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Potentiating glymphatic drainage minimizes post-traumatic cerebral oedema

Nature volume 623, pages 992–1000 (2023)Cite this article

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Abstract

Cerebral oedema is associated with morbidity and mortality after traumatic brain injury (TBI)1. Noradrenaline levels are increased after TBI2,3,4, and the amplitude of the increase in noradrenaline predicts both the extent of injury5 and the likelihood of mortality6. Glymphatic impairment is both a feature of and a contributor to brain injury7,8, but its relationship with the injury-associated surge in noradrenaline is unclear. Here we report that acute post-traumatic oedema results from a suppression of glymphatic and lymphatic fluid flow that occurs in response to excessive systemic release of noradrenaline. This post-TBI adrenergic storm was associated with reduced contractility of cervical lymphatic vessels, consistent with diminished return of glymphatic and lymphatic fluid to the systemic circulation. Accordingly, pan-adrenergic receptor inhibition normalized central venous pressure and partly restored glymphatic and cervical lymphatic flow in a mouse model of TBI, and these actions led to substantially reduced brain oedema and improved functional outcomes. Furthermore, post-traumatic inhibition of adrenergic signalling boosted lymphatic export of cellular debris from the traumatic lesion, substantially reducing secondary inflammation and accumulation of phosphorylated tau. These observations suggest that targeting the noradrenergic control of central glymphatic flow may offer a therapeutic approach for treating acute TBI.

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Fig. 1: Pan-adrenergic receptor inhibition eliminates oedema and improves functional outcomes after TBI.
Fig. 2: Post-TBI suppression of glymphatic efflux is counteracted by pan-adrenergic inhibition.
Fig. 3: Fluid transport by cervical lymphatic vessels is reduced by TBI and restored after pan-adrenergic receptor inhibition.
Fig. 4: Noradrenergic storm after TBI disrupts contraction wave entrainment but is prevented by PPA treatment.
Fig. 5: Efflux of cells and cellular debris through CLVs in the event of TBI is neuronal in origin.
Fig. 6: Brain fluid export is compromised by TBI and counteracted by pan-adrenergic inhibition.

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Data availability

All data are available in the main text figures and/or extended data figures. Source data are provided with this paper.

Code availability

The particle tracking and vessel diameter measurement codes used in this study are publicly available at Zenodo (https://doi.org/10.5281/zenodo.8165799). Data were analysed using GraphPad Prism Software (v.7). The LabVIEW program used for pressure and diameter data collection of isolated lymphatic vessels is publicly available at Zenodo (https://doi.org/10.5281/zenodo.8286107). The LabVIEW program used for pressure and diameter data collection of isolated lymphatic vessels is publicly available at Zenodo (https://doi.org/10.5281/zenodo.8286119). The Python program used for spatiotemporal analysis of contraction waves in isolated lymphatic vessels is publicly available at Zenodo (https://doi.org/10.5281/zenodo.8259778).

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Acknowledgements

We thank D. Xue for graphical support. This project has received funding from the Dr Miriam and Sheldon G. Adelson Medical Research Foundation, the NIH/NINDS/NCCIH/NHLBI (R01AT012312, R01AT011439 and U19 NS128613, R01HL122578), the Simons Foundation, Career Award at the Scientific Interface from Burroughs Wellcome Fund, the JPND, the Novo Nordisk Foundation and Lundbeck Foundation, as well as by US Army Research Office grants (MURI W911NF1910280, PRARP; W81XWH-16-1-0555 and W81XWH-22-1-0676). Opinions, interpretations, conclusions and recommendations are those of the authors and are not necessarily endorsed by the Department of Defense.

Author information

Authors and Affiliations

  1. Center for Translational Neuromedicine, University of Rochester, Rochester, NY, USA

    Rashad Hussain, Wei Wang, Arokoruba Cheetham-West, Wei Song, Weiguo Peng, Qian Sun, Sisi Peng, Virginia Plá, Hajime Hirase, Steven A. Goldman & Maiken Nedergaard

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

    Jeffrey Tithof & Douglas H. Kelley

  3. Department of Mechanical Engineering, University of Minnesota, Minneapolis, MN, USA

    Jeffrey Tithof & Daehyun Kim

  4. Center for Translational Neuromedicine, University of Copenhagen Faculty of Health and Medical Sciences, Copenhagen, Denmark

    Weiguo Peng, Björn Sigurdsson, Hajime Hirase, Pia Weikop, Steven A. Goldman & Maiken Nedergaard

  5. Department of Pharmacology, School of Medicine, Tulane University, New Orleans, LA, USA

    Jorge A. Castorena-Gonzalez

  6. Department of Medical Pharmacology and Physiology, School of Medicine, University of Missouri, Columbia, MO, USA

    Michael J. Davis

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  1. Rashad Hussain
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Contributions

R.H. and M.N. designed the study. R.H., W.W. and A.C.-W. performed the oedema and behaviour studies. W.W., W.P. and B.S. assisted with glymphatic assessment, radiotracer studies and CSF production estimations. R.H. analysed the data and designed the figures. V.P. assisted with Evans blue clearance studies and W.W. performed fluorescence microscopy. R.H. recorded two-photon microscopy data. D.H.K. and J.T. developed MATLAB code. J.T. performed particle-tracking velocimetry, generated tracking maps and videos, and helped in data analysis. R.H., M.N. and M.J.D. designed in vitro cervical lymphatic vessel experiments; M.J.D. and J.A.C.-G. performed those experiments, generated the videos, generated fast Fourier transform maps and analysed the data. R.H. and W.S. performed in vivo cervical lymphatic vessels imaging and recorded vital measurements, and D.K. and J.T. helped in the analysis. R.H. performed the GCaMP7 mouse experiments. J.T. analysed the particle debris data. Q.S., S.P. and P.W. helped in microdialysis experiments, noradrenergic estimation and analysis. R.H. performed the western blotting and immunohistochemistry. W.W. assisted with IVIS infrared imaging. H.H. helped in conceptualizing the pan-noradrenergic inhibition treatment strategies. R.H. and M.N. wrote the manuscript. S.A.G. assisted in data interpretation, critical evaluation of findings, writing and editing.

Corresponding authors

Correspondence to Rashad Hussain or Maiken Nedergaard.

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Competing interests

S.A.G. is a part-time employee and stock holder of Sana Biotechnology, a cell therapy company; he is also a co-founder of CNS2, which has licensed relevant patents. None of the work described in this paper has been funded by those companies. The other authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Effect of individual components of PPA is less efficient in reducing cerebral oedema after TBI. Locomotor and anxiety-like behaviour of post-traumatic brain injury mice is relieved by PPA treatment.

a-b, The severity of cerebral oedema in the mouse brain was estimated 3 h post-TBI with or without treatment of prazosin (Prz), propranolol (Prpl), and atipamezole (Ati). Experimental groups were compared by one-way ANOVA (n = 35 mice, F4,30 = 3.73, p = 0.014,) followed by Dunnett’s multiple comparisons test; Sham-Control vs TBI-saline (n = 7 mice each, p = 0.025), TBI-saline vs Prz (n = 6 mice, p = 0.044), Prpl (n = 7 mice, p = 0.72), and Ati (n = 8 mice, p = 0.99). c, Cerebral oedema measurement in mice 24 h post-TBI with or without PPA treatment at 23 h. Experimental groups compared by one-way ANOVA (n = 23 mice, F2,20 = 9.387, p = 0.001) followed by Dunnett’s multiple comparisons test; Sham-Control (n = 7 mice) vs TBI-saline (n = 7 mice), p = 0.0007, TBI-saline vs TBI + PPA (n = 9 mice, p = 0.036). d, Locomotion, anxiety-like behaviours, and exploration abilities at two and 12 weeks post-TBI, with or without PPA treatment. Data shown as box and whisker plot with median, and min-max values, the dots are biological replicates/mice. e, Two-week evaluation (n = 33 mice, 11 mice/group, group means were compared by one-way ANOVA followed by Tukey’s multiple comparison test); average speed (F2,30 = 12.16, p = 0.0001; Control vs TBI-saline, p = 0.014, TBI-saline vs TBI-PPA, p = 0.164), total distance travelled (F2,30 = 5.291, p = 0.0108; Control vs TBI-saline, p = 0.021, TBI-saline vs TBI-PPA, p = 0.025), number of freeze episodes (F2,30 = 3.482, p = 0.0437; Control vs TBI-saline, p = 0.034; TBI-saline vs TBI-PPA, p = 0.352), and freeze time per episode (F2,30 = 4.944, p = 0.0139; Control vs TBI-saline, p = 0.044; TBI-saline vs TBI-PPA, p = 0.019). f, Twelve-week evaluation; n = 31 mice; Control (n = 11 mice), TBI-saline (n = 10 mice), TBI + PPA (n = 10 mice), group means were compared by one-way ANOVA followed by Tukey’s multiple comparison test where applicable. Average speed (F2,28 = 0.2695, p = 0.7657), total distance travelled (F2,28 = 0.4609, p = 0.6354), number of freeze episodes (F2,28 = 11.47, p = 0.0002; Control vs TBI-saline, p = 0.0002; TBI-saline vs TBI-PPA, p = 0.0204), and freeze time per episode (F2,28 = 14.2, p < 0.0001; Control vs TBI-saline, p = 0.0001; TBI-saline vs TBI-PPA, p = 0.0004). Bar graphs show mean and SEM (b, c), box and whisker plots show median and min-max values (e, f), and the dots are biological replicates/mice.

Source Data

Extended Data Fig. 2 Transcranial live imaging of tracer movement is as reliable as ex vivo and in vitro slice imaging.

a, Representative dorsal and ventral views of brain imaged by ex vivo conventional fluorescent microscopy in control, TBI+saline, and TBI + PPA groups performed at (top) day 0 and (bottom) six months post-TBI (n = 4 biological replicates/mice at each time point). b, Regression analysis of BSA-647 fluorescence intensity for quantifying association of (top) transcranial in vivo vs ex vivo dorsal and (bottom) transcranial in vivo vs in vitro slices (R2 = 0.802 and 0.821, respectively). c, Representative images from confocal microscopy showing vascular ultrastructure, labelled with lectin (red) and BSA-647 tracer (cyan), colocalized/distributed along the blood vessels in non-injury control, TBI-saline, and TBI + PPA groups. d, Experimental scheme. e, Representative images (n = 3 biological replicates/mice). f, Quantification of transcranial time-lapse imaging of Alexa flour 647 conjugated BSA tracer signals in vivo (n = 16 mice; Control (n = 5), TBI-saline (n = 4), TBI + PPA (n = 7), 60 time points, linear regression, F2,177 = 1144, p < 0.0001). g, Mean pixel intensity of BSA-647 in different regions of the brain (n = 5 mice/group, multiple slices averaged per mouse, group means compared using one-way ANOVA followed by Tukey’s multiple comparison test where applicable): dorsal cortex (F2,12 = 17.59, p = 0.0003; Control vs TBI-saline, P = 0.0002; TBI-saline vs TBI + PPA, p = 0.0098), striatum (F2,12 = 1.621, p = 0.238), hippocampus (F2,12 = 4.413, p = 0.0366; Control vs TBI-saline, p = 0.0292; TBI-saline vs TBI + PPA, p = 0.332), lateral cortex (F2,12 = 8.807, p = 0.0044; Control vs TBI-saline, p = 0.0038; TBI-saline vs TBI + PPA, p = 0.0423), corpus callosum (F2,12 = 1.737, p = 0.217), and hypothalamus (F2,12 = 11.33, p = 0.0017; Control vs TBI-saline, p = 0.0018; TBI-saline vs TBI + PPA, p = 0.587). Data shown as scatter plot with trendline (b), line graph of group means with SEM (f), bars show mean and SEM (g), and the dots are biological replicates/mice. Scale: (a, c) 5 mm, (e) 100 µm.

Source Data

Extended Data Fig. 3 Post-TBI noradrenergic receptor inhibition downregulates IL-4, IL-6, TNFα, and CXCL10 levels within the brain.

Brain samples collected 24 h post-TBI with or without PPA treatment were analysed for cytokine/chemokine levels both in the ipsilateral and contralateral hemispheres. Data is shown as percentage increase in the chemokine/cytokine levels relative to the contralateral hemisphere. Experimental groups were compared by one-way ANOVA followed by Dunnett’s multiple comparisons test. b, G-CSF (n = 27 mice; Control (n = 8), TBI-saline (n = 9), TBI + PPA (n = 10), F2,24 = 2.583, p = 0.0964). c, GM-CSF (n = 23 mice; Control (n = 7), TBI-saline (n = 7), TBI + PPA (n = 9), F2,20 = 1.021, p = 0.3781). d, IL-1β (n = 19 mice; Control (n = 7), TBI-saline (n = 7), TBI + PPA (n = 5), F2,16 = 4.427, p = 0.0295; Control vs TBI-saline, p = 0.038, TBI-saline vs TBI + PPA, p = 0.083). e, IL-4 (n = 21 mice; 7 mice/group, F2,18 = 7.615, p = 0.0040; Control vs TBI-saline, p = 0.023, TBI-saline vs TBI + PPA, p = 0.0045). f, IL-6 (n = 24 mice; 8 mice/group, F2,21 = 4.653, p = 0.0212; Control vs TBI-saline, p = 0.032, TBI-saline vs TBI + PPA, p = 0.049). g, IL-12p70 (n = 24 mice; Control (n = 9), TBI-saline (n = 8), TBI + PPA (n = 7), F2,21 = 4.739, p = 0.020; Control vs TBI-saline, p = 0.023, TBI-saline vs TBI + PPA, p = 0.0695). h, CXCL10 (n = 28 mice; Control (n = 11), TBI-saline (n = 8), TBI + PPA (n = 9), F2,25 = 6.384, p = 0.0058; Control vs TBI-saline, p = 0.013, TBI-saline vs TBI + PPA, p = 0.010). i, KC (n = 30 mice; Control (n = 11), TBI-saline (n = 9), TBI + PPA (n = 10), F2,27 = 3.239, p = 0.0549; Control vs TBI-saline, p = 0.0495, TBI-saline vs TBI + PPA, p = 0.019). j, LIF (n = 20 mice; Control (n = 5), TBI-saline (n = 7), TBI + PPA (n = 8), F2,17 = 1.55, p = 0.241). k, MCP1 (n = 22 mice; Control (n = 8), TBI-saline (n = 6), TBI + PPA (n = 8), F2,19 = 6.328, p = 0.0078; Control vs TBI-saline, p = 0.006, TBI-saline vs TBI + PPA, p = 0.098). l, MIG (n = 27 mice; 9 mice/group, F2,24 = 1.128, p = 0.341). m, MIP2 (n = 30 mice; Control (n = 10), TBI-saline (n = 9), TBI + PPA (n = 11), F2,27 = 3.735, p = 0.0370; Control vs TBI-saline, p = 0.029, TBI-saline vs TBI + PPA, p = 0.39). n, RANTES (n = 28 mice; Control (n = 9), TBI-saline (n = 9), TBI + PPA (n = 10), F2,25 = 1.056, p = 0.3629). o, VEGF (n = 26 mice; Control (n = 9), TBI-saline (n = 8), TBI + PPA (n = 9), F2,23 = 7.53, p = 0.0031; Control vs TBI-saline, p = 0.010, TBI-saline vs TBI + PPA, p = 0.0048). p, INF-γ (n = 25 mice; Control (n = 9), TBI-saline (n = 7), TBI + PPA (n = 9), F2,22 = 1.077, p = 0.358). q, TNFα (n = 24 mice; Control (n = 8), TBI-saline (n = 7), TBI + PPA (n = 9), F2,21 = 3.908, p = 0.0361; Control vs TBI-saline, p = 0.128, TBI-saline vs TBI + PPA, p = 0.033). Data shown as bar charts of mean and SEM, and the dots are biological replicates/mice.

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Extended Data Fig. 4 Post-TBI noradrenergic receptor inhibition reduces astrocytic hypertrophy, microglial invasion, and subsequent hyper-phosphorylation of tau.

a, Schematic showing induction of injury followed by a two-week experimental window. b, Coronal sections of mouse brain showing the lesion centre were immunostained for GFAP (red) and DAPI (blue); the site of injury/damaged somatosensory cortex, enlarged ventricles both on ipsilateral and contralateral sides, and the white matter tract corpus callosum are indicated by yellow arrows, white # symbols, and a white * sign, respectively, in non-injury control, TBI, and TBI + PPA slices. c, Brain sections (bregma; AP −0.8 to 2 mm) were immunostained for microglia (Iba-1, red) and pan-nuclear marker (DAPI, blue); the bottom right corner shows the region of interest. d, Quantification of immunofluorescence of GFAP (n = 18 mice, 6 mice/group, multiple slices averaged per mouse, one-way ANOVA, F2,15 = 11.6, p = 0.0009, Tukey’s multiple comparison test; Control vs TBI-saline, p = 0.0007, TBI-saline vs TBI + PPA, p = 0.033), number of microglia (n = 12 mice, 4 mice/group, multiple slices averaged per mouse, one-way ANOVA, F2,9 = 2.879, p = 0.108) and Iba-1 immunostaining (n = 16 mice; Control (n = 4), TBI-saline (n = 6), TBI + PPA (n = 6), multiple slices averaged per mouse, one-way ANOVA, F2,13 = 14.89, p = 0.0004, Tukey’s multiple comparison test; Control vs TBI-saline, p = 0.0022; TBI-saline vs TBI + PPA, p = 0.0008). e, (Top) Schematic showing the experimental time window of western blot and immunohistochemistry experiments for detection of hyper-phosphorylation of tau protein. (Bottom) Western blot analysis was performed in whole brain homogenates for tau targets: pTauSer404, pTauThr205, and pTauSer262 (n = 3 biological replicates/mice). f, Representative images showing hyper-phosphorylation of tau at site Ser262, Tau5, and DAPI in separate sets of mice at six months after TBI, with or without NA pan-adrenergic receptor blockade. g-j, Quantification of immunostaining of pTau in the cortex, striatum, and hippocampus for targets. g, pTauSer262 (n = 13 mice; Control (n = 3), TBI-saline (n = 5), TBI + PPA (n = 5), multiple slices averaged per mouse, one-way ANOVA followed by Tukey’s multiple comparison test where applicable), Cortex: F2,10 = 5.122, p = 0.0294; Control vs TBI-saline, p = 0.029, TBI-saline vs TBI + PPA, p = 0.133, Striatum: F2,10 = 13.7, p = 0.0014; Control vs TBI-saline, p = 0.0010, TBI-saline vs TBI + PPA, p = 0.085, Hippocampus: F2,10 = 15.92, p = 0.0008; Control vs TBI-saline, p = 0.0007, TBI-saline vs TBI + PPA, p = 0.011. h, pTauT212 (n = 15 mice, 5 mice per group, multiple slices averaged per mouse, one-way ANOVA followed by Tukey’s multiple comparison test where applicable), Cortex: F2,12 = 75.42, p < 0.0001; Control vs TBI-saline, p < 0.0001, TBI-saline vs TBI + PPA, p = 0.001, Striatum: F2,12 = 22.89, p < 0.0001; Control vs TBI-saline, p < 0.0001, TBI-saline vs TBI + PPA, p = 0.079, Hippocampus: n = 11 mice, Control (n = 5), TBI-saline (n = 3), TBI + PPA (n = 3), multiple slices averaged per mouse, F2,8 = 8.088, p = 0.0120; Control vs TBI-saline, p = 0.0099, TBI-saline vs TBI + PPA, p = 0.215. i, pTauThr205 (n = 15 mice, 5 mice per group, multiple slices averaged per mouse, one-way ANOVA followed by Tukey’s multiple comparison test where applicable), Cortex: F2,12 = 62.05, p < 0.0001; Control vs TBI-saline, p < 0.0001, TBI-saline vs TBI + PPA, p = 0.0034, Striatum: F2,12 = 14.37, p = 0.0007; Control vs TBI-saline, p = 0.0005, TBI-saline vs TBI + PPA, p = 0.076, Hippocampus: n = 11 mice, Control (n = 5), TBI-saline (n = 3), TBI + PPA (n = 3), multiple slices averaged per mouse, F2,8 = 12.94, p = 0.0031; Control vs TBI-saline, p = 0.0026, TBI-saline vs TBI + PPA, p = 0.022. j, Tau5 (n = 11 mice, Control (n = 3), TBI-saline (n = 5), TBI + PPA (n = 3), multiple slices averaged per mouse, one-way ANOVA followed by Tukey’s multiple comparison test where applicable), Cortex: F2,8 = 6.605, p = 0.020; Control vs TBI-saline, p = 0.019, TBI-saline vs TBI + PPA, p = 0.915, Striatum: F2,8 = 3.39, p = 0.086; Control vs TBI-saline, p = 0.073, TBI-saline vs TBI + PPA, p = 0.571, Hippocampus: F2,8 = 5.412, p = 0.0326; Control vs TBI-saline, p = 0.029, TBI-saline vs TBI + PPA, p = 0.24. Data shown as bar charts of mean and SEM, bars show mean and SEM, and the dots are biological replicates/mice. Scale: (b) 250 µm, (c) 50 µm, (f) 25 µm.

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Extended Data Fig. 5 Western blots of programmed cell death pathway proteins Caspase 7, 3, and 9 at two weeks post-injury, with or without PPA treatment. Despite the anticipated disruption of BBB, TBI does not increase the influx of mannitol, a BBB impermeable tracer.

a-b, Brain tissue was collected from control and TBI mice with or without PPA, homogenized in RIPA buffer, and analysed for the levels of programmed cell death markers Caspase 7, 3, and 9 (n = 2 biological replicates/mice). a, Schematics showing the tissue collection from ipsilateral and contralateral hemispheres, which was homogenized, followed by protein separation by gel electrophoresis and PVC membrane transfers. b, Caspase enzymes (7, 3, 9) were detected on PVC membrane by specific primary antibodies followed by LiCOR secondary antibody incubation and imaging using Odyssey Imager. c, Schematic illustrating the vascular compartment of the brain and intravenous injection (10 µL) of radiolabelled mannitol (14C). The radiotracer 14C labelled Mannitol was injected through an intra-arterial catheter immediately after TBI, and brain samples were collected 30 min later, weighed, and dissolved overnight. Their radioactivity was then measured using a liquid scintillation counter. d, Radioactivity data (n = 21 mice; Control (n = 6), TBI-saline (n = 6), TBI + PPA (n = 9)) is shown as percentage of the total injected dose in the vasculature. Group means were compared by one-way ANOVA (F2,18 = 8.13, p = 0.003) followed by Tukey’s multiple comparison test; Control vs TBI-saline, p = 0.025; TBI-saline vs TBI + PPA, p = 0.0027; Control vs TBI + PPA, p = 0.73. Data shown as box and whisker plot with the lower and upper quartile (box limits), median and min-max values, and the dots represent biological replicates/mice.

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Extended Data Fig. 6 Post-TBI noradrenergic inhibition restores interstitial fluid flow, tracer dispersion and efflux.

a, Schematic showing fluorescent tracer Direct Blue 53 (DB53) injected into the striatum in pre-cannulated mice, with or without TBI. DB53 was detected in vivo within the live brain 3 h post-TBI by IVIS Spectrum IR imaging. b, Averaged images showing the distribution of DB53 in the brain. c, IR quantification shown as radiant efficiency is compared using one-way ANOVA; n = 19 mice; Control (n = 6), TBI-saline (n = 6), TBI + PPA (n = 9), F2,16 = 23.5, p < 0.0.0001, Tukey’s multiple comparison test; Control vs TBI-saline, p < 0.0001, TBI-saline vs TBI + PPA, p = 0.005. d, Schematic diagram illustrating the methods used to assess the efflux of tracer from the brain into the circulatory system, thus quantifying fluid transport out of the brain and oedema clearance. DB53 was injected into the left striatum, and its appearance within a femoral vein was recorded using time-lapse IVIS spectrum IR imaging. e, Representative images showing the distribution of DB53 (640-690 nm) in the femoral vein: Control (top row), TBI-saline (middle row), and TBI + PPA groups (bottom row). f, DB53 IR signals from different experimental groups are quantified, and values shown as radiant efficiency (n = 15, 5 mice per group, two-way ANOVA, F2,156 = 242.1, p < 0.0001, Tukey’s multiple comparison test; Control vs. TBI-saline, p < 0.0001; TBI saline vs. TBI + PPA, p = 0.0002). Data shown as box and whisker plot with the lower and upper quartile (box limits), median and min-max values (c) or line graph with group means and continuous SEM (f); the dots are biological replicates/mice. Scale bars: 5 mm.

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Extended Data Fig. 7 PPA administration in healthy mice results in enhanced clearance of radiotracers from CSF.

a, Experimental schematic: wild-type mice were implanted with cisterna magna cannula 24 h prior to the experiments. The awake mice were injected with radiotracers (one tracer per group), with or without PPA treatment. Blood was collected 30 min post-injection, centrifuged for plasma extraction, mixed with a scintillation cocktail (Ultima Gold, PerkinElmer), and analysed in liquid scintillation counter for C-14 and Na-22 radioactivity (LS6500 Multi-purpose Scintillation Counter, Beckman Coulter, GA, USA). b-d, Radioactivity was detected in the plasma samples injected with C-14 mannitol (left), C-14 inulin (middle), and Na-22 Sodium (right). Data analysed for mean and SEM; group means were compared using student t-test (unpaired, two-tailed): b, Mannitol (n = 14 mice, 7 mice per group, F6,6 = 3.584, p = 0.0065), c, Inulin (n = 7 mice; 3 and 4 mice in control and PPA group, respectively, F2,3 = 1.146, p = 0.014), and d, Na-22 (n = 17 mice; 10 and 7 mice in control and PPA group, respectively, F6,9 = 5.892, p < 0.0001). e, Schematic illustrating the CSF compartment of the brain and experimental timeline. f, Quantification of radiotracer within the blood plasma with or without TBI and PPA treatment (n = 20 mice, Control (n = 6), TBI-saline (n = 7), TBI + PPA (n = 7), one-way ANOVA, F2,17 = 6.29, p = 0.009, Tukey’s multiple comparison test; Control vs TBI-saline, p = 0.038, TBI-saline vs TBI + PPA, p = 0.011, Control vs TBI + PPA, p = 0.88). Bars show mean and SEM (b-d), box and whisker plots show the lower and upper quartile (box limits), median and min-max values (f), and the dots are biological replicates/mice.

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Extended Data Fig. 8 Comparison of size of cervical lymph nodes upon injury and PPA treatment. TBI alters cardiac but not respiratory rates in mice. Post traumatic linear increase in NA levels is counteracted by PPA treatment. NA treatment of CLVs ex vivo results in loss of entrainment while preemptive treatment with PPA nullifies the effect.

a, Mice, implanted with cisterna magna cannulas, were injected with a mixture of FITC dextran and fluorophore Tx Red, lymph nodes were isolated 40–60 min post-injury with or without PPA treatment, and the sizes of lymph nodes (LN) were measured in images acquired using a fluorescent dissecting microscope (MVX10, Olympus). b-c, LN diameter and tracer distribution area (n = 25 mice; Control (n = 6), TBI-saline (n = 10), TBI + PPA (n = 9), multiple cervical lymph nodes averaged per mouse) were compared using one-way ANOVA. b, Diameter; F2,22 = 0.3796, p = 0.6886. c, Tracer distribution area, F2,22 = 10.46, p = 0.0006, Tukey’s multiple comparison test, Control vs TBI-saline, p = 0.0013, TBI-saline vs TBI + PPA, p = 0.005). d-e, Respiratory and cardiac rates of anaesthetized mice (n = 28 mice; Control (n = 6), TBI-saline (n = 10), TBI + PPA (n = 9), one-way ANOVA followed by Tukey’s multiple comparison test). Recordings were obtained using a small animal physiological monitoring system (Harvard Apparatus). The recording duration was synchronized with the Thorlabs 2 P imager while performing lymphatic vessel imaging experiments. d, Heart rate (F2,25 = 6.2, p = 0.0065; Control vs TBI-saline, p = 0.0123, TBI-saline vs TBI + PPA, p = 0.933), e, respiratory rate (F2,25 = 1.101, p = 0.3482; Control vs TBI-saline, p = 0.8876; TBI-saline vs TBI + PPA, p = 0.3407). f, Semi-log curve fit of NA levels depicts a steady increase over time. g, Cumulative area under the curve of NA levels with or without injury and PPA treatment (n = 15 mice; Control (n = 7), TBI-saline (n = 4), TBI + PPA (n = 4), one-way ANOVA, F2,12 = 2.75, p = 0.0019, Tukey’s multiple comparison test; Control vs TBI-saline, p = 0.0017, TBI-saline vs TBI + PPA, p = 0.0118). h, Image of an isolated cervical lymphatic vessel with the area used for spatiotemporal map generation marked by a rectangular box. i, Spatiotemporal maps showing CLV contraction pattern in control, NA, and NA + PPA treatment. Continuous vertical bands correspond to single contraction waves that conduct over the entire length of the vessel. The intensity of each line is inversely proportional to the magnitude of the constriction. All contractions initiate at the top of the segment. Horizontal lines are diameter tracking artifacts due to small pieces of fat or connective tissue remaining on the outside of the vessel that rotated during contraction. Data shown as box and whisker plots with the lower and upper quartile (box limits), median and min-max values (b–e, g) and sigmoid line graph (f); the dots are biological replicates/mice (b–e, g). Scale: 500 µm.

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Extended Data Fig. 9 PPA treatment does not alter cardiac and respiratory rates in non-injured control mice but increases the high amplitude contraction frequency of cervical lymphatic vessels (CLV).

a-e, Group means were compared by student t-test (unpaired, two tailed). a, Heart rate (n = 9 mice; 4 control and 5 PPA, p = 0.4671). b, Respiration rate (n = 9 mice; 4 control and 5 PPA, p = 0.233). c, Mean arterial pressure (7 mice per group, p = 0.026). d, Cerebral blood flow (9 mice per group, p = 0.291). e, Intracranial pressure (8 mice per group, p = 0.0048). f, C57Bl6 mice implanted with cisterna magna cannula were injected with FITC dextran (10 µL) and recorded for contraction frequency (20–40 min post-injection). g, Contraction profile (representative segments, length 2 min) of CLV recorded in control (b) and with PPA administration (c) both under 2.5% isoflurane. h, High amplitude contractions (> 1.5-fold change in diameter) were quantified and shown as a box blot. Data (n = 17 mice, 8 control and 9 PPA mice, 1-2 representative recordings/mice), were compared using a student t-test (unpaired, two-tailed, F8,7 = 1.626, p = 0.0034). Data shown as box and whisker plots with the lower and upper quartile (box limits), median and min-max values (a–e, h) and line graph (g); the dots are biological replicates/mice. Scale: 500 µm.

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Extended Data Fig. 10 Dorsal meningeal lymphatic vessel dysfunction is evident in hit-and-run type TBI.

Mice injected with fluorescent tracer FITC-Dextran (2 kDa, green) and Tx-Red FluoSpheres (1 µm, red) were evaluated for outflow potential via dorsal meningeal lymphatic vessels, with or without injury and subsequent treatment of PPA or saline, respectively. a, Representative images showing whole mount dural lymphatic vessels. b-c, Representative images of the region of interest showing dorsal meningeal lymphatic vessels in the superior sagittal sinus (SSS, b) and transverse sagittal sinus area (TSS, c). d-e, Tracer intensity was measured (using Image J, semi-automated fluorescence intensity method) and compared among treatment groups (n = 18 mice; control (n = 7), TBI-saline (n = 5), and TBI + PPA (n = 6), multiple slices averaged per mouse, one-way ANOVA, followed by Tukey’s multiple comparison test). d, Meningeal lymphatics in SSS; FITC-Dextran (left, F2,15 = 5.182, p = 0.019; Control vs TBI-saline, p = 0.0189, TBI-saline vs TBI + PPA, p = 0.5723), Tx-Red FluoSpheres (right, F2,15 = 8.147, p = 0.0040; Control vs TBI-saline, p = 0.0053, TBI-saline vs TBI + PPA, p = 0.6775). e, Meningeal lymphatic vessels in TSS: FITC-Dextran (left, F2,15 = 8.944, p = 0.0028; Control vs TBI-saline, p = 0.0026, TBI-saline vs TBI + PPA, p = 0.0166) and Tx-Red FluoSpheres (right, F2,15 = 13.53, p = 0.0004; Control vs TBI-saline, p = 0.0003, TBI-saline vs TBI + PPA, p = 0.0291). Data shown as box and whisker plot with the lower and upper quartile (box limits), median and min-max values, and the dots are biological replicates/mice. Scale: 5 mm.

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Hussain, R., Tithof, J., Wang, W. et al. Potentiating glymphatic drainage minimizes post-traumatic cerebral oedema. Nature 623, 992–1000 (2023). https://doi.org/10.1038/s41586-023-06737-7

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