Abstract
The majority of the brain’s vasculature is composed of intricate capillary networks lined by capillary pericytes. However, it remains unclear whether capillary pericytes influence blood flow. Using two-photon microscopy to observe and manipulate brain capillary pericytes in vivo, we find that their optogenetic stimulation decreases lumen diameter and blood flow, but with slower kinetics than similar stimulation of mural cells on upstream pial and precapillary arterioles. This slow vasoconstriction was inhibited by the clinically used vasodilator fasudil, a Rho-kinase inhibitor that blocks contractile machinery. Capillary pericytes were also slower to constrict back to baseline following hypercapnia-induced dilation, and slower to dilate towards baseline following optogenetically induced vasoconstriction. Optical ablation of single capillary pericytes led to sustained local dilation and a doubling of blood cell flux selectively in capillaries lacking pericyte contact. These data indicate that capillary pericytes contribute to basal blood flow resistance and slow modulation of blood flow throughout the brain.
Similar content being viewed by others
Data availability
Source data are provided with this paper. Any additional data are available from the corresponding author upon request.
Code availability
Vasometrics54, an ImageJ/Fiji-based macro for unbiased vessel diameter measurement, can be downloaded at: https://github.com/mcdowellkonnor/ResearchMacros. All other code is available from the corresponding author upon request.
References
Kisler, K., Nelson, A. R., Montagne, A. & Zlokovic, B. V. Cerebral blood flow regulation and neurovascular dysfunction in Alzheimer disease. Nat. Rev. Neurosci. 18, 419–434 (2017).
Blinder, P. et al. The cortical angiome: an interconnected vascular network with noncolumnar patterns of blood flow. Nat. Neurosci. 16, 889–897 (2013).
Gould, I. G., Tsai, P. S., Kleinfeld, D. & Linninger, A. The capillary bed offers the largest hemodynamic resistance to the cortical blood supply. J. Cereb. Blood Flow. Metab. 37, 52–68 (2016).
Grant, R. I. et al. Organizational hierarchy and structural diversity of microvascular pericytes in adult mouse cortex. J. Cereb. Blood Flow. Metab. 39, 411–425 (2017).
Hill, R. A. et al. Regional blood flow in the normal and ischemic brain is controlled by arteriolar smooth muscle cell contractility and not by capillary pericytes. Neuron 87, 95–110 (2015).
Armulik, A., Genové, G. & Betsholtz, C. Pericytes: developmental, physiological, and pathological perspectives, problems, and promises. Dev. Cell 21, 193–215 (2011).
Peppiatt, C. M., Howarth, C., Mobbs, P. & Attwell, D. Bidirectional control of CNS capillary diameter by pericytes. Nature 443, 642–643 (2006).
Hall, C. N. et al. Capillary pericytes regulate cerebral blood flow in health and disease. Nature 508, 55–60 (2014).
Kisler, K. et al. Pericyte degeneration leads to neurovascular uncoupling and limits oxygen supply to brain. Nat. Neurosci. 20, 406–416 (2017).
Cai, C. et al. Stimulation-induced increases in cerebral blood flow and local capillary vasoconstriction depend on conducted vascular responses. Proc. Natl Acad. Sci. USA 15, E5796–E5804 (2018).
Fernández-Klett, F., Offenhauser, N., Dirnagl, U., Priller, J. & Lindauer, U. Pericytes in capillaries are contractile in vivo, but arterioles mediate functional hyperemia in the mouse brain. Proc. Natl Acad. Sci. USA 107, 22290–22295 (2010).
Wei, H. S. et al. Erythrocytes are oxygen-sensing regulators of the cerebral microcirculation. Neuron 91, 851–862 (2016).
Yemisci, M. et al. Pericyte contraction induced by oxidative-nitrative stress impairs capillary reflow despite successful opening of an occluded cerebral artery. Nat. Med. 15, 1031–1037 (2009).
Montagne, A. et al. Pericyte degeneration causes white matter dysfunction in the mouse central nervous system. Nat. Med. 24, 326–337 (2018).
Sagare, A. P. et al. Pericyte loss influences Alzheimer-like neurodegeneration in mice. Nat. Commun. 4, 2932 (2013).
Nortley, R. et al. Amyloid β oligomers constrict human capillaries in Alzheimer’s disease via signaling to pericytes. Science 365, eaav9518 (2019).
Arango-Lievano, M. et al. Topographic reorganization of cerebrovascular mural cells under seizure conditions. Cell Rep. 23, 1045–1059 (2018).
Li, Y. et al. Pericytes impair capillary blood flow and motor function after chronic spinal cord injury. Nat. Med. 23, 733–741 (2017).
Nikolakopoulou, A. M. et al. Pericyte loss leads to circulatory failure and pleiotrophin depletion causing neuron loss. Nat. Neurosci. 22, 1089–1098 (2019).
Vanlandewijck, M. et al. A molecular atlas of cell types and zonation in the brain vasculature. Nature 554, 475–480 (2018).
Andrasfalvy, B. K., Zemelman, B. V., Tang, J. & Vaziri, A. Two-photon single-cell optogenetic control of neuronal activity by sculpted light. Proc. Natl Acad. Sci. USA 107, 11981–11986 (2010).
Longden, T. A. et al. Capillary K+-sensing initiates retrograde hyperpolarization to increase local cerebral blood flow. Nat. Neurosci. 20, 717–726 (2017).
Neuhaus, A. A., Couch, Y., Sutherland, B. A. & Buchan, A. M. Novel method to study pericyte contractility and responses to ischaemia in vitro using electrical impedance. J. Cereb. Blood Flow. Metab. 37, 2013–2024 (2016).
Saponara, S. et al. Effects of commonly used protein kinase inhibitors on vascular contraction and L-type Ca2+ current. Biochem. Pharmacol. 84, 1055–1061 (2012).
Rorsman, N. J. G., Ta, C. M., Garnett, H., Swietach, P. & Tammaro, P. Defining the ionic mechanisms of optogenetic control of vascular tone by channelrhodopsin-2. Br. J. Pharmacol. 175, 2028–2045 (2018).
Narayanan, D., Xi, Q., Pfeffer, L. M. & Jaggar, J. H. Mitochondria control functional CaV1.2 expression in smooth muscle cells of cerebral arteries. Circ. Res. 107, 631–641 (2010).
Jin, L., Ying, Z. & Webb, R. C. Activation of Rho/Rho kinase signaling pathway by reactive oxygen species in rat aorta. Am. J. Physiol. Heart Circ. Physiol. 287, H1495–H1500 (2004).
Yoon, J., Choi, M., Ku, T., Choi, W. J. & Choi, C. Optical induction of muscle contraction at the tissue scale through intrinsic cellular amplifiers. J. Biophotonics 7, 597–606 (2014).
Gutiérrez-Jiménez, E. et al. The effects of hypercapnia on cortical capillary transit time heterogeneity (CTH) in anesthetized mice. J. Cereb. Blood Flow. Metab. 38, 290–303 (2018).
Watson, A.N. et al. Mild pericyte deficiency is associated with aberrant brain microvascular flow in aged PDGFRβ+/- mice. J. Cereb. Blood Flow Metab. 40, 2387–2400 (2020).
Rungta, R. L., Chaigneau, E., Osmanski, B. F. & Charpak, S. Vascular compartmentalization of functional hyperemia from the synapse to the pia. Neuron 99, 362–375 (2018).
Berthiaume, A. A. et al. Dynamic remodeling of pericytes in vivo maintains capillary coverage in the adult mouse brain. Cell Rep. 22, 8–16 (2018).
Schmid, F., Reichold, J., Weber, B. & Jenny, P. The impact of capillary dilation on the distribution of red blood cells in artificial networks. Am. J. Physiol. Heart Circ. Physiol. 308, H733–H742 (2015).
Lyons, D. G., Parpaleix, A., Roche, M. & Charpak, S. Mapping oxygen concentration in the awake mouse brain. eLife 5, e12024 (2016).
Gutiérrez-Jiménez, E. et al. Effect of electrical forepaw stimulation on capillary transit-time heterogeneity (CTH). J. Cereb. Blood Flow. Metab. 36, 2072–2086 (2016).
Alarcon-Martinez, L. et al. Interpericyte tunnelling nanotubes regulate neurovascular coupling. Nature 585, 91–95 (2020).
Li, Y., Wei, W. & Wang, R. K. Capillary flow homogenization during functional activation revealed by optical coherence tomography angiography based capillary velocimetry. Sci. Rep. 8, 4107 (2018).
Li, B., Lee, J., Boas, D. A. & Lesage, F. Contribution of low- and high-flux capillaries to slow hemodynamic fluctuations in the cerebral cortex of mice. J. Cereb. Blood Flow. Metab. 36, 1351–1356 (2016).
Stefanovic, B. et al. Functional reactivity of cerebral capillaries. J. Cereb. Blood Flow. Metab. 28, 961–972 (2007).
Desjardins, M., Berti, R., Lefebvre, J., Dubeau, S. & Lesage, F. Aging-related differences in cerebral capillary blood flow in anesthetized rats. Neurobiol. Aging 35, 1947–1955 (2014).
Grubb, S. et al. Precapillary sphincters maintain perfusion in the cerebral cortex. Nat. Commun. 11, 395 (2020).
Gonzales, A. L. et al. Contractile pericytes determine the direction of blood flow at capillary junctions. Proc. Natl Acad. Sci. USA 117, 27022–27033 (2020).
Nelson, A. R. et al. Channelrhodopsin excitation contracts brain pericytes and reduces blood flow in the aging mouse brain in vivo. Front. Aging Neurosci. 12, 108 (2020).
Kureli, G. et al. F-actin polymerization contributes to pericyte contractility in retinal capillaries. Exp. Neurol. 332, 113392 (2020).
Alarcon-Martinez, L. et al. Retinal ischemia induces α-SMA-mediated capillary pericyte contraction coincident with perivascular glycogen depletion. Acta Neuropathol. Commun. 7, 134 (2019).
Alarcon-Martinez, L. et al. Capillary pericytes express α-smooth muscle actin, which requires prevention of filamentous-actin depolymerization for detection. eLife 7, e34861 (2018).
Cipolla, M. J., Gokina, N. I. & Osol, G. Pressure-induced actin polymerization in vascular smooth muscle as a mechanism underlying myogenic behavior. FASEB J. 16, 72–76 (2002).
Shibuya, M., Hirai, S., Seto, M., Satoh, S. & Ohtomo, E. Effects of fasudil in acute ischemic stroke: results of a prospective placebo-controlled double-blind trial. J. Neurol. Sci. 238, 31–39 (2005).
Vesterinen, H. M. et al. Systematic review and stratified meta-analysis of the efficacy of RhoA and Rho kinase inhibitors in animal models of ischaemic stroke. Syst. Rev. 2, 33 (2013).
Jespersen, S. N. & Østergaard, L. The roles of cerebral blood flow, capillary transit time heterogeneity, and oxygen tension in brain oxygenation and metabolism. J. Cereb. Blood Flow. Metab. 32, 264–277 (2012).
Schmid, F., Tsai, P. S., Kleinfeld, D., Jenny, P. & Weber, B. Depth-dependent flow and pressure characteristics in cortical microvascular networks. PloS Comput. Biol. 13, e1005392 (2017).
Sengillo, J. D. et al. Deficiency in mural vascular cells coincides with blood–brain barrier disruption in Alzheimer’s disease. Brain Pathol. 23, 303–310 (2013).
Dziewulska, D. & Lewandowska, E. Pericytes as a new target for pathological processes in CADASIL. Neuropathology 32, 515–521 (2012).
McDowell, K. P., Berthiaume, A. A., Tieu, T., Hartmann, D. A. & Shih, A. Y. VasoMetrics: unbiased spatiotemporal analysis of microvascular diameter in multi-photon imaging applications. Quant. Imaging Med. Surg. 11, 969–982 (2021).
Cuttler, A. S. et al. Characterization of Pdgfrb-Cre transgenic mice reveals reduction of ROSA26 reporter activity in remodeling arteries. Genesis 49, 673–680 (2011).
Madisen, L. et al. A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing. Nat. Neurosci. 15, 793–802 (2012).
Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010).
Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L. & Luo, L. A global double-fluorescent Cre reporter mouse. Genesis 45, 593–605 (2007).
Hartmann, D.A. et al. Pericyte structure and distribution in the cerebral cortex revealed by high-resolution imaging of transgenic mice. Neurophotonics 2, 041402 (2015).
Shih, A. Y. et al. Two-photon microscopy as a tool to study blood flow and neurovascular coupling in the rodent brain. J. Cereb. Blood Flow. Metab. 32, 1277–1309 (2012).
Chen, M. et al. Simply combining fasudil and lipoic acid in a novel multitargeted chemical entity potentially useful in central nervous system disorders. RSC Adv. 5, 37266–37269 (2014).
Shin, H. K. et al. Rho-kinase inhibition acutely augments blood flow in focal cerebral ischemia via endothelial mechanisms. J. Cereb. Blood Flow. Metab. 27, 998–1009 (2007).
Asano, T. et al. Mechanism of action of a novel antivasospasm drug, HA1077. J. Pharmacol. Exp. Ther. 241, 1033–1040 (1987).
Roth, T. L. et al. Transcranial amelioration of inflammation and cell death after brain injury. Nature 505, 223–222 (2014).
Bedussi, B. et al. Clearance from the mouse brain by convection of interstitial fluid towards the ventricular system. Fluids Barriers CNS 12, 23 (2015).
Rodrigues, A. J., Evora, P. R. & Schaff, H. V. Protective effect of N-acetylcysteine against oxygen radical-mediated coronary artery injury. Braz. J. Med. Biol. Res. 37, 1215–1224 (2004).
Drew, P. J. et al. Chronic optical access through a polished and reinforced thinned skull. Nat. Methods 7, 981–984 (2010).
Shih, A. Y., Mateo, C., Drew, P. J., Tsai, P. S. & Kleinfeld, D. A polished and reinforced thinned skull window for long-term imaging and optical manipulation of the mouse cortex. J. Vis. Exp. 7, 3742 (2012).
Driscoll, J. D., Shih, A. Y., Drew, P. J., Cauwenberghs, G. & Kleinfeld, D. Two-photon imaging of blood flow in cortex. Cold Spring Harb. Protoc. 8, 759–767 (2013).
Podgorski, K. & Ranganathan, G. Brain heating induced by near-infrared lasers during multiphoton microscopy. J. Neurophysiol. 116, 1012–1023 (2016).
Roche, M. et al. In vivo imaging with a water immersion objective affects brain temperature, blood flow and oxygenation. eLife 8, e47324 (2019).
Xu, Q., Huff, L. P., Fujii, M. & Griendling, K. K. Redox regulation of the actin cytoskeleton and its role in the vascular system. Free Radic. Biol. Med. 109, 84–107 (2017).
Drew, P. J., Blinder, P., Cauwenberghs, G., Shih, A. Y. & Kleinfeld, D. Rapid determination of particle velocity from space-time images using the Radon transform. J. Comput. Neurosci. 29, 5–11 (2010).
Ke, M. T., Fujimoto, S. & Imai, T. SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction. Nat. Neurosci. 16, 1154–1161 (2013).
Emmenlauer, M. et al. XuvTools: free, fast and reliable stitching of large 3D datasets. J. Microsc. 233, 42–60 (2009).
Acknowledgements
Our work is supported by grants to A.Y.S. from the NIH/NINDS (grant nos. NS106138, NS097775) and the NIH/NIA (AG063031, AG062738), by the American Heart Association (grant no. 14GRNT20480366), by the Alzheimer’s Association NIRG award (grant no. 2016-NIRG-397149) and by an Institutional Development Award (IDeA) from the NIGMS under grant no. P20GM109040. D.A.H. is supported by awards from the NIH/NCATS (grant nos. UL1 TR001450 and TL1 TR001451) and by NIH/NINDS grant no. F30NS096868. We thank J. Costello for contributions to image analysis. We appreciate the helpful comments and discussion of M. Levy, D. Kleinfeld, A. Riegel, P. Kara and N. Bhat.
Author information
Authors and Affiliations
Contributions
Experiments were designed by A.Y.S., D.A.H. and A.-A.B. Experiments were conducted by D.A.H., A.-A.B., R.I.G., S.A.H., T.K. and A.Y.S. Data analysis was performed by D.A.H., A.-A.B., S.A.H., T.K., T.T., K.P.M. and A.Y.S. Statistics were performed by A.L.K., A.V.F. and D.A.H. The manuscript was written by A.Y.S. and D.A.H. with contributions from all authors.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Peer review information Nature Neuroscience thanks David Bennett, Turgay Dalkara and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Supplementary Information
Supplementary Figs. 1–24.
Supplementary Video 1
Line-scan data from ChR2-YFP mouse.
Supplementary Video 2
Line-scan data from YFP control mouse.
Supplementary Video 3
Hypercapnia-induced dilation of pre-capillary arterioles and capillaries.
Supplementary Video 4
Optogenetically induced constriction of pre-capillary arteriole followed by relaxation.
Supplementary Video 5
Optogenetically induced constriction of pre-capillary arteriole followed by relaxation (second example).
Supplementary Video 6
Optogenetically induced constriction of capillary followed by relaxation.
Supplementary Video 7
Optogenetically induced constriction of capillary followed by relaxation (second example).
Supplementary Video 8
Absence of precapillary arteriole constriction in YFP control mouse.
Supplementary Video 9
Absence of capillary constriction in YFP control mouse.
Supplementary Data 1
All data used to generate plots in the supplementary files and associated statistical analyses.
Source data
Source Data Fig. 1
Source data for Fig. 1.
Source Data Fig. 2
Source data and statistical details for Fig. 2.
Source Data Fig. 3
Source data and statistical details for Fig. 3.
Source Data Fig. 4
Source data and statistical details for Fig. 4.
Source Data Fig. 5
Source data and statistical details for Fig. 5.
Source Data Fig. 6
Source data and statistical details for Fig. 6.
Source Data Fig. 7
Source data and statistical details for Fig. 7.
Rights and permissions
About this article
Cite this article
Hartmann, D.A., Berthiaume, AA., Grant, R.I. et al. Brain capillary pericytes exert a substantial but slow influence on blood flow. Nat Neurosci 24, 633–645 (2021). https://doi.org/10.1038/s41593-020-00793-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41593-020-00793-2
- Springer Nature America, Inc.
This article is cited by
-
Induced pluripotent stem cell derived pericytes respond to mediators of proliferation and contractility
Stem Cell Research & Therapy (2024)
-
Cellular and molecular mechanisms of the blood–brain barrier dysfunction in neurodegenerative diseases
Fluids and Barriers of the CNS (2024)
-
Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma
Journal of Experimental & Clinical Cancer Research (2024)
-
Distal activity patterns shape the spatial specificity of neurovascular coupling
Nature Neuroscience (2024)
-
Neurovascular coupling and CO2 interrogate distinct vascular regulations
Nature Communications (2024)