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Restoration of cervical lymphatic vessel function in aging rescues cerebrospinal fluid drainage

Nature Aging volume 4, pages 1418–1431 (2024)Cite this article

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Abstract

Cervical lymphatic vessels (cLVs) have been shown to drain solutes and cerebrospinal fluid (CSF) from the brain. However, their hydrodynamical properties have never been evaluated in vivo. Here, we developed two-photon optical imaging with particle tracking in vivo of CSF tracers (2P-OPTIC) in superficial and deep cLVs of mice, characterizing their flow and showing that the major driver is intrinsic pumping by contraction of the lymphatic vessel wall. Moreover, contraction frequency and flow velocity were reduced in aged mice, which coincided with a reduction in smooth muscle actin expression. Slowed flow in aged mice was rescued using topical application of prostaglandin F2α, a prostanoid that increases smooth muscle contractility, which restored lymphatic function in aged mice and enhanced central nervous system clearance. We show that cLVs are important regulators of CSF drainage and that restoring their function is an effective therapy for improving clearance in aging.

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Fig. 1: Two-photon optical imaging with particle tracking in vivo of CSF tracers (2P-OPTIC).
Fig. 2: Intrinsic pump dysfunction in aging.
Fig. 3: Lymphatic efflux volumes are reduced in old age.
Fig. 4: Aging causes loss of α-SMA coverage in scLVs.
Fig. 5: PGF2α restores lymphatic flow and reverses the effects of aging on CSF drainage.
Fig. 6: PGF2α reverses the effects of aging on CSF drainage and increases brain clearance.

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Data availability

Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding authors upon reasonable request.

Code availability

All relevant code is available in the public ___domain repository at https://gitlab-public.circ.rochester.edu/araghuna/bulk-flow-is-not-an-artifact_raghunandan_et_al_2021.git/.

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Acknowledgements

We thank D. Xue for assistance with schematics. The study was funded by Lundbeck Foundation R386–2021–165 (to M.N.), The Novo Nordisk Foundation NNF20OC0066419 (to M.N.), NIH grants R01AT011439 (to M.N.), U19NS128613 (to M.N. and D.H.K.), R01NS100366 (to M.N.), RF1AG057575 (to M.N.), R01AT012312 (to D.H.K. and M.N.), Human Frontier Science Program RGP0036 (to M.N.), The Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (to M.N.), The Simons Foundation 811237 (to M.N.), the EU Joint Programme – Neurodegenerative Disease Research (JPND; to M.N.) and the US Army Research Office MURI W911NF1910280 (to M.N. and D.H.K.). H.M. was supported by a Cerebrovascular Research Grant from the Aneurysm and AVM Foundation. The views and conclusions contained in this article are solely those of the authors and should not be interpreted as representing the official policies, either expressed or implied, of the NIH, the Army Research Office or the US Government.

Author information

Author notes

  1. These authors contributed equally: Ting Du, Aditya Raghunandan, Humberto Mestre.

Authors and Affiliations

  1. Center for Translational Neuromedicine, Division of Glial Disease and Therapeutics, University of Rochester, Rochester, NY, USA

    Ting Du, Humberto Mestre, Virginia Plá, Guojun Liu, Antonio Ladrón-de-Guevara, Evan Newbold, Paul Tobin, Daniel Gahn-Martinez, Saurav Pattanayak, Qinwen Huang, Weiguo Peng & Maiken Nedergaard

  2. Department of Mechanical Engineering, University of Rochester, Rochester, NY, USA

    Aditya Raghunandan & Douglas H. Kelley

  3. Department of Mechanical Engineering, University of Michigan-Dearborn, Dearborn, MI, USA

    Aditya Raghunandan

  4. Department of Neurology, University of Pennsylvania, Philadelphia, PA, USA

    Humberto Mestre

  5. Center for Translational Neuromedicine, University of Copenhagen, Copenhagen, Denmark

    Virginia Plá & Maiken Nedergaard

  6. Department of Biomedical Engineering, University of Rochester, Rochester, NY, USA

    Antonio Ladrón-de-Guevara

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Contributions

T.D., H.M., M.N., A.R. and D.H.K. designed the experiments. T.D., H.M., A.R., A.L.G., E.N., P.T., D.G.-M., G.L., S.P., Q.H. and W.P. performed all in vivo experiments. A.R performed two-photon image analysis and particle tracking velocimetry measurements based on techniques developed by D.H.K. A.R., V.P. and H.M. performed the immunohistochemical staining and analysis. T.D., A.R. and H.M. analyzed the data. T.D., A.R., H.M., D.H.K. and M.N. organized the data and wrote/edited the manuscript.

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Correspondence to Maiken Nedergaard or Douglas H. Kelley.

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Extended data

Extended Data Fig. 1 Cervical Lymphatic efflux is not modulated by cardiac and respiration forces.

(a–d) Representative vessel contractions (blue curves) and downstream velocity (orange) phase-averaged waveforms compared to changes in measured cardiac (red curves) and respiration signals (green curves). (e–h) Linear regression of intrinsic rate or ejection rate with 95% confidence intervals did not reveal any correlation with heart rate or respiration rate.

Source data

Extended Data Fig. 2 Mean pixel intensity of dextran dye is highly correlated with lymphatic vessel (LV) diameter and is a poor predictor of LV transport.

(a) cLVs were labeled by the dextran (red) injected into the cheek, while microsphere particles injected into the CM appear green; (inset) the white arrows show the velocity of each particle. Scale bar: 50 μm. (b) Normalized LV diameter changes (as a percent of baseline), the mean fluorescence intensity changes (as a percent of baseline), and normalized flow speed in cervical lymph vessel before and after PGF2α. Normalized flow speed was quantified by calculating the magnitude of the mean downstream velocity in each movie frame, then dividing those values by the corresponding value for the first frame (8.48 μm/s). Treatment with PGF2α caused a 22.4% decrease in LV diameter and 41.7% decrease in mean pixel intensity, despite having a 146% increase in flow speeds. (c) Scatterplot depicting the correlation between computed z-scores of mean pixel intensity and, maximum flow speed and LV diameter. Linear regression between mean pixel intensity and max flow speed show that mean pixel intensity explains around 22% of the variance in flow speed and has a negative slope, counterintuitively suggesting that higher dye concentration correlates with lower flow speeds (P = 0.0044). Contrastingly, mean pixel intensity explains 68% of the variance in LV diameter and the relationship has a positive slope (β = 0.81), indicating that mean pixel intensity is highly correlated with LV diameter (P < 0.0001). Due to the strong correlation between mean pixel intensity and LV diameter, our dataset also suggested that wider LV diameters resulted in slower flow speeds (β = −0.69, R2 = 0.33, P = 0.0003).

Source data

Extended Data Fig. 3 Lymphatic smooth muscle actin loss impairs phasic contractions in old age.

(a) Representative images of cervical lymph vessels after labeling for nuclei (DAPI), collagen IV (Col IV) and smooth muscle actin. Orthogonal sections show 3D distribution. Scale bar: 100 μm. (b-c) Collagen IV and SMA fluorescence vary transverse to the vessel direction. Vessel width was normalized to allow direct comparison between lymph vessels. Thick lines represent mean intensity with SEM shown as shadowed area, thin lines being individual animals. If more than one lymph vessel was collected per individual, average fluorescence was calculated. Color dots are individual animals. Mann-Whitney (Interanimal, Col IV: Old vs Young, p = 0.3411, U = 6; SMA: Old vs Young, p = 0.0328, U = 1). Two-sided unpaired t-test was performed, Bar graphs show area under the curve calculated for both proteins, as mean ± SEM; n = 4–5 mice/group. (d) Representative phase-averaged expansion and contraction of lymph vessels for different age groups. Phase-averaging was performed over at least 10 cycles. (e) Representative normalized vessel wall velocity for different age groups, calculated by differentiating the curves in d. By definition, positive speeds signify expansion, and negative speeds signify contraction. Two-way ANOVA with Sidak’s multiple comparisons test was performed (d-e). Data are presented as mean ± SEM; n = 4-5 mice/group.

Source data

Extended Data Fig. 4 Cardiac and respiration rates are unaffected by PGF2α.

(a) Heart rate and (b) Respiration rates before and after PGF2α for young and old mice groups. Two-sided paired t-test was performed. Data are presented as mean ± SEM; n = 5 mice/group.

Source data

Extended Data Fig. 5 PGF2α sustains lymphatic drainage into cervical lymph nodes.

(a) CSF clearance was evaluated via an intracisternal injection of ovalbumin-conjugated to Alexa 647 (OVA- Alexa 647) with or without PGF2α in young and old mice. After 90 min sLN and dLN were dissected and taken imaging under microscope. (b) Representative images of the ex vivo lymph nodes with or without PGF2α administration in young and old mice group. Scale bar = 2 mm. Fluorescent mean pixel intensity (MPI) normalized by the area of lymph nodes for sLN (c) and dLN (d). one-way ANOVA with post hoc Tukey’s test. Data are presented as mean ± SEM; n = 4 mice/group.

Source data

Supplementary information

Supplementary Information

Descriptions of Supplementary Videos 1–7.

Reporting Summary

Supplementary Video 1

Simultaneous recording of lymph flow speed with ECG and respiration in scLVs. Two-photon microscopy imaging (left) and synchronized physiological measurements (right). Trajectories from particle tracking velocimetry are indicated by colored curves tracking the microspheres. Scale bar, 50 μm.

Supplementary Video 2

Simultaneous recording of lymph flow speed with ECG and respiration in deep cervical lymph vessels. Two-photon microscopy imaging (left) and synchronized physiological measurements (right). Trajectories from particle tracking velocimetry are indicated by colored curves tracking the microspheres. Scale bar, 50 μm.

Supplementary Video 3

Aging-induced reduction in cervical lymphatic function. cLVs in 2-month-old, 18-month-old and 22-month-old mice visualized with two-photon microscopy. Colored lines depict particle trajectories derived from particle tracking. Simultaneous changes in the vessel diameter and real-time instantaneous velocity of lymph in the downstream (efflux) direction are shown. Slower vessel contraction rates and efflux speeds are observed in aging. Scale bar, 50 μm.

Supplementary Video 4

Valve function in young and aged mice. Two-photon microscopy was used to capture healthy valve function in 2-month-old mice and dysfunctional valves in 22-month-old mice. Valve leaflets open and close to regulate flow in 2-month-old mice but are biased to remain open in 22-month-old mice, failing to regulate flow. Scale bar, 25 μm.

Supplementary Video 5

PGF2α promotes scLV function in 2-month-old mice. Two-photon microscopy of scLVs in 2-month-old mice before (left) and after (right) the administration of PGF2α. Colored lines depict trajectories from particle tracking. Simultaneous measurements of physiological signals before and after PGF indicate changes to vessel diameter and increased contraction frequency after administration of PGF2α. No change in cardiac or respiration rates were observed. Scale bar, 50 μm.

Supplementary Video 6

PGF2α rescues scLV function in 22-month-old mice. Two-photon microscopy of scLVs in 22-month-old mice before (left) and after (right) the administration of PGF2α. Colored lines depict trajectories from particle tracking. Simultaneous measurements of physiological signals before and after PGF2α show changes to vessel diameter and increased contraction frequency after the administration of PGF2α. No change in cardiac or respiration rates were observed. Scale bar, 50 μm.

Supplementary Video 7

Fluorescence macroscope imaging of CSF efflux in scLVs in mice after PGF2α treatment. Fluorescence macroscope imaging of CSF efflux in scLVs in 2-month-old or 22-month-old mice after intracisternal injection of a fluorescent CSF tracer for 60 min with or without PGF2α. Scale bar, 2 mm.

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Du, T., Raghunandan, A., Mestre, H. et al. Restoration of cervical lymphatic vessel function in aging rescues cerebrospinal fluid drainage. Nat Aging 4, 1418–1431 (2024). https://doi.org/10.1038/s43587-024-00691-3

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