which show overexpression of lysosomal, antigen presentation, and complement genes.ConclusionAPOE4 expression leads to a sex‐dependent meningeal lymphatic vascular remodeling, which is associated with an exacerbated activation of meningeal macrophages. In future studies, we will test whether the changes in meningeal lymphatics observed in mice expressing APOE4 are mediated by myeloid cell derived APOE.

Mild brain injury aggravates gliosis, toxic tau, and anxiety in the P301S model of TAUopathy

AbstractBackgroundTraumatic Brain Injury (TBI) is a known risk factor for developing a neurodegenerative disease later in life, including Alzheimer’s Disease (AD) and other forms of tauopathy such as chronic traumatic encephalopathy (CTE) and frontal‐temporal dementia (FTD). Although several studies highlight the relationship between the severity and frequency of a TBI and the development of these tauopathies, the mechanism behind this association remains unknown. Proper meningeal lymphatic function is required for clearance of waste products and protein aggregates such as amyloid beta and tau from the brain parenchyma. While post‐TBI inflammation is beneficial for clearing debris and promoting tissue repair, prolonged glial activation and decreased meningeal lymphatic drainage have been linked to worsened cognitive function.MethodA mild TBI (mTBI) was delivered to three‐month‐old young adult mice overexpressing P301S mutant human tau (PS19) through the skull on the right inferior temporal lobe of the brain. Sham and TBI injured mice were tested on behavioral tests at 5 months of age. At 7 months of age, brains were examined for tau pathology and neuroinflammation using confocal microscopy.ResultFour months after mTBI, the brains of TBI PS19 mice exhibit increased levels of microglia and astrocytes, and increased P‐S202/T‐205‐Tau in the cortex and thalamus of the ipsilateral hemisphere compared to the contralateral side and to Sham PS19 controls. These findings correlate with our behavior study showing increased hyperactivity, risk‐taking behavior, and impaired short term working memory. PS19 TBI mice also present larger ventricles than their uninjured controls suggesting a potential loss of white matter in the cortex and/or hippocampus.ConclusionP‐S202/T‐205‐Tau orginated from the cortex near the injury site can aggregate and promote the formation of more toxic tau that spreads to deeper regions of the cortex and in the thalamus primarily through the thalamocortical circuit. Also, microglia and astrocyte activation and production of inflammatory mediators has been associated with synapse pruning and engulfment of damaged neurons expressing ‘eat me signals’ leading to neurodegeneration.

Disturbed meningeal lymphatic function associated with malignancy and progression in patients with intracranial malignant tumors

Meningeal lymphatic vessels (mLVs) have proven to bear a relationship with tumor immunity and therapeutic efficacy of intracranial malignant tumors in pre-clinical animal studies. We aimed to explore the association between mLV function and intracranial malignant tumors in clinical participants. The participants were allocated to a control group or a group of patients with intracranial tumors. Dynamic enhanced magnetic resonance was used to evaluate the wash-in and wash-out functions of mLVs around the superior sagittal sinus and the sigmoid sinus. A total of 246 individuals were recruited for our study. The area under curve and wash-in rate of mLVs in the intracranial tumor group were higher than in the control group (2,749 vs. 2,110, p<0.001 and 3.72 vs. 2.87, p<0.001, respectively). The wash-out ratio of mLVs was lower in the intracranial tumor group than in the control group (0.65 vs. 0.73, p<0.001). Decreased wash-out of mLVs was associated with tumor progression (β= -0.118; p<0.001). High-grade glioma and isocitrate dehydrogenase wild type were associated with a lower mLV wash-out function (β= -0.057, p= 0.044 and β= -0.069, p= 0.047, respectively). Intracranial malignant tumors were associated with elevated wash-in function and decreased wash-out function of mLVs. High-grade glioma and isocitrate dehydrogenase wild type were associated with low mLV wash-out function, and long-term decreased mLV wash-out function was a risk factor for tumor progression. There was no funding.

Cognitive Brain Science: Glymphatic (Lymphatic) System, Circadian Stress and Chronic Insomnia

Sleep evolutionarily persists in all species, and sleep disturbance is the general pathogenesis of neurodegeneration, circadian stress, and electromagnetic load/overload. Sleep quality in human decreases with age, and disruption of normal sleep architecture often precedes the onset of neurodegenerative diseases. The lymphatic system, which cleans the brain of protein waste, is mostly active during sleep. Chronic stress remodels and restructures the lymphatic vasculature. The glymphatic system because of its dependence on glial cells — non-neuronal cells in the central nervous system (CNS) that do not produce electrical impulses but support and protect neurons. The present and functional lymphatic vascular system is found in the meninges that cover the central CNS. This unexpected (rediscovery) discovery led to a reassessment of fluid and solute drainage mechanisms in the CNS, neuroimmune interactions, and the involvement of meningeal lymph nodes in the onset and progression of neurological disorders. Various factors affecting meningeal lymphatic function, such as growth factor signaling and aging, and their effect on the continuous flow of brain-derived molecules and meningeal immune cells to the cervical lymph nodes. Discovery of the role of the lymphatic vasculature draining the CNS in various pathologies that have a strong neuroinflammatory component, including brain injuries, tumors and aging-related neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases. Neural networks that link sleep, aging, lymphatic system clearance and protein aggregation have shed new light on the pathogenesis of a wide range of neurodegenerative diseases for which lymphatic system failure may represent a therapeutically targeted end-to-end common pathway.

Plasma neurodegeneration biomarker concentrations associate with glymphatic and meningeal lymphatic measures in neurological disorders

Clearance of neurotoxic brain proteins via cerebrospinal fluid (CSF) to blood has recently emerged to be crucial, and plasma biomarkers of neurodegeneration were newly introduced to predict neurological disease. This study examines in 106 individuals with neurological disorders associations between plasma biomarkers [40 and 42 amino acid-long amyloid-β (Aβ40 and Aβ42), total-tau, glial fibrillary acidic protein (GFAP), and neurofilament light (NfL)] and magnetic resonance imaging measures of CSF-mediated clearance from brain via extra-vascular pathways (proxy of glymphatic function) and CSF-to-blood clearance variables from pharmacokinetic modeling (proxy of meningeal lymphatic egress). We also examine how biomarkers vary during daytime and associate with subjective sleep quality. Plasma concentrations of neurodegeneration markers associate with indices of glymphatic and meningeal lymphatic functions in individual- and disease-specific manners, vary during daytime, but are unaffected by sleep quality. The results suggest that plasma concentrations of neurodegeneration biomarkers associate with measures of glymphatic and meningeal lymphatic function.

Age-related alterations in meningeal immunity drive impaired CNS lymphatic drainage.

The meningeal lymphatic network enables the drainage of cerebrospinal fluid (CSF) and facilitates the removal of central nervous system (CNS) waste. During aging and in Alzheimer's disease, impaired meningeal lymphatic drainage promotes the buildup of toxic misfolded proteins in the CNS. Reversing this age-related dysfunction represents a promising strategy to augment CNS waste clearance; however, the mechanisms underlying this decline remain elusive. Here, we demonstrate that age-related alterations in meningeal immunity underlie this lymphatic impairment. Single-cell RNA sequencing of meningeal lymphatic endothelial cells from aged mice revealed their response to IFNγ, which was increased in the aged meninges due to T cell accumulation. Chronic elevation of meningeal IFNγ in young mice via AAV-mediated overexpression attenuated CSF drainage-comparable to the deficits observed in aged mice. Therapeutically, IFNγ neutralization alleviated age-related impairments in meningeal lymphatic function. These data suggest manipulation of meningeal immunity as a viable approach to normalize CSF drainage and alleviate the neurological deficits associated with impaired waste removal.

Immunohistochemical visualization of lymphatic vessels in human dura mater: methodological perspectives

BackgroundDespite greatly renewed interest concerning meningeal lymphatic function over recent years, the lymphatic structures of human dura mater have been less characterized. The available information derives exclusively from autopsy specimens. This study addressed methodological aspects of immunohistochemistry for visualization and characterization of lymphatic vessels in the dura of patients.MethodsDura biopsies were obtained from the right frontal region of the patients with idiopathic normal pressure hydrocephalus (iNPH) who underwent shunt surgery as part of treatment. The dura specimens were prepared using three different methods: Paraformaldehyde (PFA) 4% (Method #1), paraformaldehyde (PFA) 0.5% (Method #2), and freeze-fixation (Method #3). They were further examined with immunohistochemistry using the lymphatic cell marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and as validation marker we used podoplanin (PDPN).ResultsThe study included 30 iNPH patients who underwent shunt surgery. The dura specimens were obtained average 16.1 ± 4.5 mm lateral to the superior sagittal sinus in the right frontal region (about 12 cm posterior to glabella). While lymphatic structures were seen in 0/7 patients using Method #1, it was found in 4/6 subjects (67%) with Method #2, while in 16/17 subjects (94%) using Method #3. To this end, we characterized three types of meningeal lymphatic vessels: (1) Lymphatic vessels in intimate contact with blood vessels. (2) Lymphatic vessels without nearby blood vessels. (3) Clusters of LYVE-1-expressing cells interspersed with blood vessels. In general, highest density of lymphatic vessels were observed towards the arachnoid membrane rather than towards the skull.ConclusionsThe visualization of meningeal lymphatic vessels in humans seems to be highly sensitive to the tissue processing method. Our observations disclosed most abundant lymphatic vessels towards the arachnoid membrane, and were seen either in close association with blood vessels or remote from blood vessels.

Lymphatic drainage at the meningeal‐brain interface in aging and Alzheimer’s disease

AbstractBackgroundAlzheimer’s disease (AD) is the most prevalent form of dementia. We have recently shown that proper meningeal lymphatic drainage is important for brain fluid flow, learning and memory in aging and for brain amyloid beta (Aβ) clearance in mouse models of AD. Interestingly, genetic factors that increased the risk for AD are also linked to altered meningeal lymphatic function.MethodMeningeal lymphatic function was evaluated in mouse models of AD at different ages by RNA sequencing of sorted meningeal lymphatic endothelial cells (LECs) and by assessing of lymphatic vessel morphology and drainage capacity. Meningeal lymphatic dysfunction was induced by photoablation, whereas enhancement of meningeal lymphatic drainage was achieved by increasing the levels of VEGF‐C. The transcriptional signatures of brain blood endothelial cells and microglia were determined by single‐cell RNA sequencing upon manipulation of meningeal lymphatic drainage. Changes in Aβ plaque clearance by monoclonal antibodies were assessed upon prolonged manipulation of meningeal lymphatic drainage. Integrated analysis was performed using murine and human cell RNA‐seq data, as well as AD summary statistics.ResultWe show that a progressive deposition of meningeal Aβ with aging leads to gene expression changes in meningeal lymphatic endothelial cells and an accelerated reduction in meningeal lymphatic vessel coverage in the 5xFAD mouse model. Interestingly, modulating meningeal lymphatic function in AD transgenic mice affects the clearance of Aβ by monoclonal antibodies, a feature that is closely associated with the activation signatures in brain blood endothelial cells and microglia. Furthermore, genes that are highly expressed in lymphatic vasculature are associated with increased risk for AD and altered levels of Aβ in the CSF of AD patients. Integrated analysis of murine and human microglia transcriptomes revealed an association between the signatures of activated microglia from mice with defective meningeal lymphatic function and activated microglia isolated from the AD brain. We are now turning our attention to the interaction between the main genetic risk factor for AD, expression of the apolipoprotein E4, and changes in brain drainage by the meningeal lymphatic vascular system.ConclusionCollectively, our data underline the notion that a dysfunctional meningeal lymphatic vasculature may impact on AD pathophysiology and therapeutic outcome.

Improving the Function of Meningeal Lymphatic Vessels to Promote Brain Edema Absorption after Traumatic Brain Injury.

Brain edema is the most common and fatal complication after traumatic brain injury (TBI). Meningeal lymphatic vessels (MLVs) are the conduits that transport cerebrospinal fluid (CSF) and macromolecules to deep extracranial cervical lymph nodes (dCLNs). After TBI, the drainage function of MLVs can become impaired. However, the scenario in which the improvement of the function of MLVs can promote brain edema absorption after TBI has not been reported. Therefore, the purpose of this study was to investigate the effects of ketoprofen, 9-cis retinoic acid (RA) and vascular endothelial cell growth factor-C (VEGF-C), which promote the proliferation of peripheral lymphatic vessels, on the cerebellar medullary cistern injection of TBI rats, as well as their mechanism of action on brain edema after TBI. In the experiment, we found that ketoprofen, 9-cisRA, and VEGF-C can improve the function of MLVs, promote the extracranial drainage of CSF and the absorption of brain edema, weaken the neuroinflammatory response, reduce reactive oxygen species (ROS) production, maintain the structural integrity of MLVs, and improve neurological function. In addition, ketoprofen, 9-cisRA, and VEGF-C upregulated the lymphatic-specific proteins VEGF receptor (VEGFR)3, PROX1, forkhead box protein C2 (FOXC2), and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1). These results indicate that ketoprofen, 9-cisRA, and VEGF-C may maintain the integrity of the meningeal lymphatic wall and promote lymphatic proliferation by upregulating the expression of lymphatic vessel-specific proteins, improve meningeal lymphatic function after TBI, promote CSF drainage and brain edema absorption, reduce the immune response of the nervous system, and reduce ROS formation, thereby improving prognoses. These findings may provide new ideas for the treatment of brain edema after TBI.

Age-associated suppression of exploratory activity during sickness is linked to meningeal lymphatic dysfunction and microglia activation.

Peripheral inflammation triggers a transient, well-defined set of behavioral changes known as sickness behavior1-3, but the mechanisms by which inflammatory signals originating in the periphery alter activity in the brain remain obscure. Emerging evidence has established meningeal lymphatic vasculature as an important interface between the central nervous system (CNS) and the immune system, responsible for facilitating brain solute clearance and perfusion by cerebrospinal fluid (CSF)4,5. Here, we demonstrate that meningeal lymphatics both assist microglial activation and support the behavioral response to peripheral inflammation. Ablation of meningeal lymphatics results in a heightened behavioral response to IL-1β-induced inflammation and a dampened transcriptional and morphological microglial phenotype. Moreover, our findings support a role for microglia in tempering the severity of sickness behavior with specific relevance to aging-related meningeal lymphatic dysfunction. Transcriptional profiling of brain myeloid cells shed light on the impact of meningeal lymphatic dysfunction on microglial activation. Furthermore, we demonstrate that experimental enhancement of meningeal lymphatic function in aged mice is sufficient to reduce the severity of exploratory abnormalities but not pleasurable consummatory behavior. Finally, we identify dysregulated genes and biological pathways, common to both experimental meningeal lymphatic ablation and aging, in microglia responding to peripheral inflammation that may result from age-related meningeal lymphatic dysfunction.

Effects of increased intracranial pressure on cerebrospinal fluid influx, cerebral vascular hemodynamic indexes, and cerebrospinal fluid lymphatic efflux

The glymphatic-lymphatic fluid transport system (GLFTS) consists of glymphatic pathway and cerebrospinal fluid (CSF) lymphatic outflow routes, allowing biological liquids from the brain parenchyma to access the CSF along with perivascular space and to be cleaned out of the skull through lymphatic vessels. It is known that increased local pressure due to physical compression of tissue improves lymphatic transport in peripheral organs, but little is known about the exact relationship between increased intracranial pressure (IICP) and GLFTS. In this study, we verify our hypothesis that IICP significantly impacts GLFTS, and this effect depends on severity of the IICP. Using a previously developed inflating balloon model to induce IICP and inject fluorescent tracers into the cisterna magna, we found significant impairment of the glymphatic circulation after IICP. We further found that cerebrovascular occlusion occurred, and cerebrovascular pulsation decreased after IICP. IICP also interrupted the drainage of deep cervical lymph nodes and dorsal meningeal lymphatic function, enhancing spinal lymphatic outflow to the sacral lymph nodes. Notably, these effects were associated with the severity of IICP. Thus, our findings proved that the intensity of IICP significantly impacts GLFTS. This may have translational applications for preventing and treating related neurological disorders.

Functional Investigation of Meningeal Lymphatic System in Experimental Intracerebral Hemorrhage.

Promotion of hematoma resolution in a timely manner reduces intracerebral hemorrhage (ICH) brain injury induced by toxic blood components and subsequent neuroinflammation. The meningeal lymphatic system is responsible for clearance of macromolecules and pathogenic substances from the central nervous system; however, its role in intraparenchymal hematoma clearance and ICH outcomes is unknown. In the present study, we aimed to understand the contribution of the meningeal lymphatic system to ICH pathologies and to test whether pharmacological enhancement of meningeal lymphatic function promotes hematoma resolution and brain recovery after ICH. Immunofluorescence of whole-mount meninges was used to measure complexity and coverage level of meningeal lymphatic vasculature following ICH induction. Fluorescent microbeads and PKH-26-labeled erythrocytes were used to evaluate drainage function of the meningeal lymphatic system. Visudyne treatment, deep cervical lymph node ligation, and VEGF (vascular endothelial growth factor)-C injection were performed to manipulate meningeal lymphatic function. Neurobehavioral performance and hematoma volume were assayed by the cylinder test and histological measurements. Iron deposition, residual erythrocytes, neuronal loss, and astrogliosis were assessed by immunohistochemistry and antibody-based fluorescence staining. Meningeal lymphangiogenesis and enhanced lymphatic drainage occurred during the late phase of ICH. Ablation and blockage of meningeal lymphatic vessels impeded hematoma clearance, whereas pharmacological enhancement of their function reduced hematoma volume, improved behavioral performance, and reduced brain residual erythrocytes, iron deposition, neuronal loss, and astroglial activation. Early enhancement of meningeal lymphatic function is beneficial for ICH recovery. Targeting the meningeal lymphatic system is therefore a potential therapeutic approach for treating ICH.

Abstract 100: Meningeal Lymphatic System Modulates Intra-parenchymal Hematoma Clearance And Brain Recovery In Experimental Intracerebral Hemorrhage

Background: Intracerebral hemorrhage (ICH) brain injury is induced by toxic blood components and subsequent neurologic deficits can be alleviated by promoting hematoma resolution. The meningeal lymphatic system has been shown to mediate substances clearance from the central nervous system, including macromolecules and pathogenic metabolites. However, it remains unclear whether this system plays a role in hematoma clearance and ICH outcomes. In this study, we investigated the correlation between meningeal lymphatic function and ICH pathologies. Pharmacological promotion of meningeal lymphatic function was also used to determine whether targeting meningeal lymphatic system can facilitate ICH recovery. Methods: Whole-mount meninges were used to evaluate the complexity and coverage rate of meningeal lymphatic vessels. Meningeal lymphatic drainage was evaluated by injecting fluorescent microbeads and PKH26-labeled erythrocytes into the cisterna magna and brain parenchyma, respectively. Hematoma volume was measured in fresh coronal brain sections. Neurobehavioral performance was evaluated by cylinder test. Pathological outcomes including iron deposition, residual erythrocytes, neuronal loss, and astrogliosis were detected by immunohistochemistry or immunofluorescence. Results: Meningeal lymphangiogenesis and increased lymphatic drainage occur during the late phase of ICH. Depriving the function of meningeal lymphatic vessels impeded hematoma clearance, whereas boosting meningeal lymphatic drainage and lymphangiogenesis improved hematoma clearance rate and behavioral function as well as reduced iron deposition, neuronal loss, and astrogliosis in the ICH brain. Conclusion: Early enhancement of meningeal lymphatic function is beneficial for ICH recovery, implicating the meningeal lymphatic system as a potential therapeutic target for treating ICH.

DSCR1 upregulation enhances dural meningeal lymphatic drainage to attenuate amyloid pathology of Alzheimer's disease.

Highly developed meningeal lymphatics remove waste products from the brain. Disruption of meningeal lymphatic vessels in a mouse model of amyloid pathology (5XFAD) accelerates the accumulation of amyloid plaques in the meninges and brain, and causes learning and memory deficits, suggesting that clearance of toxic wastes by lymphatic vessels plays a key role in neurodegenerative diseases. Here, we discovered that DSCR1 (Down syndrome critical region 1, known also as RCAN1, regulator of calcineurin 1) facilitates the drainage of waste products by increasing the coverage of dorsal meningeal lymphatic vessels. Furthermore, upregulation of DSCR1 in 5XFAD mice diminishes Aβ pathology in the brain and improves memory defects. Surgical ligation of cervical lymphatic vessels afferent to dcLN blocks the beneficial effects of DSCR1 on Aβ accumulation and cognitive function. Interestingly, intracerebroventricular delivery of AAV1-DSCR1 to 5XFAD mice is sufficient to rebuild the meningeal lymphatic system and re-establish cognitive performance. Collectively, our data indicate that DSCR1 facilitates the growth of dorsal meningeal lymphatics to improve drainage efficiency and protect against Alzheimer's disease (AD) pathologies, further highlighting that improving meningeal lymphatic function is a feasible treatment strategy for AD. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Meningeal lymphatics affect microglia responses and anti-Aβ immunotherapy.

Alzheimer's disease (AD) is the most prevalent cause of dementia1. Although there is no effective treatment for AD, passive immunotherapy with monoclonal antibodies against amyloid beta (Aβ) is a promising therapeutic strategy2,3. Meningeal lymphatic drainage has an important role in the accumulation of Aβ in the brain4, but it is not known whether modulation of meningeal lymphatic function can influence the outcome of immunotherapy in AD. Here we show that ablation of meningeal lymphatic vessels in 5xFAD mice (a mouse model of amyloid deposition that expresses five mutations found in familial AD) worsened the outcome of mice treated with anti-Aβ passive immunotherapy by exacerbating the deposition of Aβ, microgliosis, neurovascular dysfunction, and behavioural deficits. By contrast, therapeutic delivery of vascular endothelial growth factor C improved clearance of Aβ by monoclonal antibodies. Notably, there was a substantial overlap between the gene signature of microglia from 5xFAD mice with impaired meningeal lymphatic function and the transcriptional profile of activated microglia from the brains of individuals with AD. Overall, our data demonstrate that impaired meningeal lymphatic drainage exacerbates the microglial inflammatory response in AD and that enhancement of meningeal lymphatic function combined with immunotherapies could lead to better clinical outcomes.

CNS-Draining Meningeal Lymphatic Vasculature: Roles, Conundrums and Future Challenges

A genuine and functional lymphatic vascular system is found in the meninges that sheath the central nervous system (CNS). This unexpected (re)discovery led to a reevaluation of CNS fluid and solute drainage mechanisms, neuroimmune interactions and the involvement of meningeal lymphatics in the initiation and progression of neurological disorders. In this manuscript, we provide an overview of the development, morphology and unique functional features of meningeal lymphatics. An outline of the different factors that affect meningeal lymphatic function, such as growth factor signaling and aging, and their impact on the continuous drainage of brain-derived molecules and meningeal immune cells into the cervical lymph nodes is also provided. We also highlight the most recent discoveries about the roles of the CNS-draining lymphatic vasculature in different pathologies that have a strong neuroinflammatory component, including brain trauma, tumors, and aging-associated neurodegenerative diseases like Alzheimer’s and Parkinson’s. Lastly, we provide a critical appraisal of the conundrums, challenges and exciting questions involving the meningeal lymphatic system that ought to be investigated in years to come.

Live Imaging of Intracranial Lymphatics in the Zebrafish.

The recent discovery of meningeal lymphatics in mammals is reshaping our understanding of fluid homeostasis and cellular waste management in the brain, but visualization and experimental analysis of these vessels is challenging in mammals. Although the optical clarity and experimental advantages of zebrafish have made this an essential model organism for studying lymphatic development, the existence of meningeal lymphatics has not yet been reported in this species. Examine the intracranial space of larval, juvenile, and adult zebrafish to determine whether and where intracranial lymphatic vessels are present. Using high-resolution optical imaging of the meninges in living animals, we show that zebrafish possess a meningeal lymphatic network comparable to that found in mammals. We confirm that this network is separate from the blood vascular network and that it drains interstitial fluid from the brain. We document the developmental origins and growth of these vessels into a distinct network separated from the external lymphatics. Finally, we show that these vessels contain immune cells and perform live imaging of immune cell trafficking and transmigration in meningeal lymphatics. This discovery establishes the zebrafish as a important new model for experimental analysis of meningeal lymphatic development and opens up new avenues for probing meningeal lymphatic function in health and disease.