Functional aspects of meningeal lymphatics in ageing and Alzheimer's disease

Abstract

Ageing is a major risk factor for many neurological pathologies, but its mechanisms remain unclear. Unlike other tissues, the parenchyma of the central nervous system (CNS) lacks lymphatic vasculature and waste products are removed partly through a paravascular route. (Re)discovery and characterization of meningeal lymphatic vessels has prompted an assessment of their role in waste clearance from the CNS. Here we show that meningeal lymphatic vessels drain macromolecules from the CNS (cerebrospinal and interstitial fluids) into the cervical lymph nodes in mice. Impairment of meningeal lymphatic function slows paravascular influx of macromolecules into the brain and efflux of macromolecules from the interstitial fluid, and induces cognitive impairment in mice. Treatment of aged mice with vascular endothelial growth factor C enhances meningeal lymphatic drainage of macromolecules from the cerebrospinal fluid, improving brain perfusion and learning and memory performance. Disruption of meningeal lymphatic vessels in transgenic mouse models of Alzheimer's disease promotes amyloid-β deposition in the meninges, which resembles human meningeal pathology, and aggravates parenchymal amyloid-β accumulation. Meningeal lymphatic dysfunction may be an aggravating factor in Alzheimer's disease pathology and in age-associated cognitive decline. Thus, augmentation of meningeal lymphatic function might be a promising therapeutic target for preventing or delaying age-associated neurological diseases.

Extended Data Figure 1. Ablation of meningeal lymphatics leads to decreased CSF macromolecule drainage without affecting meningeal/brain blood vasculature or brain ventricular volume

a, Seven days after meningeal lymphatic ablation, a volume of 5 μL of fluorescent ovalbumin-Alexa647 (OVA-A647) was injected intra-cisterna magna (i.c.m.), into the CSF, and drainage of tracer into the deep cervical lymph nodes (dCLNs) was assessed 2 h later. Representative images of OVA-A647 (red) drained into the dCLNs stained for LYVE-1 (green) and with DAPI (blue; scale bar, 200 μm). b, Quantification of OVA-A647 area fraction (%) in the dCLNs showed significantly less amount of tracer in the Visudyne/photoconversion group than in control groups (mean ± s.e.m., n = 6 per group; one-way ANOVA with Bonferroni’s post-hoc test; a and b is representative of 2 independent experiments; significant differences between vehicle/photoconversion and Visudyne/photoconversion groups were observed in a total of 5 independent experiments). c, Seven days after meningeal lymphatic ablation, mice from the 3 groups were submitted to magnetic resonance venography (MRV) or angiography (MRA) and 24 h later to T2-weighted MRI to assess blood-brain barrier integrity after i.v. injection of the contrast agent gadolinium (Gd) at a dose of 0.3 mmol/Kg. d, Representative 3D reconstructions of intracranial veins and arteries of mice from each group (scale bar, 5 mm). e–h, No significant changes between groups were observed for (e) venous vessel volume, (f) superior sagittal sinus (SSS) diameter, (g) arterial vessel volume and (h) basilar artery diameter (mean ± s.e.m., n = 5 in vehicle/photoconversion and in Visudyne/photoconversion, n = 4 in Visudyne; one-way ANOVA with Bonferroni’s post-hoc test). i, Using the Lymph4D software, it was possible to measure changes in signal intensity gain in MRI sequences 1–5 (relative to baseline) in the hippocampus of mice from each group (scale bar, 3 mm). j, Quantification of the signal intensity gain (relative to baseline) in the hippocampus over 5 MRI acquisition sequences showed no differences between groups (mean ± s.e.m., n = 5 in vehicle/photoconversion and in Visudyne/photoconversion, n = 4 in Visudyne; repeated measures two-way ANOVA with Bonferroni’s post-hoc test). k, Mice were subjected to T2-weighted MRI to assess volume changes in brain ventricles 7 days after injection of vehicle or Visudyne and photoconversion. l, Representative images of 3D reconstruction of brain ventricles of mice from the two groups (scale bar, 1 mm). m, No differences were detected in the volume of the brain ventricles after meningeal lymphatic ablation (mean ± s.e.m., n = 5 per group; two-tailed Mann-Whitney test).

Extended Data Figure 2. Intracranial pressure measurements and assessment of CSF drainage and brain influx

a, Intracranial pressure (ICP) was measured in four different steps of intra-cisterna magna (i.c.m.) injection of 2 μL or 5 μL of tracer solution: pre-injection, during injection, post-injection (with syringe inside the cisterna magna) and post-injection (with syringe out of the cisterna magna). A significant increase in ICP for each volume was observed during injection when compared to pre-injection and post-injection (syringe in). Significantly higher ICP values post-injection (syringe in) were observed when compared to ICP values pre-injection. A significant decrease in ICP for each volume was observed post-injection (syringe out) when compared to all other steps of i.c.m. injection. No significant differences in ICP values were observed between groups injected with 2 μL or 5 μL of tracer for any of the analyzed steps of the i.c.m. injection method (mean ± s.e.m., n = 7 per group; repeated measures two-way ANOVA with Bonferroni’s post-hoc test; *vs pre-injection; ^#vs during injection; ^&vs post-injection (syringe in); data was pooled from 2 independent experiments). b, ICP was measured 30, 60 and 120 min post injection (p.i.) of 2, 5 or 10 μL of tracer solution into the CSF and compared to ICP values in non-injected mice. Significant differences were observed between ICP values of non-injected mice and mice injected with 2 μL of tracer at 30 min and 120 min post-injection (mean ± s.e.m., n = 5 per group; one-way ANOVA with Bonferroni’s post-hoc test). c, Seven days after meningeal lymphatic ablation, a volume of 2 μL of fluorescent OVA-A647 was injected into the CSF and drainage of tracer into the dCLNs was assessed 2 h later. d, Representative images of OVA-A647 (red) drained into the dCLNs, stained for LYVE-1 (green) and with DAPI (blue; scale bar, 200 μm). e, Quantification of OVA-A647 area fraction (%) in the dCLNs showed significantly less amount of tracer in the Visudyne/photoconversion group than in control groups. f, Representative brain sections stained with DAPI (blue) showing OVA-A647 (red) influx into the brain parenchyma of mice from Visudyne/photoconversion and control groups (scale bar, 5 mm; inset scale bar, 1 mm). g, Quantification of OVA-A647 area fraction (%) in brain sections showing a significant decrease in the Visudyne/photoconversion group when compared to control groups. Data in e and g is presented as mean ± s.e.m., n = 6 per group; one-way ANOVA with Bonferroni’s post-hoc test was used in e and g; c–g is representative of 2 independent experiments.

Extended Data Figure 3. Impaired brain perfusion by CSF macromolecules is observed in lymphatic ligated and in Prox1^+/− mice and does not correlate with AQP4 levels

a, Adult mice were submitted to surgical ligation of the lymphatic vessels afferent to the dCLNs. One week after the procedure, 5 μL of OVA-A647 was injected into the CSF (i.c.m.) and mice were transcardially perfused 2 h later. Representative brain sections stained with DAPI (blue) showing OVA-A647 (red) influx into the brain parenchyma of ligated and sham-operated mice (scale bar, 5 mm; inset scale bar, 2 mm). b, Quantification of OVA-A647 area fraction (%) in brain sections showed a significant decrease in the ligation group. c, Representative sections of dCLNs stained with DAPI (blue) and for LYVE-1 (green), showing OVA-A647 (red) coverage in the ligation and sham-operated groups (scale bar, 200 μm). d, Quantification of OVA-A647 area fraction (%) in the dCLNs showed a significant decrease in the ligation group. Data in b and d is presented as mean ± s.e.m., n = 8 per group; two-tailed Mann-Whitney test was used in b and d; data in a–d was pooled from 2 independent experiments and is representative of 3 independent experiments. e, Wild-type (WT) and Prox1^+/− mice (2–3 months-old) were injected with 5 μL of OVA-A647 into the CSF (i.c.m.) and transcardially perfused 2 h later. f, Representative brain sections stained with DAPI (blue) showing OVA-A647 (red) influx into the brain parenchyma of Prox1^+/− and WT mice (scale bar, 5 mm). g, Quantification of OVA-A647 area fraction (%) in brain sections showed a significant decrease in Prox1^+/− mice. h, Representative sections of dCLNs stained with DAPI (blue) and for LYVE-1 (green), showing OVA-A647 (red) coverage in the dCLNs of Prox1^+/− and WT mice (scale bar, 500 μm). i, Quantification of OVA-A647 area fraction (%) in the dCLNs showed a significant decrease in Prox1^+/− mice. Data in g and i is presented as mean ± s.e.m., n = 15 in WT, n = 12 in Prox1^+/−; two-tailed Mann-Whitney test was used in g and i; data in e–i was pooled from 2 independent experiments. j, Rate of brain paravascular influx of the contrast agent gadolinium (Gd), injected i.c.m. at 1, 10 or 25 mM (in saline), was assessed in adult mice (3 months-old) by T1-weighted magnetic resonance imaging (MRI). k, Representative MRI images obtained using Lymph4D software showing brain signal intensity for different concentrations of injected Gd (scale bar, 3 mm). Experiment in j and k was performed once. l, Adult mice were subjected to meningeal lymphatic ablation by Visudyne photoconversion. One week later, T1-weighted MRI acquisition was performed after i.c.m. injection of 5 μL of Gd (25 mM in saline). Using the Lymph4D software, it was possible to measure the rate of contrast agent influx into the delineated brain cortical region of mice from both groups (scale bar, 3 mm). Images in sequence 2 and subsequent were obtained by subtraction of sequence 1. m, Quantification of the signal intensity gain (relative to sequence 1) in the brain cortex revealed a significant decrease in the Visudyne/photoconversion group, when compared to vehicle/photoconversion. n, o, Coronal sections of the brain of vehicle- or Visudyne-treated mice (n = 4 per group) were aligned and stacked into 2D colormaps (concatenated from 16 MRI sequences) showing (n) contrast of Gd signal intensity and (o) isotropic diffusion coefficient (scale bars, 3 mm). p, Area fraction quantification of high, medium and low values of isotropic diffusion coefficient in the four 2D stacks, in Visudyne relative to vehicle. Data in m and p is presented as mean ± s.e.m., n = 4 per group; repeated measures two-way ANOVA with Bonferroni’s post-hoc test was used in m and one-way ANOVA with Bonferroni’s post-hoc test was used in p; l–p is representative of 2 independent experiments. q, Representative confocal images of DAPI (blue) and aquaporin 4 (AQP4, green) staining and OVA-A647 (red) levels in brain sections from vehicle- and Visudyne-treated mice (scale bar, 500 μm). r, Quantification of area fraction (%) of AQP4 in the brains of mice treated with vehicle or Visudyne showing no differences between groups. s, Images showing representative staining for AQP4⁺ astrocytic endfeet (red) and CD31⁺ blood vessels (green) in the brain cortex of mice from vehicle and Visudyne groups (scale bar, 50 μm). t–v, No changes were observed in the area of (t) AQP4⁺ astrocytic endfeet and of (u) CD31⁺ blood vessels or in (v) the ratio between area of AQP4⁺ and of CD31⁺. Data in r and t–v is presented as mean ± s.e.m., n = 7 per group; two-tailed Mann-Whitney test was used in r and t–v; data in q–v was pooled from 2 independent experiments and is representative of 3 independent experiments.

Extended Data Figure 4. Ablation of meningeal lymphatic vessels impairs efflux of macromolecules from the brain

a, Seven days after meningeal lymphatic ablation, 1 μL of fluorescent OVA-A647 (0.5 mg/mL in artificial CSF) was stereotaxically injected (coordinates from bregma, AP = +1.5 mm, ML = −1.5 mm, DV = +2.5 mm) into the brain parenchyma. b, Representative brain sections rostral and caudal to the injection site, stained for glial fibrillary acidic protein (GFAP, in green), demonstrating OVA-A647 (red) coverage of the brain parenchyma in the Visudyne/photoconversion group and the control groups (scale bar, 5 mm). c, Quantification of OVA-A647 area fraction (%) in the injected brain hemisphere showing a significantly higher level in the Visudyne/photoconversion group, when compared to both control groups (mean ± s.e.m., n = 6 per group; one-way ANOVA with Bonferroni’s post-hoc test). d, Seven days after meningeal lymphatic ablation, 1 μL of fluorescent Aβ₄₂-HiLyte647 (0.05 μg/mL in artificial CSF) was stereotaxically injected (coordinates from bregma, AP = +1.5 mm, ML = −1.5 mm, DV = +2.5 mm) into the brain parenchyma. e, Representative brain sections rostral and caudal to the injection site, stained for GFAP (green), demonstrating Aβ₄₂-HiLyte647 (red) coverage of the brain parenchyma in the Visudyne/photoconversion group and the control groups (scale bar, 5 mm). f, Quantification of Aβ₄₂-HiLyte647 area fraction (%) in the injected brain hemisphere showing a significantly higher level in the Visudyne/photoconversion group, when compared to both control groups (mean ± s.e.m., n = 6 per group; one-way ANOVA with Bonferroni’s post-hoc test). g, Seven days after meningeal lymphatic ablation, 1 μL of fluorescent low density lipoprotein (LDL)-BODIPY FL (0.1 mg/mL in artificial CSF) was stereotaxically injected (coordinates from bregma, AP = +1.5 mm, ML = −1.5 mm, DV = +2.5 mm) into the brain parenchyma. h, Representative brain sections rostral and caudal to the injection site, stained for GFAP (red), demonstrating LDL-BODIPY FL (green) coverage of the brain parenchyma in the Visudyne/photoconversion group and the control groups (scale bar, 5 mm). i, Quantification of LDL-BODIPY FL area fraction (%) in the injected brain hemisphere showing a significantly higher level in the Visudyne/photoconversion group, when compared to both control groups (mean ± s.e.m., n = 6 per group; one-way ANOVA with Bonferroni’s post-hoc test).

Extended Data Figure 5. Behavioral assessment and hippocampal RNA-seq analysis after impairing meningeal lymphatic function

a, b, No differences in (a) total distance and in (b) time in center of the open field arena were observed between vehicle/photoconversion, Visudyne and Visudyne/photoconversion groups (mean ± s.e.m., n = 9 per group; one-way ANOVA with Bonferroni’s post-hoc test). c, d, Performance of mice from the 3 groups was also identical both in the (c) training and in the (d) novel location task of the novel location recognition paradigm (mean ± s.e.m., n = 9 per group; two-way ANOVA with Bonferroni’s post-hoc test). e, f, Mice performance in the contextual fear conditioning paradigm showed no differences between groups in the (e) context test, but a statistically significant difference in the (f) cued test (mean ± s.e.m., n = 9 per group; one-way ANOVA with Bonferroni’s post-hoc test). g, The cognitive performance of adult mice was assessed in the Morris water maze (MWM) test, one week after sham surgery or surgical ligation of the lymphatics afferent to the dCLNs. h–j, Ligated mice presented a significant increase in the (h) latency to platform during acquisition, when compared to sham-operated mice. No significant differences between groups were observed in the (i) % of time spent in the target quadrant in the probe trial or in the (j) reversal (mean ± s.e.m., n = 8 in sham, n = 9 in ligation; repeated measures two-way ANOVA with Bonferroni’s post-hoc test was used in h and j; two-tailed Mann-Whitney test was used in i). k, Vehicle or Visudyne injection with photoconversion were performed twice within two weeks interval. Total RNA was extracted from the hippocampus of mice from both groups and sequenced (RNA-seq). RNA-seq principal component (PC) analysis did not show a differential clustering of samples from vehicle and Visudyne groups. l, Heatmap showing relative expression levels of genes in vehicle/photoconversion and in Visudyne/photoconversion samples. m, After meningeal lymphatic ablation (twice within two weeks interval) and MWM performance, total RNA was extracted from the hippocampus of mice from vehicle/photoconversion or Visudyne/photoconversion groups and sequenced. RNA-seq principal component (PC) analysis demonstrating a differential clustering of samples from vehicle and Visudyne groups. A total of 2138 genes were down-regulated and 1599 genes were up-regulated in the hippocampus after meningeal lymphatic ablation and MWM performance. n, Heatmap showing relative expression levels of genes in vehicle/photoconversion and in Visudyne/photoconversion samples (color scale bar values represent standardized rlog-transformed values across samples for l and n). o, Neurological disease, neuronal activity and synaptic plasticity related GO and KEGG terms enriched upon Visudyne treatment, as measured by the –log10(adj. P-value). p, GO and KEGG terms related with metabolite generation and processing, glycolysis and mitochondrial respiration and oxidative stress that were enriched, as measured by the –log10(adj. P-value), upon Visudyne treatment and MWM performance. q, r, Heatmap showing relative expression levels of genes involved in two of the significantly altered GO terms related to (q) Excitatory synapse and (r) Learning or memory. s–v, Heatmaps showing relative expression levels of genes involved in four of the significantly altered GO terms related to (s) NADH dehydrogenase complex, (t) Generation of precursor metabolites and energy, (u) Cellular response to oxidative stress and (v) Cellular response to nitrogen compound. Datasets in k–v all consist of n = 5 per group; in k and m P-values were corrected for multiple hypothesis testing with the Benjamini–Hochberg false discovery rate procedure; in l and n–v functional enrichment of differential expressed genes was performed using gene sets from GO and KEGG and determined with Fisher’s exact test; color scale bar values in n and q–v represent standardized rlog-transformed values across samples.

Extended Data Figure 6. Characterization of meningeal lymphatics in young and old mice and improvement of lymphatic function by viral-mediated expression of mVEGF-C

a, OVA-A647 was injected into the CSF (i.c.m.) of young-adult (3 months of age) and old (20–24 months of age) mice. Representative brain sections stained with DAPI (blue) showing degree of OVA-A647 (red) influx into the parenchyma (scale bar, 5 mm; inset scale bar, 2 mm). b, Quantification of OVA-A647 area fraction (%) in brain sections (mean ± s.e.m., n = 6 in 3 months, n = 8 in 20–24 months; two-tailed Mann-Whitney test; representative of 2 independent experiments). c, Representative images of DAPI (blue) and LYVE-1 (green) staining in meningeal whole-mounts of young-adult (2 months-old) and old (20–24 months-old) male and female mice (scale bar, 1 mm). d, Measurement of LYVE-1⁺ vessel diameter and area fraction showed a significant decrease in both parameters in old mice, when compared to young-adults, in both females and males. e, Representative images of DAPI (blue) and LYVE-1 (green) staining in dCLNs 2 h after injection of OVA-A594 (red) into the CSF of young-adult and old mice from both genders (scale bar, 200 μm). f, Quantification of OVA-A594 area fraction (%) in the dCLNs of mice from different ages and genders showed a significant decrease in 20–24 months-old female and male mice. Data in d and f is presented as mean ± s.e.m., n = 9 per group at 2 months, n = 7 per group at 20–24 months for male and female; two-way ANOVA with Bonferroni’s post-hoc test was used in d and f; data was pooled from 2 independent experiments. g, Representative dot and contour plots showing the gating strategy used to isolate meningeal lymphatic endothelial cells (LECs) by fluorescence-activated cell sorting (FACS) from the meninges of young-adult and old mice (n = 3 per group, pooled from 2 independent experiments). h, Adult mice were injected i.c.m. with 2 μL of AAV1-CMV-EGFP (EGFP) or AAV1-CMV-mVEGF-C (mVEGF-C), both at 10¹³ genome copies (GC)/mL, and transcardially perfused with saline 2 or 4 weeks later. i, Representative brain coronal sections of mice showing EGFP⁺ infected cells (green) in the pia mater, surrounding the GFAP⁺ glia limitans (red) of the brain parenchyma, at 2 and 4 weeks post injection (scale bar, 5 mm; inset scale bar, 200 μm). j, Representative insets from meningeal whole-mounts stained for CD31 (blue), EGFP (green) and LYVE-1 (red; scale bar, 200 μm). Green cells are observed in the cerebellar meninges, pineal gland and transverse sinus in the EGFP group at 2 and 4 weeks, but not in the same regions of the meninges in the mVEGF-C group. k, Representative images of LYVE-1⁺ lymphatic vessels (red) and LYVE-1⁻CD31⁺ blood vessels (blue) in the superior sagittal sinus of mice treated with either EGFP or mVEGF-C, for 2 or 4 weeks (scale bar, 200 μm). l, m, Mice treated with AAV1 expressing mVEGF-C presented a significant increase in (l) lymphatic vessel diameter, but not in (m) coverage by blood vessels. Data in l and m is presented as mean ± s.e.m., n = 4 per group at 2 weeks, n = 3 per group at 4 weeks; two-way ANOVA with Bonferroni’s post-hoc test was used in l and m; data in h–m is representative of 2 independent experiments. n, Representative images of blood flow (mm/s) and arterial and venous blood oxygenation (% of sO₂) readings obtained by Photoacoustic imaging of brain/meningeal vasculature of old mice (20–22 months-old) treated for 1 month with EGFP or mVEGF-C virus (both at 10¹³ GC/mL). o, p, The different treatments did not affect (n) blood flow or (p) blood oxygenation in the brain/meninges of old mice (mean ± s.e.m., n = 5 per group; two-tailed Mann-Whitney test was used in n and two-way ANOVA with Bonferroni’s post-hoc test was used in p; data results from a single experiment). q, Old mice (20–22 months-old) were injected i.c.m. with 2 μL of viral vectors expressing EGFP or mVEGF-C. One month later, T1-weighted MRI acquisition was performed after i.c.m. injection of 5 μL of gadolinium (25 mM in saline). Using the Lymph4D software, it was possible to measure the rate of contrast agent influx into the delineated brain hippocampal region of mice from both groups (scale bar, 3 mm). Images in sequence 2 and subsequent were obtained by subtraction of sequence 1. r, Quantification of the signal intensity gain (relative to sequence 1) in the hippocampus revealed a significant increase in the mVEGF-C group, when compared to EGFP (mean ± s.e.m., n = 4 per group; repeated measures two-way ANOVA with Bonferroni’s post-hoc; data was pooled from 2 independent experiments).

Extended Data Figure 7. Treatment with VEGF-C ameliorates meningeal lymphatic function, brain perfusion by CSF macromolecules and cognitive performance in old mice

a, Hydrogel alone (vehicle) or containing recombinant human VEGF-C (200 ng/mL) was applied on top of a thinned skull surface of adult (3 months-old) and old mice (20–24 months-old). Gels were re-applied two weeks later. Four weeks after the initial treatment, 5 μL of OVA-A647 (in artificial CSF) was injected into the CSF (i.c.m.) and mice were transcardially perfused 2 h later. b, Representative images of DAPI (blue) staining and LYVE-1⁺ vessels (in green) in the superior sagittal sinus after transcranial delivery of VEGF-C (scale bar, 50 μm). c, Treatment with VEGF-C resulted in significant increase of lymphatic vessel diameter in the superior sagittal sinus in both adult and old mice. d, Representative sections of dCLNs stained with DAPI (blue) and for LYVE-1 (green) showing drained OVA-A647 (red; scale bar, 200 μm). e, Quantification of OVA-A647 (red) area fraction (%) in the dCLNs showed increased drainage in old mice treated with VEGF-C, when compared to vehicle-treated age-matched mice. f, Representative brain sections stained with DAPI (blue) showing OVA-A647 (red) influx into the brain parenchyma (scale bar, 5 mm). g, Influx of OVA-A647 into the brain parenchyma of old mice was significantly increased after transcranial delivery of VEGF-C. Data in c, e and g are presented as mean ± s.e.m., n = 12 in vehicle at 3 months, n = 11 in VEGF-C at 3 months, n = 8 in vehicle at 20–24 months and n = 9 in VEGF-C at 20–24 months; two-way ANOVA with Bonferroni’s post-hoc test was used in c, e and g; data in a–g was pooled from 2 independent experiments. h, Hydrogel alone (vehicle) or containing recombinant human VEGF-C156S (200 ng/mL) was applied on top of a thinned skull surface of old mice. Gels were re-applied two weeks later. i, Whole-mounts of brain meninges were stained for LYVE-1 (green) and CD31 (red). Images show insets of lymphatic vessels near the superior sagittal sinus (scale bar, 100 μm). j, Old mice that received VEGF-C156S treatment showed increased diameter of LYVE-1⁺ vessels in the superior sagittal sinus. k, Representative sections of dCLNs stained with DAPI (blue) and for LYVE-1 (green) showing levels of OVA-A647 (red) drained from the CSF (scale bar, 200 μm). l, Quantification of OVA-A647 area fraction (%) in the dCLNs showed a significant increase in VEGF-C156S group when compared to vehicle. m, Representative images of OVA-A647 (red) in brain sections also stained with DAPI (blue; scale bar, 5 mm). n, Quantification of OVA-A647 area fraction (%) in brain sections showed a significant increase in brain influx of the tracer in old mice treated with VEGF-C156S. Data in j, l and n is presented as mean ± s.e.m., n = 7 mice per group; two-tailed Mann-Whitney test was used in j, l and n; data in h–n was pooled from 2 independent experiments. o, Young-adult (2 months), middle-aged (12–14 months) or old (20–22 months) mice were injected with viral vectors expressing EGFP or mVEGF-C. One month after injection, learning and memory was assessed using the MWM test. p, Injection of mVEGF-C virus in young-adult mice did not alter their performance in the acquisition, probe trial or reversal of the MWM (mean ± s.e.m., n = 8 in EGFP and n = 9 in mVEGF-C; repeated measures two-way ANOVA with Bonferroni’s post-hoc test was used in the acquisition and reversal; two-tailed Mann-Whitney test was used in the probe trial; data was obtained in a single experiment). q, Injection of mVEGF-C virus in middle-aged mice did not alter their performance in the acquisition and in the probe trial, but significantly improved their performance in the reversal (mean ± s.e.m., n = 12 in EGFP and n = 14 in mVEGF-C; repeated measures two-way ANOVA with Bonferroni’s post-hoc test was used in the acquisition and reversal, two-tailed Mann-Whitney test was used in the probe trial; data was pooled from 2 independent experiments). r, Injection of mVEGF-C virus in old mice did not alter their performance in the probe trial, but significantly improved their performance in the acquisition and in the reversal (mean ± s.e.m., n = 25 in EGFP and n = 25 in mVEGF-C; repeated measures two-way ANOVA with Bonferroni’s post-hoc test was used in the acquisition and reversal; two-tailed Mann-Whitney test was used in the probe trial; data was pooled from 3 independent experiments). s–u, Treatment of (s) young-adult mice with mVEGF-C did not affect the % of allocentric navigation strategies used in the MWM. The % of allocentric navigation strategies was significantly higher in (t) middle-aged mice treated with mVEGF-C during the reversal and in (u) old mice treated with mVEGF-C during the acquisition and reversal, when compared to their age-matched EGFP-treated counterparts. Data in s–u is presented as mean ± s.e.m.; n = 8 in EGFP and n = 9 in mVEGF-C at 2 months in s; n = 12 in EGFP and n = 14 in mVEGF-C at 12–14 months in t; n = 25 per group at 20–22 months in u; repeated measures two-way ANOVA with Bonferroni’s post-hoc test was used in s–u; data in s was obtained from a single experiment, data in t was pooled from 2 independent experiments and data in u was pooled from 3 independent experiments. v, Insets of the hippocampal dentate gyrus (granular zone, GZ), stained with DAPI (blue) and for Ki67 (in red), in mice injected with viral vectors expressing EGFP or mVEGF-C at 2, 12–14 and 20–22 months (scale bar, 200 μm). w, Aging induced a significant decrease in Ki67⁺ proliferating cells in the dentate gyrus. Expression of mVEGF-C in the meninges at the analyzed ages did not affect the number of Ki67⁺ cells in the dentate gyrus (mean ± s.e.m., n = 5 per group; two-way ANOVA with Bonferroni’s post-hoc test).

Extended Data Figure 8. Expression of mVEGF-C in the meninges of J20 mice does not ameliorate lymphatic drainage or brain amyloid pathology

a, J20 mice were injected i.c.m. with 2 μL of AAV1-CMV-EGFP or AAV1-CMV-mVEGF-C (10¹³ GC/mL) at 6–7 months. One month after injection, the mice were tested in the open field (OF) and in the MWM. b, c, Total distance and % of time in the center of the OF arena was not ameliorated by treatment of J20 mice with mVEGF-C. d–f, No statistically significant differences were observed in the (d) acquisition, in the (e) probe trial or in the (f) reversal of the MWM test after 1 month of mVEGF-C. Data in b–f is presented as mean ± s.e.m., n = 11 in EGFP, n = 12 in mVEGF-C; two-tailed Mann-Whitney test was used in b, c and e and repeated measures two-way ANOVA with Bonferroni’s post-hoc test was used in d and f; data results from a single experiment. g, J20 mice were treated with EGFP or mVEGF-C and, 1 month later, CSF, meninges and brain were collected for analysis. h, Representative images of DAPI (blue) and LYVE-1⁺ lymphatic vessels (green) in the superior sagittal sinus of mice treated with either EGFP or mVEGF-C (scale bar, 500 μm). i, AAV1-mediated expression of mVEGF-C did not affect meningeal lymphatic vessel diameter. j, Levels of Aβ in the CSF measured by ELISA remained unaltered after mVEGF-C treatment. k, Representative images of dorsal hippocampus (scale bar, 500 μm) of J20 mice of EGFP or mVEGF-C groups stained with DAPI (cyan) and for IBA1 (green) and Aβ (red). l–n, No changes were observed in amyloid plaque (l) size, (m) number or (n) coverage between the groups. Data in i, j and l–n is presented as mean ± s.e.m., n = 6 per group; two-tailed Mann-Whitney test was used in i, j and l–n; data in g–n results from a single experiment. o, J20 mice (2–3 months-old) and 5xFAD mice (3–4 months-old), and respective age-matched WT littermate controls, were injected with fluorescent OVA-A647 (i.c.m.) in order to measure drainage into the dCLNs. p, Representative images of DAPI (blue) and LYVE-1 (green) staining in dCLNs of WT and J20 mice (scale bar, 200 μm) 2 h after injection of OVA-A647 (red). q, Quantification of OVA-A647 area fraction (%) in the dCLNs shows equal levels of tracer in mice from both genotypes (mean ± s.e.m., n = 5 per group; two-tailed Mann-Whitney test; representative of 2 independent experiments). r, Representative images of DAPI (blue) and LYVE-1 (green) staining in dCLNs of WT and 5xFAD mice (scale bar, 200 μm) 2 h after injection of OVA-A594 (red). s, Quantification of OVA-A594 area fraction (%) in the dCLNs shows equal levels of tracer in mice from both genotypes (mean ± s.e.m., n = 11 per group; two-tailed Mann-Whitney test; data was pooled from 2 independent experiments). t, Representative images of DAPI (blue) and LYVE-1 (green) staining in meningeal whole-mounts of WT and 5xFAD mice at 3–4 months (scale bar, 1 mm). u, Measurement of LYVE-1⁺ vessel diameter, area fraction and number of sprouts (per mm of vessel) showed no differences between genotypes (mean ± s.e.m., n = 7 per group; two-tailed Mann-Whitney test; data was pooled from 2 independent experiments).

Extended Data Figure 9. Meningeal lymphatic ablation in AD transgenic mice worsens amyloid pathology without affecting blood vessel function

a, Representative images of blood flow (mm/s) and arterial and venous blood oxygenation (% of sO₂) readings obtained by Photoacoustic imaging of brain/meningeal vasculature of 5xFAD mice one week after vehicle/photoconversion, Visudyne or Visudyne/photoconversion. b, c, The different treatments did not affect (b) blood flow or (c) blood oxygenation in the brain/meninges of 5xFAD mice (mean ± s.e.m., n = 5 per group; one-way ANOVA with Bonferroni’s post-hoc test was used in b and two-way ANOVA with Bonferroni’s post-hoc test was used in c; data results from a single experiment). d, Representative flow cytometry dot and contour plots showing the gating strategies used to determine the frequency of specific immune cell populations, using a myeloid or lymphoid panel of markers, in the meninges of 5xFAD after prolonged (1.5 months) meningeal lymphatic ablation. e, Analysis of specific immune cell populations in the meninges of 5xFAD mice from the different groups showed a significant increase in macrophages in the Visudyne/photoconversion group when compared to the control groups. A significant increase in neutrophils was observed in Visudyne group, but not in vehicle/photoconversion group, when compared to Visudyne/photoconversion group (mean ± s.e.m., n = 5 per group; two-way ANOVA with Holm-Sidak’s post-hoc test; *vs vehicle/photoconversion; ^#vs Visudyne; data results from a single experiment). f, 4–5 months-old J20 mice were submitted to meningeal lymphatic ablation by injection (i.c.m.) of Visudyne or vehicle as a control, followed by a photoconversion step. This procedure was repeated every 3 weeks, for a total of 3 months, to achieve prolonged meningeal lymphatic ablation. g, Staining with DAPI (blue) and for LYVE-1 (green) and Aβ (red) in meningeal whole-mounts of J20 mice showing marked amyloid deposition in mice from the Visudyne group (scale bar, 500 μm). h, Representative brain sections of J20 mice at 7–8 months stained with DAPI (cyan) and for Aβ (red; scale bar, 500 μm) showing degree of amyloid deposition after meningeal lymphatic ablation. i–k, Quantification of amyloid plaque (i) size, (j) number and (k) coverage in the dorsal hippocampus of J20 mice showed a statistically significant increase in coverage in the Visudyne group, when compared to vehicle. Data in i–k is presented as mean ± s.e.m., n = 5 in vehicle, n = 6 in Visudyne; two-tailed Mann-Whitney test was used in i–k; experiments in f–k were performed once. l, m, Sections of human brain cortex, containing meningeal layers (leptomeninges) attached, from (l) non-AD brain (scale bar, 500 μm; inset scale bar, 200 μm) and (m) AD brain (left image scale bar, 100 μm; right image scale bar, 500 μm) were stained with DAPI (blue), for the astrocyte marker GFAP (green) and for Aβ (red). Data in l and m results of n = 8 non-AD samples and n = 9 AD samples and is representative of 2 independent experiments.

Figure 1. Impairing meningeal lymphatics affects brain CSF influx and ISF diffusion and worsens cognitive function

a, Seven days after lymphatic ablation mice were injected with 5 μL of ovalbumin-Alexa647 (OVA-A647) into the cisterna magna (i.c.m.). b, Representative images of meningeal whole-mounts stained for LYVE-1/CD31 (scale bar, 1 mm). c, d, Quantification of area fraction (%) occupied by (c) LYVE-1⁺ lymphatic vessels and (d) LYVE-1⁻CD31⁺ blood vessels. e, Representative brain sections showing 4′,6-diamidino-2-phenylindole (DAPI) and OVA-A647 (scale bar, 5 mm; inset scale bar, 1 mm). f, Quantification of OVA-A647 area fraction. Data in c, d and f is presented as mean ± s.e.m., n = 6 per group; one-way ANOVA with Bonferroni’s post-hoc test was used in c, d and f; a–f is representative of 2 independent experiments; significant differences between vehicle/photoconversion and Visudyne/photoconversion were replicated in 5 independent experiments. g, Gadolinium (Gd) was injected (i.c.m.) and T1-weighted magnetic resonance imaging (MRI) acquisition was performed 7 days after meningeal lymphatic ablation. h, Representative images of sequence 1 and of Gd intensity gain in subsequent sequences (hippocampus delineated in red; scale bar, 3 mm). i, Quantification of the Gd signal intensity gain over 16 sequences (relative to sequence 1) in hippocampus. Data in i is presented as mean ± s.e.m., n = 4 per group; repeated measures two-way ANOVA with Bonferroni’s post-hoc test; g–i is representative of 2 independent experiments. j, Meningeal lymphatic ablation was performed twice and two weeks after the last intervention, open field (OF), novel location recognition (NLR), contextual fear conditioning (CFC) and Morris water maze (MWM) behavioral tests were performed (Extended data Fig. 5 for OF, NLR and CFC). k, Latency to platform (acquisition). l, Time spent (%) in the target quadrant (probe). m, Latency to platform (reversal). n, o, Allocentric navigation strategies (%) used in the MWM (n) acquisition and (o) reversal. Data in k–m and n, o are presented as mean ± s.e.m., n = 9 per group; repeated measures two-way ANOVA with Bonferroni’s post-hoc test was used in k, m, n and o; one-way ANOVA with Bonferroni’s post-hoc test was used in l; significant differences between vehicle/photoconversion and Visudyne/photoconversion were replicated in 3 independent experiments.

Figure 2. Improving meningeal lymphatic function in aged mice increases brain perfusion and alleviates cognitive deficits

a, Principal component (PC) analysis plot for RNA-seq of lymphatic endothelial cells (LECs) from meninges of young-adult and aged mice. 230 genes up- and 377 genes down-regulated in meningeal LECs at 20–24 months. b, Expression of Pecam1, Lyve1, Prox1, Flt4, Pdpn and Ccl21a. c, Gene sets obtained by functional enrichment of differentially expressed genes in meningeal LECs at 20–24 months. d, Heatmap showing relative expression level of genes involved in Transmembrane receptor protein tyrosine kinase signaling pathway (color scale bar values represent standardized rlog-transformed values across samples). Data in a–d consists of n = 3 per group (individual RNA samples result from LECs pooled from 10 meninges over 2 independent experiments); data in b is presented as mean ± s.e.m. with two-way ANOVA with Bonferroni’s post-hoc test; in a–c P-values were corrected for multiple hypothesis testing with the Benjamini–Hochberg false discovery rate procedure; in c and d functional enrichment of differential expressed genes performed using gene sets from GO and KEGG and determined with Fisher’s exact test. e, Old mice were injected (i.c.m.) with 2 μL of AAV1-CMV-EGFP (EGFP) or AAV1-CMV-mVEGF-C (mVEGF-C), at 10¹³ genome copies (GC)/mL. One month later, OVA-A647 was injected i.c.m. f, Insets of the superior sagittal sinus showing DAPI/LYVE-1/CD31 (scale bar, 200 μm). g, h, Quantification of (g) diameter of LYVE-1⁺ lymphatic vessels and of (h) area fraction (%) of LYVE-1⁻CD31⁺ blood vessels. i, Representative sections of deep cervical lymph nodes (dCLNs) showing DAPI/LYVE-1/OVA-A647 (scale bar, 200 μm). j, Quantification of LYVE-1 and OVA-A647 area fraction in dCLNs. k, Representative brain coronal sections showing DAPI/OVA-A647 (scale bar, 5 mm). l, Quantification of OVA-A647 area fraction in brain sections. Data in g, h, j and l are presented as mean ± s.e.m., n = 5 in EGFP, n = 6 in mVEGF-C; two-tailed Mann-Whitney test was used in g, h, j and l; e–l is representative of 2 independent experiments. m, Old mice were injected with EGFP or mVEGF-C viruses (i.c.m.) after ligation of the lymphatics afferent to the dCLNs or sham surgery. One month later, learning and memory was assessed in the NLR and MWM tests and mice were injected (i.c.m.) with OVA-A647. n, o, Time with the object (%) was assessed in the NLR (n) training and (o) novel location tasks. p, Latency to platform (acquisition). q, Time spent (%) in the target quadrant (probe). r, Latency to platform (reversal). s, Representative sections of dCLNs showing DAPI/LYVE-1/OVA-A647 (scale bar, 200 μm). t, Quantification of OVA-A647 area fraction in dCLNs. Data in n–r and t is presented as mean ± s.e.m., n = 9 in sham + EGFP and ligation + EGFP, n = 10 in sham + mVEGF-C and ligation + mVEGF-C; two-way ANOVA with Bonferroni’s post-hoc test was used in n, o, q and t; repeated measures two-way ANOVA with Bonferroni’s post-hoc test was used in p and r; m–t results from 2 independent experiments.

Figure 3. Ablation of meningeal lymphatics aggravates amyloid pathology in AD transgenic mice

a, Young-adult 5xFAD mice were submitted to meningeal lymphatic ablation or control procedures. Procedures were repeated 3 weeks later and amyloid pathology was assessed 6 weeks after initial treatment. b, Staining for CD31/LYVE-1/Aβ in meninges (scale bar, 2 mm; inset scale bar, 500 μm). c, Orthogonal view of IBA⁺ macrophages clustering around an amyloid plaque in meninges of a 5xFAD with ablated lymphatics (scale bar, 200 μm). d, Representative images of DAPI/Aβ in the hippocampus of 5xFAD mice from each group (scale bar, 500 μm). e–g, Quantification of amyloid plaque (e) size, (f) number and (g) coverage in the hippocampus of 5xFAD mice. Data in e–g is presented as mean ± s.e.m., n = 10 per group; one-way ANOVA with Bonferroni’s post-hoc test was used in e–g; a–g is representative of 2 independent experiments. h, Staining for amyloid pathology was performed in human non-AD and AD brains (Extended data Fig. 9) and different meningeal layers. i, j, Meningeal superior sagittal sinus tissue of (i) non-AD or (j) AD patients stained with DAPI/Aβ (scale bar, 2 mm). k, l, Meningeal dura mater tissue of (k) non-AD or (l) AD patients, stained for IBA1/Aβ (scale bars, 1 mm; orthogonal view inset scale bars, 50 μm). Data in h–l results of n = 8 non-AD samples and n = 9 AD samples and is representative of 2 independent experiments.