Rapid lymphatic efflux limits cerebrospinal fluid flow to the brain

The relationships between cerebrospinal fluid (CSF) and brain interstitial fluid are still being elucidated. It has been proposed that CSF within the subarachnoid space will enter paravascular spaces along arteries to flush through the parenchyma of the brain. However, CSF also directly exits the subarachnoid space through the cribriform plate and other perineural routes to reach the lymphatic system. In this study, we aimed to elucidate the functional relationship between CSF efflux through lymphatics and the potential influx into the brain by assessment of the distribution of CSF-infused tracers in awake and anesthetized mice. Using near-infrared fluorescence imaging, we showed that tracers quickly exited the subarachnoid space by transport through the lymphatic system to the systemic circulation in awake mice, significantly limiting their spread to the paravascular spaces of the brain. Magnetic resonance imaging and fluorescence microscopy through the skull under anesthetized conditions indicated that tracers remained confined to paravascular spaces on the surface of the brain. Immediately after death, a substantial influx of tracers occurred along paravascular spaces extending into the brain parenchyma. We conclude that under normal conditions a rapid CSF turnover through lymphatics precludes significant bulk flow into the brain. Electronic supplementary material The online version of this article (10.1007/s00401-018-1916-x) contains supplementary material, which is available to authorized users.


Fig. S2. Characterization of paravascular location of tracer and spread between arteries
and veins at the brain surface. a-b, Ex vivo images of tracer spread 60 min after i.c.v. infusion of 2.5 µL 200 µM P40D680 in an SMMHC-GFP mouse suggesting spreading of tracer (white) between an artery (A) and a vein (V) at the brain surface (a, scale bars: 500 µm) and a location of tracer outside of the smooth muscle cell layer of the artery (b, SMMHC-GFP, green, scale bars: 100 µm). c-d, Ex vivo images of tracer spread 2 min after i.c.v. infusion of 2.5 µL 200 µM P40D680 in an SMMHC-GFP mouse indicate spreading between artery (A) and vein (V) is already occurring at this point at the brain surface (c, scale bars: 250 µm), while image of 100 µm brain section shows no tracer penetration into the brain parenchyma (d, scale bars: 100 µm).  Noise was measured as standard deviation (SD) of the background signal collected from two ROIs placed independently outside the animals to the left and right (ROI 3, ROI 4). Circular ROIs for brain parenchyma SI and noise measurements were selected at identical sizes. PVS for cortical surface vessels was investigated in the territory of the middle cerebral artery. Target structures were identified close to the brain surface and presenting hyperintense when compared with adjacent brain tissue (ROI 5, ROI 6). Segmentation was performed manually. SNR was calculated for brain parenchyma and PVS for each time point investigated as follows: SNR = ((Mean SIhemisphere_1 / Mean SDhemisphere_1) + (Mean SIhemisphere_2 / Mean SDhemisphere_2)) / 2 SNR for the different timepoints were normalized to the time of death (set to t=0).

Video S1
Model of the relationship between CSF outflow and PVS spread. During awake conditions, tracer-filled CSF rapidly flows through the basal cisterns and effluxes along perineural routes to lymphatic vessels outside the skull. The rapid washout from the subarachnoid space leads to minimal spread of tracers to the PVS of the brain when assessed ex vivo. During anesthetized conditions, tracer-filled CSF is slower to efflux and is retained over time in the basal cisterns.
This results in greater presence of tracers in the PVS when assessed ex vivo.

Video S2
Video showing spread of tracer along brain surface blood vessels after infusion of 2.5 µL 200 µM P40D800 into the contralateral ventricle. Time after end of infusion indicated. Scale bar: 1000 µm. Images are acquired at 1 frame per 15 s at 20x magnification.

Video S3
Video showing spread of tracer along both surface arteries and veins 60 min after i.c.v. infusion of 2.5 µL 200 µM P40D800. Dorsal middle cerebral vein, dorsal rostral cerebral vein and branches of the MCA showing tracer signal are indicated in the video. Images are acquired at 2.5 frames per s at variable magnification.

Video S4
Video showing pressure wave propagating along MCA branches and subsequent tracer spread to penetrating blood vessels following overdose with ket/med 60 min after infusion of 2.5 µL 200 µM P40D800 into the contralateral ventricle. Time relative to last breath indicated. Scale bar: 500 µm. Images are acquired at 1 frame per 2.5 s at 30x magnification.

Video S5
Representative video (n=5) showing spread of tracer after infusion of 5 µL of a Gadospin D solution at 25 mM gadolinium into the cisterna magna acquired with a sequence of T1-weighted MRI acquisitions. The mouse was kept alive under ket/med and showed no spread of the tracer towards the PVS. Images are acquired at 1 frame (color-coded MIP) per 4.5 min.

Video S6
Representative video (n=5) showing spread of tracer after infusion of 5 µL of a Gadospin D solution at 25 mM gadolinium into the cisterna magna acquired with a sequence of T1-weighted MRI acquisitions. The mouse was overdosed with ket/med and death is indicated as t=0. Strong enhancements of signal are detectable 9 min after death at the circle of Willis and at the middle cerebral artery. Smaller branches of the middle cerebral artery start to be visible 18 min after death.
Images are acquired at 1 frame (color-coded MIP) per 4.5 min.

Video S7
Video showing continued spread of tracer to penetrating blood vessels after transcardiac perfusion with ice-cold PBS 60 min after infusion of 2.5 µL 200 µM P40D800 into the contralateral ventricle. Time relative to start of video indicated. Scale bar: 1000 µm. Images are acquired at 1 frame per 2.5 s at 20x magnification.