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Glymphatic influx and clearance are accelerated by neurovascular coupling

Nature Neuroscience volume 26, pages 1042–1053 (2023)Cite this article

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A Publisher Correction to this article was published on 04 September 2023

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Abstract

Functional hyperemia, also known as neurovascular coupling, is a phenomenon that occurs when neural activity increases local cerebral blood flow. Because all biological activity produces metabolic waste, we here sought to investigate the relationship between functional hyperemia and waste clearance via the glymphatic system. The analysis showed that whisker stimulation increased both glymphatic influx and clearance in the mouse somatosensory cortex with a 1.6-fold increase in periarterial cerebrospinal fluid (CSF) influx velocity in the activated hemisphere. Particle tracking velocimetry revealed a direct coupling between arterial dilation/constriction and periarterial CSF flow velocity. Optogenetic manipulation of vascular smooth muscle cells enhanced glymphatic influx in the absence of neural activation. We propose that impedance pumping allows arterial pulsatility to drive CSF in the same direction as blood flow, and we present a simulation that supports this idea. Thus, functional hyperemia boosts not only the supply of metabolites but also the removal of metabolic waste.

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Fig. 1: Functional hyperemia increases periarterial CSF influx.
Fig. 2: Perivascular CSF flow kinetics is closely controlled by arterial diameter.
Fig. 3: Perivascular spaces—transfer of CSF tracers and dynamic changes.
Fig. 4: Functional hyperemia does not increase glymphatic influx during wakefulness.
Fig. 5: Arterial diameter changes and not neuronal activation per se increases glymphatic influx.
Fig. 6: Laser stimulation in optogenetic mice increases CSF downstream velocity.
Fig. 7: Functional hyperemia increases glymphatic clearance.
Fig. 8: Impedance pumping models reproduce characteristics of flows driven by functional hyperemia and optogenetic stimulation.

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Data availability

Source data are provided with this paper. Source data and Extended Data are also available in the figshare repository at https://doi.org/10.6084/m9.figshare.22340581.v2. We used the Allen Brain Atlas: Mouse Brain for anatomical reference.

Code availability

All relevant codes are available from the corresponding author upon request.

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Acknowledgements

This project has received funding from the Novo Nordisk Foundation (NNF20OC0066419) and Lundbeckfonden (R386-2021-165), by the U.S. National Institute under Awards R01AT012312RF1AG057575, R01AT011439, U19 NS128613, the U.S. Army under award MURI W911NF1910280 and the Simon and Adelson foundations (to M.N.). S.H-R. is recipient of a Lundbeck Ph.D. Fellowship (R230-2016-2135). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. We would like to thank D. Xue (Center for Translational Neuromedicine, University of Copenhagen, Denmark) for expert graphical design and K.L. Turner (Department of Biomedical Engineering, Pennsylvania State University, USA) for help with the (HbT) conversion.

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  1. These authors contributed equally: Stephanie Holstein-Rønsbo, Yiming Gan.

Authors and Affiliations

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

    Stephanie Holstein-Rønsbo, Martin Kaag Rasmussen, Björn Sigurdsson, Felix Ralf Michael Beinlich, Laura Rose, Verena Untiet & Maiken Nedergaard

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

    Yiming Gan & Douglas H. Kelley

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

    Michael J. Giannetto, Lauren M. Hablitz & Maiken Nedergaard

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Contributions

M.N. and S.H-R. conceived of the study and wrote the manuscript. S.H-R. planned and carried out the experiments, analyzed and interpreted data. Y.G. and D.H.K. analyzed data and developed the impedance pumping model. M.K.R., M.G. and V.U. planned and executed experiments. F.R.M.B. planned experiments and analyzed data. L.H. analyzed data. B.S. and L.R. developed analyses and analyzed data. All authors have reviewed the manuscript.

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Correspondence to Maiken Nedergaard.

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Extended data

Extended Data Fig. 1 Whisker stimulation causes Ca2+ and hemodynamic responses in contralateral barrel cortex.

a. Adult wild-type mice were head-plated under ketamine/xylazine anesthesia and exposed to unilateral whisker stimulation (5 Hz 10 ms, 10 psi, 30 seconds per stimulation). Macroscopic imaging was utilized to map neuronal activation (Ca2+ signaling) in GCaMP mice. b. The Ca2+ response in the stimulated and unstimulated hemisphere across whisker stimulation in Thy1-GCaMP6S mice (n = 8 mice). c. Representative images of the Ca2+-response to three different whisker stimulation protocols (1, 5 or 10 Hz with 10 ms pulse length). The image is averaged across five stimulations. d. Ca2+ traces from barrel field cortex in response to 30 sec whisker stimulation protocols in Thy1-GCaMP6S mice (frequencies: 1, 5 or 10 Hz and pulse lengths: 5, 10 or 15 ms) (n = 7 for 5 and 10 ms, n = 5 for 15 ms). e. Area under the curve (AUC) of tested pulse lengths (5, 10 or 15 ms, 5 Hz) (n = 7 mice). One-way ANOVA w Tukey’s correction: P = 0.0097, 5 vs. 10 ms; P = 0.16, 5 vs. 15 ms; P = 0.99, 10 vs. 15 ms. f, AUC of traces from Fig. 1c comparing the effect of the three stimulation protocols on Ca2+-signaling (left) or cerebral blood flow (right) (n = 7). One-way ANOVA w Dunnett’s correction: Ca2+: P = 0.005, 1 vs. 5 Hz; P = 0.17, 1 vs. 10 Hz; P = 0.006, 5 vs. 10 Hz. CBF: P = 0.04, 1 vs. 5 Hz; P = 0.04, 5 vs. 10 Hz. g. The total hemoglobin (HbT) change in barrel cortex in response to unilateral whisker stimulation (5 Hz 10 ms, 10 psi). Regions of interest (ROI) are depicted above barrel and motor cortex in both hemispheres. h. The hemodynamic response during whisker stimulation in barrel cortex (pink and orange) and in control regions (green and blue). The gray dashed line represents the HbT response in the stimulated barrel cortex (orange), when elevating the air pressure from 10 to 20 psi. Right: AUC of the HbT in barrel field cortex, when applying air pressure of 10 or 20 psi (n = 6 mice). Two-tailed paired t-test: P = 0.013. I. Hemodynamic response in the same mice before application of any whisker stimulation. Data are represented as mean ± SEM. *=P < 0.05, **=P < 0.01.

Extended Data Fig. 2 CSF tracer influx is symmetric across the hemispheres in control non-stimulated mice.

a. Fluorescence signal (mean pixel intensity (MPI)) of the CSF tracer (70 kDa dextran) in each hemisphere of the control mice (n = 7). b. Average fluorescent signal change across 90 seconds in left and right hemisphere of control mice receiving no whisker stimulation (n = 7). Normalized according to tracer intensity at stimulation start (t = 30 seconds). c. Front-tracking of the CSF tracer spread during macroscopic imaging demonstrates higher velocity in the stimulated hemisphere (n = 8). Two mice from Fig. 1e were not eligible for front-tracking analysis. Two-tailed paired t-test: P = 0.033. d. Left: Group overlay of the fluorescent signal displaying the parenchymal influx of the CSF tracer in ex vivo brain sections spanning the barrel cortex area (anterior/posterior (A/P): 0.3 to -1.7) in control mice receiving no whisker stimulations. Each hemisphere was analyzed (regions of interest are outlined by white dotted lines). Right: Percentage distribution of tracer (3 kDa dextran) in the two hemispheres (49.7 ± 1.1% vs. 50.3 ± 1.1%). The total tracer signal in each brain was set to 100% (n = 6). Two-tailed paired t-test: P = 0.77. Data are represented as mean ± SEM. *=P < 0.05.

Extended Data Fig. 3 Puncture of the perivascular space membrane increases CSF flow velocity.

a–c. Absolute values of the average change in pial artery diameter (a), downstream CSF flow velocity (b) and cross-stream CSF flow velocity (c) during functional hyperemia (n = 7). d. The downstream velocity increases dramatically in mice with punctured perivascular space membrane (n = 5). Mean CSF flow velocity: 70.5 ± 3.8 µm/s.

Extended Data Fig. 4 CSF tracer moves from arterial to venous perivascular spaces over time.

a. Adult NG2-dsRed mice were intracisternally injected (70 kDa dextran) under ketamine/xylazine anesthesia. At 30 or 120 minutes after infusion start, mice were intracardially perfused with lectin (WGA-647) followed by 4% paraformaldehyde (PFA). b. Representative section from 120 min group (-2.4 mm bregma), inset = lateral cortex. Analysis pipeline from left to right: vessels were scored as positive for perivascular CSF tracer (yellow circles). Vessels were excluded if there was no tracer deeper than 200 µm from the brain surface (arrowheads). Using NG2-dsRed, vessels were scored as arteries (red) or veins (blue) if they had banded or diffuse dsRed signal, respectively. Finally, the number of arteries and veins with tracer was counted for each brain region. c–e. Number of vessels (c) (scored as arteries/veins as well as uncategorized), arteries (d), or veins (e) with CSF tracer at each brain subregion (left), and pooled into cortical, deep, and total (right) (3 brain sections/mouse; -1.8, -2.4, and -3.2 mm bregma) (n = 3). f. Representative images of tracer distribution in arteries from lateral cortex at 30 minutes (top) and 120 minutes (bottom). WGA-647 labeling is high in arteries (arrows), but low in veins. NG2-dsRed signal is banded in arteries (arrows) and diffuse in veins. CSF tracer (70 kDa dextran) concentration in arterial perivascular spaces is high at 30 minutes (arrow, top panel), and in part phagocytosed by perivascular macrophages around arteries and veins at 120 minutes (bottom panel). g. Model diagram depicting high CSF tracer at arterial perivascular spaces at 30 minutes, and high CSF tracer at venous perivascular spaces at 120 minutes post infusion. Data are represented as mean ± SEM.

Extended Data Fig. 5 Tracer inflow and parenchymal spread is larger in anesthetized compared to awake mice.

a. Overlay of Figs. 1e and 4d: CSF tracer signal (mean pixel intensity (MPI)) across 30 minutes in the stimulated and unstimulated hemisphere of both anesthetized (n = 10) and awake mice (n = 8) in response to whisker stimulations. Gray bars: 30 sec whisker stimulation. b. Parenchymal spread of CSF tracer in the cortices (ROI outlined by white dotted lines) in anesthetized (n = 7) and awake mice (n = 8) measured as the %Area coverage (12.6 ± 1.8% vs. 4.6 ± 1.5%). Two-way ANOVA with Sidak correction, P = 0.0029. Data are represented as mean ± SEM. **=P < 0.01.

Extended Data Fig. 6 Optogenetic stimulation increases total tracer influx.

a. Representative image of optogenetic stimulation above the middle cerebral artery in Sm22-Cre:Ai32-ChR2 mice. b. The individual traces from the optogenetic mice shown in Fig. 5c (n = 6). Blue bars: laser stimulation (30 seconds each; 10 Hz 50 ms). c. Average change in tracer signal (mean pixel intensity (MPI)) during and between stimulations in control animals (Sm22-Cre−/−:Ai32-ChR2) (n = 4). Two-way ANOVA with Sidak correction, P = 0.83, stim; P = 0.99, unstim. Data are represented as mean ± SEM.

Source data

Supplementary information

Reporting Summary

Supplementary Video 1

Adult wild-type mice were head-plated and implanted with an intracisternal catheter under KX anesthesia. CSF tracer (70 kDa dextran) was injected into the cisterna magna before exposure to unilateral whisker stimulation for 30 min (5 Hz (10 ms), 10 psi for 30 s with 60 s interval). Transcranial macroscopic imaging was used to map CSF tracer transport. The video is played at 200× speed. Scalebar, 1 mm.

Supplementary Video 2

Adult wild-type mice were head-plated, and a cranial window was inserted above the MCA under KX anesthesia. Microspheres (1 µm) were injected into the cisterna magna before exposure to unilateral whisker stimulation. Two-photon imaging was used to map CSF flow velocity in the perivascular space of the MCA. The microspheres move in response to the cardiac pulsations of the middle cerebral artery. Video is played at 10× speed. Scalebar, 40 µm.

Supplementary Video 3

Adult optogenetic mice (Sm22-Cre:Ai32-ChR2) were head-plated under KX anesthesia and intracisternally injected with CSF tracer (2,000 kDa, FITC) before exposure to unilateral photostimulation (473 nm, 10 Hz, 50 ms, 30 s every minute) for 30 min during macroscopic imaging. Photoactivation of ChR2 in smooth muscle cells leads to arterial constriction. The video is played at 12× speed. Scalebar, 1 mm.

Supplementary Video 4

Vasodilation can drive net flow via impedance pumping. In our simulation, we applied periodic dilation forces to elastic membranes modeling the artery wall, in the regions shaded red. The resulting dilations drove cerebrospinal fluid flow (the velocity is indicated by the size of the arrows) in the modeled perivascular space (bounded by rigid membranes, shown in blue). Simulated tracers (green) following fluid motion revealed long-term directional motion to the right.

Supplementary Video 5

Vasoconstriction can drive net flow in the same direction as vasodilation. We performed a simulation identical to the one depicted in Video 4 but applying vasoconstriction forces instead of vasodilation forces. Net flow again proceeded to the right, because in impedance pumping, the flow direction depends on the speed of the waves traveling along the elastic membrane, the pulse frequency and the locations of wave reflectors, none of which change when dilation is replaced with constriction.

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Holstein-Rønsbo, S., Gan, Y., Giannetto, M.J. et al. Glymphatic influx and clearance are accelerated by neurovascular coupling. Nat Neurosci 26, 1042–1053 (2023). https://doi.org/10.1038/s41593-023-01327-2

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