Abstract
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.
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Data availability
The source data and custom code are available in public repository G-Node: https://doi.org/10.12751/g-node.zcg4cy/.
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Acknowledgements
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).
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C.C., S.A.Z. and M.J.L. designed research; S.A.Z., C.C., S.G., L.T., C.H., K.J.T., X.Z., B.O.H., R.M.N. and M.L. performed research; C.C. and K.K. contributed new reagents/analytic tools; S.A.Z., C.C., S.G. and B.K.L. analyzed data; S.A.Z., C.C., A.D. and M.J.L. wrote the paper.
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Extended data
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.
Extended Data Fig. 2 Comparison of control puff and pinacidil puff.
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.
Extended Data Fig. 5 Immunohistochemical analysis of mural cell structural change with age.
(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.
Extended Data Fig. 6 In vivo vascular structural changes with aging.
(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.
Extended Data Fig. 9 Max projection and individual planes in Fig. 3a.
The joint of 1st and 2nd order capillary in Fig. 3a can be identified by examining individual planes.
Supplementary information
Supplementary Table 1
P values of all the comparisons in the figures and Extended Data figures.
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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
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DOI: https://doi.org/10.1038/s43587-022-00354-1
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