Brain capillary pericytes exert a substantial but slow influence on blood flow

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.

Figure 1:. Capillary pericytes contact the vast majority of mouse cerebrovasculature.

a,b, Thick tissue section from cerebral cortex shows genetically-labeled mural cells (PDGFRβ-tdTomato) in green and α-SMA immunostaining in red. c, Proportion of vascular length associated with α-SMA-positive mural cells (SMCs and ensheathing pericytes) versus α-SMA low or undetectable mural cells (capillary pericytes). A total of 138 mm of cortical vascular length was quantified across 4 volumetric data sets from 2 mice. d, Drop in α-SMA expression as pre-capillary arterioles transition into capillaries. The inset shows a penetrating arteriole offshoot with α-SMA termination occurring at the second order branch. e, Histogram showing proportion of branch orders at which α-SMA decrease occurs in the upper 350 μm of cortex. N = 24 α-SMA termini (2 mice, 11 penetrating arteriole networks). f, Schematic depicting microvascular zones, with SMCs on pial and penetrating arterioles (red), ensheathing pericytes on pre-capillary arterioles (yellow), capillary pericytes on capillaries (green), and stellate SMCs on venules (blue). Expression of α-SMA decreases sharply between pre-capillary arteriole and capillary zones. g,h, Contractile ability is established for ensheathing pericytes, but remains uncertain for capillary pericytes.

Figure 2:. Optogenetic activation of capillary pericytes constricts capillaries and reduces blood flow.

a, In vivo two-photon image from PDGFRβ-ChR2-YFP mouse with microvascular branch orders. b, Two-photon line-scans (red) used to simultaneously stimulate ChR2-YFP and measure capillary vasodynamics. c, Two-photon excitation of ChR2-YFP allows focal activation of pericytes below the brain surface. d, Example of capillary pericyte in ChR2-YFP mouse with line-scan path. Colored segments correspond to the same colored regions at the top of panel (e). e, Line-scan data resulting from repeatedly scanning the path shown in panel (d). Upper image shows beginning of line-scan, and lower image shows the end of a continuous 60 s scan. f, Left: Example of diameter portion of line-scan over 60 s of stimulation, comparing YFP control and ChR2-YFP. Dotted yellow lines demarcate diameter at baseline. Right: Time-course of diameter change during stimulation of capillaries. ChR2-YFP: n=155 capillaries (10 mice); controls: n=145 capillaries (9 mice). g, Left: Example of blood flow portion of line-scan. Right: Time-course for RBC velocity change during stimulation of capillaries. ChR2-YFP: n=160 capillaries (10 mice); controls: n=152 capillaries (9 mice). h, Change in RBC velocity at 60 s relative to baseline. F(1,290)=26.8, ***p<0.0001, one-way repeated-measures ANOVA (two-tailed). ChR2-YFP: n=157 capillaries (10 mice); controls: n=152 capillaries (9 mice). i, Time-course of RBC flux change during stimulation of capillaries. j, Change RBC flux at 60 s relative to baseline. F(1,186)=42.48, ***p<0.0001 by one-way repeated-measures ANOVA (two-tailed). ChR2-YFP: n=118 capillaries (10 mice); controls: n=87 (9 mice) capillaries. All time-course and scatter plots in this figure show mean ± S.E.M.

Figure 3:. Topical administration of fasudil prevents hemodynamic changes induced by optogenetic stimulation of capillary pericytes.

a-c, Left column: Fasudil inhibits reduction in capillary diameter (a), RBC velocity (b), and RBC flux (c) during optogenetic stimulation of capillary pericytes in ChR2-YFP mice. Right column: Change in diameter (a), RBC velocity (b), and RBC flux (c) at 60 s of stimulation relative to baseline. P values are from one-way repeated-measures ANOVA (two-tailed) adjusted by Tukey post hoc test (overall ANOVA test p values <0.015 for a-c; F(2,174)=6.63, for panel c; other panels had higher F values). (a) Capillary diameter: p=0.7146 (vehicle vs. 1mM fasudil), *p=0.0098 (vehicle vs. 10mM fasudil), p=0.1855 (1mM fasudil vs. 10mM fasudil). Vehicle: n=154 (10 mice); 1mM fasudil: n=60 (3 mice); 10mM fasudil: n=60 (3 mice). (b) RBC velocity: **p=0.008 (vehicle vs. 1mM fasudil), *p=0.0181 (vehicle vs. 10mM fasudil), p=0.9679 (1mM fasudil vs. 10mM fasudil). Vehicle: n=157 (10 mice); 1mM fasudil: n=57 (3 mice); 10mM fasudil: n=61 (3 mice). (c) RBC flux: p=0.118 (vehicle vs. 1mM fasudil), **p=0.0021 (vehicle vs. 10mM fasudil), p=0.3654 (1mM fasudil vs. 10mM fasudil). Vehicle: n=118 (10 mice); 1mM fasudil: n=45 (3 mice); 10mM fasudil: n=27 (3 mice). All time-course and scatter plots show mean ± S.E.M.

Figure 4:. Distinct contractile dynamics of mural cells in different microvascular zones.

a-d, Change in diameter during optogenetic stimulation of mural cells in different microvascular zones. Pial arterioles (a): n=25 (5 ChR2-YFP mice), n=26 (4 control mice); pre-capillary arterioles (b): n=43 (10 ChR2-YFP mice), n=33 (9 control mice); capillaries (c): n=155 (10 ChR2-YFP mice), 145 (9 control mice); venules (d): n=15 (4 ChR2-YFP mice), n=16 (3 control mice). e, Diameter change from baseline at 60 s of stimulation (25 s for arteriole SMC). For vessel type (statistics not shown on graph): F(3,429)=16.51, overall effect ***p<0.0001; ***p<0.0001 aSMC vs EP, ***p<0.0001 aSMC vs CP, ***p<0.0001 aSMC vs vSMC, p=0.66 EP vs CP, **p=0.007 EP vs vSMC, *p=0.02 CP vs vSMC. For genotype (ChR2 vs control): F(1,429)=21.53, overall effect ***p<0.0001, ***p<0.0001 for aSMC, **p=0.0017 for EP, *p=0.029 for CP, p=0.9986 for vSMC. For interaction between vessel type and genotype: F(3,429)=11.92, ***p<0.0001. Two-way repeated-measures ANOVA (two-tailed). Tukey post hoc analyses were used to compare between vessel types or genotypes. Same n as panel (a). f, Histogram showing distribution of baseline diameters for different vessel types. Pial arterioles: n=155 (10 mice); pre-capillary arterioles: n=43 (10 mice), capillaries: n=25 (5 mice). g, Left: Absolute diameter change from baseline for capillaries (green), pre-capillary arterioles (yellow), and arterioles (red) during optogenetic stimulation of vessels in ChR2-YFP mice. Right: Magnified view of differences in early constriction phase. h, Rate of diameter change during first 10 s of optogenetic stimulation of pre-capillary arterioles and capillaries. F(1,186)=37.86, ***p<0.0001, one-way repeated-measures ANOVA (two-tailed). Pre-capillary arterioles: n=43 (9 mice); capillaries: n=155 (10 mice). i, Top: Experimental time-course for hypercapnia studies. Bottom: Change in pre-capillary arteriole and capillary diameter at the end of the hypercapnic phase (hypercap), and 9 min after return to normocapnia (recovery). Pre-capillary: n=27 (5 mice); capillary: n= 51 (5 mice). j, Time-course of vessel diameter change following transition from hypercapnia to normocapnia. Same n as panel (i). k, Rate of diameter change after hypercapnia. ***p<0.001, linear mixed effects modeling (two-tailed). Same n as panel (i). All time-course data and scatter plots in this figure show mean ± S.E.M.

Figure 5:. Dilation after optogenetic vasoconstriction is faster in pre-capillary arterioles than capillaries.

a,b, Example of vessel constriction followed by post-stimulus relaxation with optogenetic stimulation of ensheathing pericytes (a) vs. capillary pericytes (b). c,d, Time-course of change in pre-capillary arteriole and capillary diameter during and after optogenetic stimulation in PDGFRβ-ChR2-YFP mice (c) and PDGFRβ-YFP controls (d). PDGFRβ-ChR2-YFP: Ensheathing pericyte: n=20 (6 mice); capillary pericyte: n=49 (6 mice). PDGFRβ-YFP (control): Ensheathing pericyte: n=13 (3 mice); capillary pericyte: n=61 (3 mice). e, Change in absolute vessel diameter in the first 80 s of post-stimulus relaxation for PDGFRβ-ChR2-YFP mice. Same n as in panel (c). f, Rate of diameter change after optogenetic stimulation. F(72)=197.73, ***p<0.001 by linear mixed effects modeling (two-tailed). Ensheathing pericyte: n=20 (6 mice); capillary pericytes n=49 (6 mice). All time-course and scatter plots in this figure show mean ± S.E.M.

Figure 6.. Ablation of individual capillary pericytes produces local capillary dilation and increased RBC flux.

a, Before and 3 days after ablation of two capillary pericytes (arrowheads) in a PDGFRβ-mT/mG mouse. b, Sham controls involve identical scan parameters as for cell ablation, but with the laser focus away from the pericyte somata. c,d, Magnified image of the boxed region in panel (a), showing precise location of laser irradiation (yellow line). Line-scans for assessment of capillary vasodynamics in the adjoined capillary segment before and after pericyte ablation. e,f, Magnified image of boxed region in panel (b) and corresponding line-scan data. g-i, Fold change in capillary diameter and vasodynamic parameters 3 days following pericyte ablation or sham irradiation. For panel (g), F(1,95)=21.46, ***p<0.0001 by one-way repeated-measures ANOVA (two-tailed); ablation: n=45 capillaries (6 mice); sham: n=57 capillaries (6 mice). For panel (h), F(1,88)=1.3, p=0.26 by one-way repeated-measures ANOVA (two-tailed); ablation: n=41 capillaries (6 mice); sham: n=54 capillaries (6 mice). For panel (i), F(1,81)=9.79, **p=0.002 by one-way repeated-measures ANOVA (two-tailed); ablation: n=39 capillaries (6 mice); sham: n=49 capillaries (6 mice). j, Cumulative distribution plot of RBC flux at baseline and 3-days post-ablation. A typical range for capillary RBC flux based on past literature is provided for comparison (see text for references). N=42 capillaries (6 mice) k, Schematic showing the ablation of a bridging pericyte. l, Top: A bridging pericyte targeted for ablation (yellow line across soma) with process reaching across the parenchyma to contact a distant vessel (arrowhead). Bottom: After ablation, pericyte contact is lost on two vessels, proximal and distal to the ablated soma. m, Change in capillary diameter as a consequence of bridging pericyte ablation. “Off-territory” is a neighboring capillary that retains pericyte coverage. P=0.9501 (proximal vs distal), ***p=0.002 (proximal vs off-territory), ***p=0.0007 (distal vs off-territory); Kruskal-Wallis test: X²(2)=19.45 overall, with adjusted p values shown after Tukey post hoc test. N=10 bridging pericyte ablations (7 mice).

Figure 7.. Pericytes control heterogeneity of basal capillary vasodynamics

a, Images of i.v. dye channel collected 3 days apart from the same cortical region. One small diameter capillary and one larger diameter capillary are highlighted. b-d, Scatterplots of capillary vasodynamic parameters collected at baseline vs. 3 days. Pearson’s correlation: Lumen diameter: R²=0.62, ***p<0.001; n=57 capillaries (6 mice), RBC velocity: R²=0.39, ***p<0.001; n=56 capillaries (6 mice), and RBC flux: R²=0.17, **p<0.01; n=49 capillaries (6 mice). e,f, Example images and line-scan data collected before and 3 days following capillary pericyte ablation. A small baseline diameter capillary (e) and large baseline diameter capillary (f) is shown. g,h, Fold change in capillary diameter (g) and RBC flux (h) in small and large diameter capillaries after pericyte ablation. For (g), small baseline diameter: F(1,44)=11.51, **p=0.0015, ablation: n=25 (6 mice), sham: n=26 (6 mice); large baseline diameter: F(1,44)=6.28, *p=0.016, ablation: n=20 (6 mice), sham: n=31 (6 mice), by repeated-measures ANOVA (two-tailed). For (h), small baseline diameter: F(1,42)=6.83, *p=0.0124, ablation: n=24 (6 mice), sham: n=25 (6 mice); large baseline diameter: p=0.14, ablation: n=15 (6 mice), sham: n=24 (6 mice) by one-way repeated-measures ANOVA (two-tailed). All data shown as mean ± S.E.M.