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.

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Change history

  • 05 November 2018

    Change history: In this Article, Extended Data Fig. 9 was appearing as Fig. 2 in the HTML, and in Fig. 2, the panel labels ‘n’ and ‘o’ overlapped the figure; these errors have been corrected online.

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Acknowledgements

We thank S. Smith for editing the manuscript, J. Roy for MRI expertise, N. Al Hamadani for animal care, G. Oliver (Feinberg School of Medicine, Northwestern University, Chicago) for Prox1+/− mice. This work was supported by grants from the National Institutes of Health/National Institute on Aging (AG034113 and AG057496), the Cure Alzheimer’s Fund, Owens Family Foundation and the Thomas H. Lowder Family Foundation (awarded to J.K.), the Hobby Foundation (awarded to A.V. and S.T.A.) and American Cancer Society (IRG 81-001-26 awarded to J.M.M.). We thank all members of the Kipnis Laboratory and the BIG center for their valuable comments during numerous discussions of this work.

Reviewer information

Nature thanks D. Holtzman and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

  1. These authors contributed equally: Sandro Da Mesquita, Antoine Louveau

Affiliations

  1. Center for Brain Immunology and Glia (BIG), University of Virginia, Charlottesville, VA, USA

    • Sandro Da Mesquita
    • , Antoine Louveau
    • , Igor Smirnov
    • , Christian Contarino
    • , Daniel Raper
    • , Kenneth E. Viar
    • , Romie D. Powell
    • , Wendy Baker
    • , Nisha Dabhi
    • , Robin Bai
    • , Christopher C. Overall
    •  & Jonathan Kipnis
  2. Department of Neuroscience, University of Virginia, Charlottesville, VA, USA

    • Sandro Da Mesquita
    • , Antoine Louveau
    • , Igor Smirnov
    • , Christian Contarino
    • , Daniel Raper
    • , Kenneth E. Viar
    • , Romie D. Powell
    • , Wendy Baker
    • , Nisha Dabhi
    • , Robin Bai
    • , Christopher C. Overall
    •  & Jonathan Kipnis
  3. Virginia Image and Video Analysis Laboratory, Department of Electrical and Computer Engineering, University of Virginia, Charlottesville, VA, USA

    • Andrea Vaccari
    •  & Scott T. Acton
  4. Department of Biomedical Engineering, University of Virginia, Charlottesville, VA, USA

    • Andrea Vaccari
    • , R. Chase Cornelison
    • , Kathryn M. Kingsmore
    • , Rui Cao
    • , Song Hu
    • , Jennifer M. Munson
    •  & Scott T. Acton
  5. Department of Mathematics, University of Trento, Povo, Italy

    • Christian Contarino
  6. Center for Public Health Genomics, University of Virginia, Charlottesville, VA, USA

    • Suna Onengut-Gumuscu
    • , Emily Farber
    •  & Stephen S. Rich
  7. Department of Neurosurgery, University of Virginia Health System, Charlottesville, VA, USA

    • Daniel Raper
  8. Department of Biomedical Engineering and Mechanics, College of Engineering, Virginia Tech, Blacksburg, VA, USA

    • Jennifer M. Munson
  9. Department of Pathology, University of Virginia, Charlottesville, VA, USA

    • M. Beatriz Lopes

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Contributions

S.D.M. designed and performed the experiments, analysed and interpreted the data and wrote the manuscript; A.L. designed and performed the experiments and participated in manuscript preparation; A.V. developed the software (Lymph4D) for MRI data processing and analysis; I.S. performed surgeries and behavioural testing; C.C. performed T2-weighted MRI, magnetic resonance angiography (MRA) and magnetic resonance venography (MRV) acquisition and data analysis; R.C.C. made hydrogels for transcranial peptide delivery; K.M.K. carried out brain T1-weighted MRI acquisition; S.O.-G. and E.F. carried out RNA-seq experiments; D.R., K.E.V., R.D.P., W.B., N.D. and R.B. assisted with experimental procedures; R.C. and S.H. carried out photoacoustic imaging; S.S.R. and J.M.M. provided resources and were involved in experimental design; M.B.L. provided human tissue samples; C.C.O. helped with RNA-seq raw data analysis, data interpretation and manuscript writing; S.T.A. participated in the development of the software (Lymph4D), provided resources and intellectual contributions; J.K. designed the experiments, provided intellectual contributions, oversaw data analysis and interpretation and wrote the manuscript.

Competing interests

J.K. is an Advisor to PureTech Health/Ariya.

Corresponding authors

Correspondence to Sandro Da Mesquita or Jonathan Kipnis.

Extended data figures and tables

  1. Extended Data Fig. 1 Ablation of meningeal lymphatic vessels 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 OVA-A647 was injected i.c.m. into the CSF, and drainage of tracer into the 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 with photoconversion group than in control groups. Data are mean ± s.e.m., n = 6 per group, one-way ANOVA with Bonferroni’s post hoc test. a, b, Data are representative of two independent experiments; significant differences between vehicle with photoconversion and visudyne with photoconversion groups were observed in a total of five independent experiments. c, Seven days after meningeal lymphatic ablation, mice from the three groups were subjected 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 Gd at a dose of 0.3 mmol kg−1. d, Representative 3D reconstructions of intracranial veins and arteries of mice from each group. Scale bar, 5 mm. eh, No significant changes between groups were observed for venous vessel volume (e), superior sagittal sinus (SSS) diameter (f), arterial vessel volume (g) and basilar artery diameter (h). Data are mean ± s.e.m., n = 5 in vehicle with photoconversion and in visudyne with 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. Data are mean ± s.e.m., n = 5 in vehicle with photoconversion and in visudyne with 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 seven 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. Data are mean ± s.e.m., n = 5 per group, two-tailed Mann–Whitney U-test. Source data

  2. Extended Data Fig. 2 ICP measurements and assessment of CSF drainage and brain influx.

    a, ICP was measured in four different steps of 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 analysed steps of the i.c.m. injection method. Data are mean ± s.e.m., n = 7 per group; repeated-measures two-way ANOVA with Bonferroni’s post hoc test, *versus pre-injection, #versus during injection, &versus post-injection (syringe in); data were pooled from two 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. Data are 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 bars, 200 μm. e, Quantification of OVA-A647 area fraction (%) in the dCLNs showed significantly less amount of tracer in the visudyne with 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 with photoconversion and control groups. Scale bar, 5 mm and 1 mm (inset). g, Quantification of OVA-A647 area fraction (%) in brain sections showing a significant decrease in the visudyne with photoconversion group when compared to control groups. Data in e and g are mean ± s.e.m., n = 6 per group, one-way ANOVA with Bonferroni’s post hoc test; cg, Data are representative of two independent experiments. Source data

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

    a, Adult mice were subjected 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 and 2 mm (inset). 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 are mean ± s.e.m., n = 8 per group, two-tailed Mann–Whitney U-test; data in ad were pooled from two independent experiments and are representative of three 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 wild-type 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 wild-type 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 are mean ± s.e.m., n = 15 wild-type mice, n = 12 Prox1+/− mice, two-tailed Mann–Whitney U-test; data in ei were pooled from two independent experiments. j, Rate of brain paravascular influx of the contrast agent Gd, injected i.c.m. at 1, 10 or 25 mM (in saline), was assessed in adult mice (3 months old) by T1-weighted MRI. k, Representative MRI images obtained using Lymph4D software showing brain signal intensity for different concentrations of injected Gd. Scale bar, 3 mm. Experiments in j and k were 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 with photoconversion group, when compared to vehicle with photoconversion. n, o, Coronal sections of the brain of vehicle- or visudyne-treated mice (n = 4 per group) were aligned and stacked into 2D colour maps (concatenated from 16 MRI sequences) showing contrast of Gd signal intensity (n) and isotropic diffusion coefficient (o). 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 are mean ± s.e.m., n = 4 per group, repeated-measures two-way ANOVA with Bonferroni’s post hoc test (m); one-way ANOVA with Bonferroni’s post hoc test (p). lp, Data are representative of two 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 shows that there are 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. tv, No changes were observed in the area of AQP4+ astrocytic endfeet (t) and CD31+ blood vessels (u) or in the ratio between area of AQP4+ and of CD31+ (v). Data in r, tv are mean ± s.e.m., n = 7 per group, two-tailed Mann–Whitney U-test; data in qv were pooled from two independent experiments and representative of three independent experiments. Source data

  4. Extended Data Fig. 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−1 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 with 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 with photoconversion group compared to both control groups. Data are 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 amyloid-β42 (Aβ42)–HiLyte647 (0.05 μg ml−1 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 with 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 with photoconversion group compared to both control groups. Data are 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 LDL–BODIPY FL (0.1 mg ml−1 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 with 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 with photoconversion group compared to both control groups. Data are mean ± s.e.m., n = 6 per group, one-way ANOVA with Bonferroni’s post hoc test. Source data

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

    a, b, No differences in total distance travelled (a) and time in centre of the open field arena (b) were observed between vehicle with photoconversion, visudyne and visudyne with photoconversion groups. Data are mean ± s.e.m., n = 9 per group, one-way ANOVA with Bonferroni’s post hoc test. c, d, Performance of mice from the three groups was also identical both during the training session (c) and during the novel location task (d) of the NLR paradigm. Data are mean ± s.e.m., n = 9 per group, two-way ANOVA with Bonferroni’s post hoc test. e, f, Performance of mice in the CFC paradigm showed no differences between groups in the context test (e), however, there was a statistically significant difference in the cued test (f). Data are 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 MWM test, one week after sham surgery or surgical ligation of the lymphatic vessels afferent to the dCLNs. h, Ligated mice showed a significant increase in the latency to platform during acquisition compared to sham-operated mice. i, j, No significant differences between groups were observed in the percentage of time spent in the target quadrant in the probe trial (i) or in latency to platform in the reversal (j). Data are mean ± s.e.m., n = 8 sham-operated mice, n = 9 mice with ligation; repeated-measures two-way ANOVA with Bonferroni’s post hoc test (h, j), two-tailed Mann–Whitney U-test (i). k, Vehicle or visudyne injection experiments with photoconversion were performed twice within a two-week 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, Heat map showing relative expression levels of genes in vehicle with photoconversion and in visudyne with photoconversion samples. m, After meningeal lymphatic ablation (twice within a two-week interval) and MWM performance, total RNA was extracted from the hippocampus of mice from vehicle with photoconversion or visudyne with photoconversion groups and sequenced. RNA-seq principal component analysis demonstrating a differential clustering of samples from vehicle and visudyne groups. A total of 2,138 genes were downregulated and 1,599 genes were upregulated in the hippocampus after meningeal lymphatic ablation and MWM performance. n, Heat map showing relative expression levels of genes in vehicle with photoconversion and in visudyne with photoconversion samples. Colour scale bar values represent standardized rlog-transformed values across samples (l, n). o, Neurological disease, neuronal activity- and synaptic plasticity-related GO and KEGG terms are enriched upon visudyne treatment, as measured by the –log10(adjusted P value). p, GO and KEGG terms related to metabolite generation and processing, glycolysis and mitochondrial respiration and oxidative stress were enriched, as measured by the –log10(adjusted P value), upon visudyne treatment and MWM performance. q, r, Heat map showing relative expression levels of genes involved in two of the significantly altered GO terms related to excitatory synapse (q) and learning or memory (r). sv, Heat maps showing relative expression levels of genes involved in four of the significantly altered GO terms related to NADH dehydrogenase complex (s), generation of precursor metabolites and energy (t), cellular response to oxidative stress (u) and cellular response to nitrogen compound (v). Datasets in kv all consist of n = 5 per group; k, m, P values were corrected for multiple hypothesis testing with the Benjamini–Hochberg false-discovery rate procedure; in l, nv, functional enrichment of differential expressed genes was performed using gene sets from GO and KEGG and determined with Fisher’s exact test. Colour scale bar values in n, qv represent standardized rlog-transformed values across samples. Source data

  6. Extended Data Fig. 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 bars, 5 mm and 2 mm (inset). b, Quantification of OVA-A647 area fraction (%) in brain sections. Data are mean ± s.e.m., n = 6 mice in 3 months, n = 8 mice in 20-24 months, two-tailed Mann–Whitney U-test; representative of two 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-month-old female and male mice. Data in d, f are mean ± s.e.m., n = 9 per group at 2 months, n = 7 per group at 20–24 months for male and female mice, two-way ANOVA with Bonferroni’s post hoc test; data were pooled from two independent experiments. g, Representative dot and contour plots showing the gating strategy used to isolate meningeal LECs by FACS from the meninges of young–adult and old mice. n = 3 per group, pooled from two 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 GC ml−1, 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 bars, 5 mm and 200 μm (inset). 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-1CD31+ 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 lymphatic vessel diameter (l), but not in coverage by blood vessels (m). Data in l and m are 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; data in h-m are representative of two independent experiments. n, Representative images of blood flow (mm s−1) and arterial and venous blood oxygenation (percentage of sO2) readings obtained by photoacoustic imaging of brain/meningeal vasculature of old mice (20–22 months old) treated for one month with eGFP or mVEGF-C virus (both at 1013 GC ml−1). o, p, The different treatments did not affect blood flow (o) or blood oxygenation (p) in the brain/meninges of old mice. Data are mean ± s.e.m., n = 5 per group; two-tailed Mann–Whitney U-test (o), two-way ANOVA with Bonferroni’s post hoc test (p); data obtained 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 Gd (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 compared to eGFP. Data are mean ± s.e.m., n = 4 per group; repeated-measures two-way ANOVA with Bonferroni’s post hoc test; data were pooled from two independent experiments. Source data

  7. Extended Data Fig. 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−1) was applied on top of a thinned skull surface of adult mice (3 months old) and old mice (20–24 months old). Gels were reapplied 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 a significant increase in 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 bars, 200 μm. e, Quantification of OVA-A647 (red) area fraction (%) in the dCLNs showed increased drainage in old mice treated with VEGF-C 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 mean ± s.e.m., n = 12 mice treated with vehicle at 3 months, n = 11 mice treated with VEGF-C at 3 months, n = 8 mice treated with vehicle at 20–24 months and n = 9 mice treated with VEGF-C at 20–24 months; two-way ANOVA with Bonferroni’s post hoc test; data in ag were pooled from two independent experiments. h, Hydrogel alone (vehicle) or containing recombinant human VEGF-C156S (200 ng ml−1) was applied on top of a thinned skull surface of old mice. Gels were reapplied 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 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 are mean ± s.e.m., n = 7 mice per group; two-tailed Mann–Whitney U-test; data in hn were pooled from two 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. Data are mean ± s.e.m., n = 8 mice treated with eGFP and n = 9 mice treated with mVEGF-C; repeated-measures two-way ANOVA with Bonferroni’s post hoc test was used in the acquisition and reversal; two-tailed Mann–Whitney U-test was used in the probe trial; data were 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. Data are mean ± s.e.m., n = 12 mice treated with eGFP and n = 14 mice treated with mVEGF-C; repeated-measures two-way ANOVA with Bonferroni’s post hoc test was used in the acquisition and reversal, two-tailed Mann–Whitney U-test was used in the probe trial; data were pooled from two 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. Data are mean ± s.e.m., n = 25 mice treated with eGFP and n = 25 mice treated with mVEGF-C; repeated-measures two-way ANOVA with Bonferroni’s post hoc test was used in the acquisition and reversal; two-tailed Mann–Whitney U-test was used in the probe trial; data were pooled from three independent experiments. s, Treatment of young–adult mice with mVEGF-C did not affect the percentage of allocentric navigation strategies used in the MWM. t, u, The percentage of allocentric navigation strategies was significantly higher in middle-aged mice treated with mVEGF-C during the reversal (t) and in old mice treated with mVEGF-C during the acquisition and reversal (u) compared to age-matched eGFP-treated mice. Data in su are mean ± s.e.m., n = 8 mice treated with eGFP and n = 9 mice treated with mVEGF-C at 2 months (s), n = 12 mice treated with eGFP and n = 14 mice treated with mVEGF-C at 12–14 (t), n = 25 per group at 20–22 months (u); su, repeated-measures two-way ANOVA with Bonferroni’s post hoc test; data were obtained from a single experiment (s), pooled from two (t) and three (u) independent experiments. v, Insets of the hippocampal dentate gyrus (granular zone (GZ)), stained with DAPI (blue) and for Ki-67 (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, Ageing induced a significant decrease in Ki-67+ proliferating cells in the dentate gyrus. Expression of mVEGF-C in the meninges at the analysed ages did not affect the number of Ki-67+ cells in the dentate gyrus. Data are mean ± s.e.m., n = 5 per group, two-way ANOVA with Bonferroni’s post hoc test. Source data

  8. Extended Data Fig. 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−1) 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 percentage of time in the centre of the open field arena was not ameliorated by treatment of J20 mice with mVEGF-C. df, No statistically significant differences were observed in the acquisition (d), in the probe trial (e) or in the reversal (f) of the MWM test after one month of mVEGF-C treatment. Data in bf are mean ±  ± s.e.m., n = 11 mice treated with eGFP, n = 12 mice treated with mVEGF-C; two-tailed Mann–Whitney U-test (b, c, e), repeated-measures two-way ANOVA with Bonferroni’s post hoc test (d, f); data were from a single experiment. g, J20 mice were treated with eGFP or mVEGF-C and, one 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 amyloid-β in the CSF measured by ELISA remained unaltered after mVEGF-C treatment. k, Representative images of dorsal hippocampi of J20 mice treated with eGFP or mVEGF-C stained with DAPI (cyan) and for IBA1 (green) and amyloid-β (red). Scale bar, 500 μm. ln, No changes were observed in amyloid plaque size (l), number (m) or coverage (n) between the groups. Data in i, j, ln are mean ± s.e.m., n = 6 per group, two-tailed Mann–Whitney U-test; data in gn obtained from a single experiment. o, J20 mice (2–3 months old), 5xFAD mice (3–4 months old) and respective age-matched wild-type 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 wild-type and J20 mice 2 h after injection of OVA-A647 (red). Scale bar, 200 μm. q, Quantification of OVA-A647 area fraction (%) in the dCLNs shows equal levels of tracer in mice from both genotypes. Data are mean ± s.e.m., n = 5 per group, two-tailed Mann–Whitney U-test, representative of two independent experiments. r, Representative images of DAPI (blue) and LYVE-1 (green) staining in dCLNs of wild-type and 5xFAD mice 2 h after injection of OVA-A594 (red). Scale bars, 200 μ μm. s, Quantification of OVA-A594 area fraction (%) in the dCLNs shows equal levels of tracer in mice from both genotypes. Data are mean ± s.e.m., n = 11 per group, two-tailed Mann–Whitney U-test, data were pooled from two independent experiments. t, Representative images of DAPI (blue) and LYVE-1 (green) staining in meningeal whole-mounts of wild-type 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. Data are mean ± s.e.m., n = 7 per group, two-tailed Mann–Whitney U-test; data were pooled from two independent experiments. Source data

  9. Extended Data Fig. 9 Meningeal lymphatic ablation in transgenic mice with Alzheimer’s disease worsens amyloid pathology without affecting blood vessel function.

    a, Representative images of blood flow (mm s−1) and arterial and venous blood oxygenation (percentage of sO2) readings obtained by photoacoustic imaging of brain/meningeal vasculature of 5xFAD mice one week after vehicle with photoconversion, visudyne or visudyne with photoconversion. b, c, The different treatments did not affect blood flow (b) or blood oxygenation (c) in the brain/meninges of 5xFAD mice. Data are mean ± s.e.m., n = 5 per group, one-way ANOVA with Bonferroni’s post hoc test (b), two-way ANOVA with Bonferroni’s post hoc test (c), data were 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 with photoconversion group compared to the control groups. A significant increase in neutrophils was observed in visudyne group, but not in vehicle with photoconversion group compared to visudyne with photoconversion group. Data are mean ± s.e.m., n = 5 per group; two-way ANOVA with Holm–Sidak’s post hoc test, *versus vehicle with photoconversion, #versus visudyne; data obtained from a single experiment. f, J20 mice aged 4–5 months were subjected 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 three weeks, for a total of three months, to achieve prolonged meningeal lymphatic ablation. g, Staining with DAPI (blue) and for LYVE-1 (green) and amyloid-β (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 of age stained with DAPI (cyan) and for amyloid-β (red) showing degree of amyloid deposition after meningeal lymphatic ablation. Scale bar, 500 μm. ik, Quantification of amyloid plaque size (i), number (j) and coverage (k) in the dorsal hippocampus of J20 mice showed a statistically significant increase in coverage in the visudyne group compared to vehicle. Data in ik are mean ± s.e.m., n = 5 vehicle-treated mice, n = 6 visudyne-treated mice; two-tailed Mann–Whitney U-test; experiments in fk were performed once. l, m, Sections of human brain cortex, containing meningeal layers (leptomeninges) attached, from the brain of a control (l) (scale bars, 500 μm and 200 μm (inset)) and the brain of a patient with Alzheimer’s disease (m) (scale bars, 100 μm (left) and 500 μm (right)) were stained with DAPI (blue), for the astrocyte marker GFAP (green) and for amyloid-β (red). Data in l and m are from n = 8 controls and n = 9 patients with Alzheimer’s disease and are representative of two independent experiments. Source data

  10. Extended Data Table 1 Demographic data of patients with and without Alzheimer’s disease

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https://doi.org/10.1038/s41586-018-0368-8

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