The microvascular inflow tract, comprising the penetrating arterioles, precapillary sphincters and first-order capillaries, is the bottleneck for brain blood flow and energy supply. Exactly how aging alters the structure and function of the microvascular inflow tract remains unclear. By in vivo four-dimensional two-photon imaging, we reveal an age-dependent decrease in vaso-responsivity accompanied by a decrease in vessel density close to the arterioles and loss of vascular mural cell processes, although the number of mural cell somas and their alpha smooth muscle actin density were preserved. The age-related reduction in vascular reactivity was mostly pronounced at precapillary sphincters, highlighting their crucial role in capillary blood flow regulation. Mathematical modeling revealed impaired pressure and flow control in aged mice during vasoconstriction. Interventions that preserve dynamics of cerebral blood vessels may ameliorate age-related decreases in blood flow and prevent brain frailty.
This is a preview of subscription content, access via your institution
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 digital issues and online access to articles
$119.00 per year
only $9.92 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
The source data and custom code are available in public repository G-Node: https://doi.org/10.12751/g-node.zcg4cy/.
Aanerud, J. et al. Brain energy metabolism and blood flow differences in healthy aging. J. Cereb. Blood Flow Metab. 32, 1177–1187 (2012).
Moeini, M. et al. Compromised microvascular oxygen delivery increases brain tissue vulnerability with age. Sci. Rep. 8, 8219 (2018).
De Silva, T. M. & Faraci, F. M. Contributions of aging to cerebral small vessel disease. Annu. Rev. Physiol. 82, 275–295 (2020).
Chen, J. J. Functional MRI of brain physiology in aging and neurodegenerative diseases. NeuroImage 187, 209–225 (2019).
Esopenko, C. & Levine, B. Aging, neurodegenerative disease, and traumatic brain injury: the role of neuroimaging. J. Neurotrauma 32, 209–220 (2015).
Chen, R.-L., Balami, J. S., Esiri, M. M., Chen, L.-K. & Buchan, A. M. Ischemic stroke in the elderly: an overview of evidence. Nat. Rev. Neurol. 6, 256–265 (2010).
Bell, R. D. et al. Pericytes control key neurovascular functions and neuronal phenotype in the adult brain and during brain aging. Neuron 68, 409–427 (2010).
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 (2019).
Hall, C. N. et al. Capillary pericytes regulate cerebral blood flow in health and disease. Nature 508, 55–60 (2014).
Grubb, S. et al. Precapillary sphincters maintain perfusion in the cerebral cortex. Nat. Commun. 11, 395 (2020).
Zambach, S. A. et al. Precapillary sphincters and pericytes at first-order capillaries as key regulators for brain capillary perfusion. Proc. Natl Acad. Sci. USA 118, e2023749118 (2021).
West, K. L. et al. BOLD hemodynamic response function changes significantly with healthy aging. NeuroImage 188, 198–207 (2019).
Diaz-Otero, J. M., Garver, H., Fink, G. D., Jackson, W. F. & Dorrance, A. M. Aging is associated with changes to the biomechanical properties of the posterior cerebral artery and parenchymal arterioles. Am. J. Physiol. Heart Circ. Physiol. 310, H365–H375 (2016).
Fonck, E. et al. Effect of aging on elastin functionality in human cerebral arteries. Stroke 40, 2552–2556 (2009).
Bolton, T. B. Mechanisms of action of transmitters and other substances on smooth muscle. Physiol. Rev. 59, 606–718 (1979).
Boswell-Smith, V., Spina, D. & Page, C. P. Phosphodiesterase inhibitors. Br. J. Pharmacol. 147, S252–S257 (2006).
Chappie, T. A., Helal, C. J. & Hou, X. Current landscape of phosphodiesterase 10A (PDE10A) inhibition. J. Med. Chem. 55, 7299–7331 (2012).
Mandalà, M., Pedatella, A. L., Morales Palomares, S., Cipolla, M. J. & Osol, G. Maturation is associated with changes in rat cerebral artery structure, biomechanical properties and tone. Acta Physiol. 205, 363–371 (2012).
Toth, P. et al. Resveratrol treatment rescues neurovascular coupling in aged mice: role of improved cerebromicrovascular endothelial function and downregulation of NADPH oxidase. Am. J. Physiol. Heart. Circ. Physiol. 306, H299–H308 (2014).
Faraci, F. M., Brian, J. E. Jr & Heistad, D. D. Response of cerebral blood vessels to an endogenous inhibitor of nitric oxide synthase. Am. J. Physiol. 269, H1522–H1527 (1995).
Davenport, A. P. et al. Endothelin. Pharmacol Rev. 68, 357–418 (2016).
Bennett, H. C. & Kim, Y. Pericytes across the lifetime in the central nervous system. Front. Cell Neurosci. 15, 627291 (2021).
Ximerakis, M. et al. Single-cell transcriptomic profiling of the aging mouse brain. Nat. Neurosci. 22, 1696–1708 (2019).
Kirst, C. et al. Mapping the fine-scale organization and plasticity of the brain vasculature. Cell 180, 780–795 (2020).
Young, A. P. et al. Endothelin B receptor dysfunction mediates elevated myogenic tone in cerebral arteries from aged male Fischer 344 rats. Geroscience 43, 1447–1463 (2021).
Springo, Z. et al. Aging impairs myogenic adaptation to pulsatile pressure in mouse cerebral arteries. J. Cereb. Blood Flow Metab. 35, 527–530 (2015).
Toth, P. et al. Age-related autoregulatory dysfunction and cerebromicrovascular injury in mice with angiotensin II-induced hypertension. J. Cereb. Blood Flow Metab. 33, 1732–1742 (2013).
Ranki, H. J., Crawford, R. M., Budas, G. R. & Jovanovic, A. Ageing is associated with a decrease in the number of sarcolemmal ATP-sensitive K+ channels in a gender-dependent manner. Mech. Ageing Dev. 123, 695–705 (2002).
Tricarico, D. & Camerino, D. C. ATP-sensitive K+ channels of skeletal muscle fibers from young adult and aged rats: possible involvement of thiol-dependent redox mechanisms in the age-related modifications of their biophysical and pharmacological properties. Mol. Pharmacol. 46, 754–761 (1994).
Taddei, S. et al. Age-related reduction of NO availability and oxidative stress in humans. Hypertension 38, 274–279 (2001).
Donato, A. J. et al. Vascular endothelial dysfunction with aging: endothelin-1 and endothelial nitric oxide synthase. Am. J. Physiol. Heart. Circ. Physiol. 297, H425–H432 (2009).
Van Der Loo, B. et al. Enhanced peroxynitrite formation is associated with vascular aging. J. Exp. Med. 192, 1731–1744 (2000).
Cernadas, M. A. R. et al. Expression of constitutive and inducible nitric oxide synthases in the vascular wall of young and aging rats. Circ. Res. 83, 279–286 (1998).
Van Guilder, G. P., Westby, C. M., Greiner, J. J., Stauffer, B. L. & Desouza, C. A. Endothelin-1 vasoconstrictor tone increases with age in healthy men but can be reduced by regular aerobic exercise. Hypertension 50, 403–409 (2007).
Del Ry, S., Maltinti, M., Giannessi, D., Cavallini, G. & Bergamini, E. Age-related changes in endothelin-1 receptor subtypes in rat heart. Exp. Aging Res. 34, 251–266 (2008).
Ungvari, Z., Tarantini, S., Donato, A. J., Galvan, V. & Csiszar, A. Mechanisms of vascular aging. Circ. Res. 123, 849–867 (2018).
Castelli, V. et al. Neuronal cells rearrangement during aging and neurodegenerative disease: metabolism, oxidative stress and organelles Dynamic. Front. Mol. Neurosci. 12, 132 (2019).
Banks, W. A., Reed, M. J., Logsdon, A. F., Rhea, E. M. & Erickson, M. A. Healthy aging and the blood–brain barrier. Nature Aging 1, 243–254 (2021).
Nikolakopoulou, A. M. et al. Pericyte loss leads to circulatory failure and pleiotrophin depletion causing neuron loss. Nat. Neurosci. 22, 1089–1098 (2019).
We acknowledge Niels Bohr Institute Electronics & Mechanical Workshop, University of Copenhagen, where we receive great technical support from S. Stausgaard-Petersen, J. H. Fagerlund and their colleagues. We acknowledge the Core Facility for Integrated Microscopy, Faculty of Health and Medical Sciences, University of Copenhagen, where we used spinning disc confocal microscopy in our in vitro studies. This study was supported by the Lundbeck Foundation (R273-2017-1791 and R345-2020-1440), the Danish Medical Research Council (1133-00016A), the Alice Brenaa Foundation, Augustinus Foundation (19-2858), Carl og Ellen Hertz Familielegat (19.19.2), A. P. Møller Foundation (20-L-0243), Helsefonden (21-B-0441), the Novo Nordisk foundation (0064289) and a Nordea Foundation Grant to the Center for Healthy Aging (02-2017-1749).
The authors declare no competing interests.
Peer review information
Nature Aging thanks Stephanie Bonney, Yongsoo Kim and Anusha Mishra 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.
Extended Data Fig. 1 Comparison of absolute diameter changes, cross-sectional area changes and peak diameters by different stimulations in adult and old mice.
(a–c) Absolute diameter changes, cross-sectional area changes and peak diameters and peak diameters of vasodilation by whisker pad stimulation (WP stim). Adult: N = 17 animals, n = 34 vessels. Old: N = 17 animals, n = 25 vessels. (d–f) Absolute diameter changes, cross-sectional area changes and peak diameters and peak diameters of vasodilation by KATP channel opener pinacidil puff. Adult: N = 9 animals, n = 15 vessels. Old: N = 11 animals, n = 17 vessels. (g–i) Absolute diameter changes, cross-sectional area changes and peak diameters and peak diameters of vasodilation by papaverine puff. Adult: N = 8 animals, n = 17 vessels. Old: N = 5 animals, n = 8 vessels. (j–l) Absolute diameter changes, cross-sectional area changes and peak diameters and peak diameters of vasoconstriction by L-NAME intravenous infusion (4 min post). Adult: N = 5 animals, n = 5 vessels. Old: N = 8 animals, n = 8 vessels. (m–o) Absolute diameter changes, cross-sectional area changes and peak diameters and peak diameters of vasoconstriction by ET1 puff. Adult: N = 5 animals, n = 10 vessels. Old: N = 7 animals, n = 15 vessels. Linear mixed effect models were used to test for differences among vessel segments, followed by Tukey post hoc tests for pairwise comparisons. Data are given as mean ± SEM. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.
The control puff experiments were performed with the same concentration of fluorescent dye but without an active compound. The initial ‘constricting’ artifact can be perfectly overlaid by both control puff and pinacidil puff, using the time of puffing as a time lock and normalizing the negative peak at the moment of puffing. (a) Vessel diameter change by control puff. (b) Vessel diameter change by pinacidil puff. (c-f) Overlay of the vessel diameter change curves at each capillary order, with (c) as penetrating arteriole, (d) as 1st order capillary, (e) as 2nd order capillary and (f) as 3rd order capillary. Data are given as mean ± SEM.
Extended Data Fig. 3 Comparison of vasodilation elicited by papaverine puff, pinacidil puff, and whisker pad (WP) stimulation.
(a–b) Relative diameter change elicited by the three stimulations in (a) adult and (b) old mice. (c–d) Absolute diameter change elicited by the three stimulations in (c) adult and (d) old mice. (e–f) Peak diameter elicited by the three stimulations in (e) adult and (f) old mice. WP stimulation: Adult: N = 31 animals, n = 56 vessels. Old: N = 26 animals, n = 53 vessels. Pinacidil: Adult: N = 9 animals, n = 15 vessels. Old: N = 11 animals, n = 17 vessels. Papaverine: Adult: N = 8 animals, n = 17 vessels. Old: N = 5 animals, n = 8 vessels. Linear mixed effect models were used to test for differences among vessel segments, followed by Tukey post hoc tests for pairwise comparisons. Data are given as mean ± SEM. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.
Extended Data Fig. 4 Representative immunohistochemical images of staining CD31 and Hoechst in the adult and old mouse brains.
This is to identify the vascular endothelial cell somata. Maximum intensity projection of an (left) adult and (right) old brain image stack. Red: NG2; Green: CD31; Gray: Hoechst. Scale bar: 20 μm.
(a) Image processing procedure for the z-stack was to free-hand draw the background area and then calculate the mean background fluorescence intensity for each image plane. The background intensity was normalized to the peak value of the z-stack. Finally, the z-stack images were projected onto one image by maximal projection. (b) The semi-manual customized code is to identify the whole vessel surface and pericyte-covered area. Upper: Hand-drawn delineation of the vessel region in the green and red imags. Lower: The NG2 image was used to select the mural cell-positive pixels. (c) Non-vascular nucleus volumetric density. The non-vascular nucleus is defined as nuclei with a distance to the nearby vessels. (d) Endothelial cell soma density obtained by dividing the number of endothelial cell somas and the examined vessel length. Adult: N = 3 animals, n = 25 vessels. Old: N = 3 animals, n = 26 vessels. Linear mixed effect models were used to test for differences among vessel segments, followed by Tukey post hoc tests for pairwise comparisons. Data are given as mean ± SEM. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.
(a–c) Total vessel number per brain volume in adult and old somatosensory cortex. (a) General total vessel number, (b) capillary order dependent total vessel number, and (c) cortical depth dependent total vessel number. (d–f) Mean vessel length in adult and old somatosensory cortex. (d) General mean vessel length, (e) capillary order dependent mean vessel length, and (f) cortical depth dependent mean vessel length. (g–i) Total vessel volume per brain volume in adult and old somatosensory cortex. (g) General total vessel volume, (h) capillary order dependent total vessel volume, and (i) cortical depth dependent total vessel volume. Adult: N = 6 animals, n = 52 vessels. Old: N = 4 animals, n = 29 vessels. Linear mixed effect models were used to test for differences among vessel segments, followed by Tukey post hoc tests for pairwise comparisons. Data are given as mean ± SEM. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001. L1 – L5 denote cortical sublayers.
Extended Data Fig. 7 Math modeling indicates that vascular blood flow and pressure are affected by aging.
(a–b) Mathematical modeling of pressure distribution in 3D reconstructed vasculature from 3 adult (a) and 3 old (b) mouse brains. (c) Summary of the averaged pressures in penetrating arteriole (PAs), pre-capillary sphincters, and first- to third-order capillaries in the vascular networks under resting-state, with whisker pad stimulation (WPstim), and after ET1 puff. (d) Correlation of mural cell coverage and blood pressure summarized for all three states. The dashed line connects the adult and old measurements at the same vascular segment for easy comparison. (e) Mathematical modeling of flow distribution in 3D reconstructed vasculature from 3 adult and 3 old mouse brains. Only one representative image from each group is shown. (f) Summary of the average flow in PAs, precapillary sphincters, and first- to third-order capillaries in the vascular networks under resting-state, WP stim, and after ET1 puff. Adult: N = 3 animals, n = 3 vessels; Old: N = 3 animals, n = 3 vessels. Linear mixed effect models were used to test for differences among vessel segments, followed by Tukey post hoc tests for pairwise comparisons. Data are given as mean ± SEM. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.
Extended Data Fig. 8 Diagram of vascular mural cell phenotypes at the microvascular inflow tract (MIT) and the mechanisms that contribute to changes in function and morphology with MIT aging.
(1) Vascular mural cell phenotypes at the MIT. Arteriole vascular smooth muscle cells (VSMCs) have spindle-shaped cell bodies. Like contractile bands, they cover the circumferences of the penetrating arterioles in a single layer. We previously reported the existence of precapillary sphincters displaying indentation of the vessel lumen and strong expression of αSMA at 50% of the joints between penetrating arterioles and first-order capillaries. They are important in maintaining and regulating capillary blood flow. Ensheathing pericytes cover capillaries up to the third order. Ensheathing pericytes have short longitudinal processes with dense circumferential offshoots woven together to create a meshed circumferential coverage of the capillary that covers 95% of the capillary endothelium. VSMCs, precapillary sphincters and ensheathing pericytes do express αSMA. A stepwise decrease in αSMA content is observed along the first, second, third, and up to the fourth order of capillary. This αSMA-positive arteriole-proximal capillary segment is termed the microvascular inflow tract (MIT)—here, the pericyte coverage across capillary junctions is ~90%; further downstream, the capillary junctional pericyte coverage is ~45%. Mesh pericytes exhibit mesh-like morphology and are found downstream of ensheathing pericytes and the αSMA terminus; they have lesser endothelial coverage (71.6%) than ensheathing pericytes, and their longitudinal processes are longer and have a less dense network of circumferential offshoots. In higher-order capillaries, thin-strand pericytes display thin and long longitudinal processes, with only a few circumferential offshoots that extend along and cover 51.3% of the capillary endothelium. (2) Changes in morphology and signaling investigated in this study contribute to vascular aging at the MIT. The resting-state diameter of vessels at the MIT increases with age, but the coverage by mural cells decreases. KATP channels are downregulated and are less densely expressed by aging. The probability of KATP-channel opening is also reduced with age. NO production and bioavailability maybe reduced by aging, and our results suggest that ongoing NO-dependent mechanisms are attenuated in old mice. Although ET1 signaling and synthesis are increased in old relative to adult mice, ETA receptors are downregulated with age. Furthermore, aging is accompanied by fragmented elastin, deposition of collagens, and vessel stiffness. All the above age-related changes at the MIT contribute to reduced responsivity to both vasodilators and vasoconstrictors, indicating the existence of a compensating mechanism to keep cerebral blood flow and oxygen supply at a physiologically healthy level with healthy aging.
The joint of 1st and 2nd order capillary in Fig. 3a can be identified by examining individual planes.
About this article
Cite this article
Cai, C., Zambach, S.A., Grubb, S. et al. Impaired dynamics of precapillary sphincters and pericytes at first-order capillaries predict reduced neurovascular function in the aging mouse brain. Nat Aging 3, 173–184 (2023). https://doi.org/10.1038/s43587-022-00354-1