Supplementary Figure 1 The average sigmoid parametric curves of capillary dilation time courses and average time to 50% peak capillary diameter dilation computed from individual capillary responses averaged per animal in control Pdgfrb +/+ mice (top, red traces, n = 12 mice, 37 individual capillaries) and pericyte-deficient Pdgfrb +/ mice (bottom, blue traces, n = 9 mice, 33 individual capillaries). The time to 50% peak dilation corresponds to the time at which the sigmoid curve reaches 50% of its maximal value, as illustrated by the intercepts of the horizontal dashed line at 0.5 on the ordinate with the curves (diameter increase) and the vertical dashed line intercepts with the abscissa (time to 50% peak dilation). Arrows below the abscissa illustrate time intercepts showing 50% peak capillary diameter dilation per mouse in each group. As in Fig. 1c, diameter changes are expressed relative to the respective basal capillary diameter prior to stimulation (value set as 0) and maximal diameter after stimulation (value set as 1). The average sigmoid curves were derived from individual sigmoid curves fitted to individual capillary responses per animal. The sigmoid fits for individual capillary responses with their intercepts with the horizontal dashed line at 0.5 on the ordinate (diameter increase) and the vertical dashed line intercepts (time to 50% peak dilation) with the abscissa are not shown because of the high degree of overlap, which makes difficult to distinguish responses between individual capillaries. However, data (circles) in Fig. 1d were derived from averaging the individual capillary responses per mouse (time to 50% peak dilation) from sigmoid fits of individual vessel dilation time courses per animal, and then the individual times for 50% peak dilation values were averaged per animal.
Supplementary Figure 2 Pericyte capillary coverage in 1- to 2-month-old Pdgfrb +/ mice and age-matched littermate controls. (a) Representative confocal microscopy images of pericyte coverage (CD13, magenta) of lectin-positive brain endothelial capillary profiles (< 6 m in diameter; lectin, blue) in the stimulated cortical S1 area in Pdgfrb +/+ and Pdgfrb +/ mice. (b) Quantification of CD13-positive pericyte coverage of cortical capillaries in Pdgfrb +/+ and Pdgfrb +/ mice from a. Mean ± 95% CI; n = 5 Pdgfrb +/+ and 6 Pdgfrb +/ mice per group (t-test, single tail, equal variance: t = 11.36, p = 6.1e-7). In each animal 5 randomly selected fields from the cortex were analyzed in 6 non-adjacent sections (~100 μm apart) and averaged per mouse to obtain individual values (circles) as illustrated. (c) Vascular smooth muscle cell actin (SM yellow) and pericyte marker CD13 in Pdgfrb +/+ control mice illustrating that pericytes lining lectin-positive endothelial capillary profiles are largely negative for SM An SM -positive arteriole (arrow) is shown for comparison.
Supplementary Figure 3 Baseline vessel diameters, RBC flow velocity and thickness of the arteriole smooth muscle cell layer in 1- to 2-month-old pericyte-deficient Pdgfrb +/ mice and age-matched littermate controls. (a) Average baseline in vivo vessel diameters (mean ± 95% CI) prior to stimulation determined for 21 total arterioles from 9 Pdgfrb +/+ mice, 27 total arterioles from 11 Pdgfrb +/ mice, 57 total capillaries from 10 Pdgfrb +/+ mice and 40 total capillaries from 10 Pdgfrb +/ mice (arterioles: t-test, equal variance: t = 0.27, p = 0.79; capillaries: t-test, equal variance: t = 1.04, p = 0.31). (b) Average (mean ± 95% CI) baseline in vivo RBC velocity acquired prior to stimulation in 14 total arterioles from 6 Pdgfrb +/+ mice and 23 total arterioles from 10 Pdgfrb +/ mice, and 32 total capillaries from 9 Pdgfrb +/+ mice and 36 total capillaries from 12 Pdgfrb +/ mice (ttest, equal variance: arterioles: t = 0.23 p = 0.82, capillaries: t = 0.33, p = 0.74). (c) Representative images of the smooth muscle cell layer (SMα, red) thickness and lack of pericyte coverage (CD13, magenta) on arterioles. (d) Thickness of the arteriolar smooth muscle cell layer determined by confocal microscopy analysis. Mean ± 95% CI from 42 arterioles from 7 Pdgfrb +/+ mice and 45 arterioles from 6 Pdgfrb +/ mice (t-test, equal variance: t = 1.14, p = 0.28).Two-tail t-tests used.
Supplementary Figure 4 Astrocyte coverage of capillary wall, astrocyte and microglia numbers and capillary length in 1- to 2- month-old pericyte-deficient Pdgfrb +/ mice and age-matched littermate controls. (a) Confocal microscopy of immunolabeled aquaporin-4-positive astrocytic endfoot coverage of microvessels (lectin), GFAP-positive astrocytes, and Iba1-positive microglia in the S1 cortical region. (b-d) Quantification (mean ± 95% CI) of aquaporin-4-positive (AQP4+) astrocytic endfoot coverage (t-test, equal variance: t = 0.24, p = 0.82) (b), the number of GFAP-positive astrocytes (t-test, equal variance: t = 0.93, p = 0.38) (c), and Iba1- positive microglia (t-test, equal variance: t = 0.26, p = 0.80) (d) from 5 Pdgfrb +/+ and 5 Pdgfrb +/ mice at 1-2 months of age; in each animal 6 randomly selected fields from the cortex were analyzed in 5 nonadjacent sections (~100 m apart) and data were averaged per mouse to obtain individual values (circles) as illustrated. (e) 3D reconstructions of 880 x 330 x 1000 m sections of Pdgfrb +/+ and Pdgfrb +/ vasculature in the S1 somatosensory cortex region. Scale bar = 200 m. Insets: Single 50 m thick slices horizontally through cortex cortex layer IV. (f) Comparison of vascular density with depth in cortex of Pdgfrb +/ and Pdgfrb +/+ mice for all vessels <35 m diameter (top; t-test, single tail, equal variance: t = 1.68, p = 0.07), and vascular density of capillaries (< 6 m diameter; t-test, single tail, equal variance: t = 1.77, p = 0.06) and larger vessels between 6-35 m (bottom; t-test, two tail, equal variance: t = 0.10, p = 0.92) measured in Pdgfrb +/ and Pdgfrb +/+ mice. Lines represent average values ± 95% CI from n = 4 mice per group. T-tests based on average vessel density through all cortical layers per mouse.
Supplementary Figure 5 Capillary length, pericyte coverage and vessel dilation in young Meox2 +/ mice. (a) Representative image of pericyte coverage (CD13, magenta; left) of lectin-positive brain endothelial capillary profiles (< 6 m in diameter; lectin, blue; middle) and overlay (right) in Meox2 +/- and Meox2 +/+ control mice. (b,c) Quantification (mean ± 95% CI) of total capillary length calculated from the length of lectinpositive endothelial profiles < 6 m in diameter (b, Mann-Whitney U test, single tail, p = 0.05), and pericyte coverage of lectin-positive brain endothelial capillary profiles (c, Mann-Whitney U test, p = 1.00) in 3 control Meox2 +/+ and 3 Meox2 +/- mice (b), and 5 control Meox2 +/+ and 3 Meox2 +/- mice (c). In each animal 6 randomly selected fields from the cortex were analyzed in 5 nonadjacent sections (~100 m apart) and data were averaged per mouse to obtain individual values (circles) as illustrated. (d, e) Average time (mean ± CI) to 50% peak capillary diameter (d, Mann-Whitney U test, p = 0.79) (d) or 50% peak arteriole diameter (e, Mann-Whitney U test, p = 0.55) was determined after an electrical hind limb stimulation (10 s, 10 Hz, 2 ms pulse duration) in capillaries from 7 control Meox2 +/+ (16 total capillaries) mice and 3 Meox2 +/- mice (15 total capillaries), and arterioles from 6 control Meox2 +/+ mice (13 total arterioles) and 3 Meox2 +/- mice (12 total arterioles). Sigmoid parametric fit analysis was performed as for figure 1 c-e. Bootstrapped in panels b-e.
Supplementary Figure 6 Lactate and glucose plasma levels in 1- to 2-month-old pericyte-deficient Pdgfrb +/ mice and age-matched littermate controls. (a) Serum lactate levels (mean + 95% CI) in 5 Pdgfrb +/+ and 5 Pdgfrb +/ mice (t-test, equal variance: t = 0.51, p = 0.63). (b) Serum glucose levels (mean ± 95% CI.) in 6 Pdgfrb +/+ and 4 Pdgfrb +/ mice (t-test, equal variance: t = 0.08, p = 0.94).
Supplementary Figure 7 Cortical neuronal activity in 1- to 2-month-old pericyte-deficient Pdgfrb +/ littermate controls. mice and age-matched (a) Representative pseudo-colored voltage-sensitive dye (VSD) image sequences of cortical neuronal activity in the S1 region in response to a 300 ms mechanical hind limb stimulus in Pdgfrb +/+ and Pdgfrb +/ mice. Hind limb region (HL) is indicated by dashed line. Scale bar = 0.5 mm. (b-d) Representative VSD intensity traces (b), time to peak (Mann-Whitney U test, p = 0.84; bootstrapped) (c) and peak fluorescence change (t-test, two tailed, equal variance: t = 0.11, p = 0.92) (d) in Pdgfrb +/+ and Pdgfrb +/ mice. Arrow in (b) indicates peak VSD signal for Pdgfrb +/ (blue) and littermate control Pdgfrb +/+ (red) mice. Mean ± 95% CI, from n = 5 mice per group.
Supplementary Figure 8 Cerebral blood flow response and pericyte coverage in 6- to 8-month-old pericyte-deficient Pdgfrb +/ mice and age-matched littermate controls. (a) Cerebral blood flow (CBF) response to an electrical hind limb stimulus (60 s, 7 Hz, 2 ms pulse duration) determined by laser doppler flowmetry (LDF) as a percentage of baseline change in 4 Pdgfrb +/ mice and 4 Pdgfrb +/+ littermate controls at 6-8 months of age. Circles denote individual values derived from 3 independent LDF measurements per mouse; mean + 95% CI (Mann-Whitney U test, p = 0.03; bootstrapped). (b) Representative confocal microscopy images of pericyte coverage (CD13, magenta) of lectin-positive brain endothelial capillary profiles (< 6 m in diameter; lectin, blue) in the S1 cortical area in Pdgfrb +/+ and Pdgfrb +/ mice at 6-8 mo of age. (c) Quantification (mean ± 95% CI) of CD13-positive pericyte coverage of cortical capillaries in 4 Pdgfrb +/+ and 4 Pdgfrb +/ mice as in b (t-test, single tail, equal variance: t = 4.89, p = 0.001). In each animal 6 randomly selected fields from the cortex were analyzed in 5 nonadjacent sections (~100 m apart) and data were averaged per mouse to obtain individual values (circles) as illustrated.
Supplementary Table 1 Primary Antibody/Lectin (Manufacture, catalog #, dilution used) Pericyte marker Goat anti-cd13 (R&D Systems, AF2335, 1:100) Endothelial marker Fluorescein-conjugated Lycoperiscon esculentum (tomato) lectin (Vector Laboratories FL-1171, 1:200) Neuronal markers Mouse anti-smi-312 (Biolegend, 837904, 1:100) Rabbit anti-neun (Millipore, ABN78, 1:100) Microglia marker Rabbit anti-iba1 (Biocare medical, CP290A, 1:1000) Secondary antibody (Manufacture, catalog #, dilution used) Cy3-conjugated bovine anti-goat (Jackson ImmunoResearch, 805-165-180, 1:100) None Donkey anti-mouse, Alexa fluor 488 (Invitrogen, S11223, 1:500) Donkey anti-rabbit, Alexa fluor 568 (Invitrogen, A10042, 1:500) Donkey anti-rabbit, Alexa fluor 488 (Invitrogen, A-21206, 1:500) Application Immuofluorescent pericyte marker in Supplementary Figure 2a-c, 10, 54, Supplementary Figure 5a-c, and Supplementary Figure 8 b-c. 61 Fluorescent endothelial marker and length quantification in the cortex and hippocampus in Supplementary Figure 2a-c, Supplementary Figure 4a, b and e, Supplementary Figure 5a-c, 12, 62 and Supplementary Figure 8 b-c. Immuofluorescent detection of pan-axonal Neurofilament marker in Figure 6a, c, k and m. 63 Immuofluorescent detection of neuronal nuclear antigen A60 in Figure 6a, b, k and l. 62 Immunofluorescent detection of microglia in Supplementary Figure 4a and d. 12 Astrocyte markers Mouse anti-gfap (GA5) (Millipore, MAB360, 1:200) Rabbit anti-aqp4 (H-80) (Santa Cruz, SC-20812, 1:50) Smooth muscle cell marker Cy3-conjugated anti-smα (1A4) (Sigma Aldrich, C6198, 1:100) Others TUNEL (Roche, 11 684 795 910) Donkey anti-mouse, Alexa fluor 488 (Invitrogen, S11223, 1:500) Donkey anti-rabbit, Alexa fluor 568 (Invitrogen, A10042, 1:500) None In situ cell death detection kit, fluorescein Immunofluorescent detection of astrocytes in Supplementary 12, 64 Figure 4a and c. Immunofluorescent detection of AQP-4 along vasculature in Supplementary Figure 4a-b. 12 Immunofluorescent detection of smooth muscle cells in Supplementary Figure 2, and Supplementary Figure 3 c-d. 65 Immunofluorescent detection of DNA fragmentation in Supplementary Figure 6 a and k. 12 Additional References 61. Efron, B. & Tibshirani, R. An introduction to the bootstrap. (Chapman & Hall, 1993). 62. Winkler, E. A., Sengillo, J. D., Bell, R. D., Wang, J. & Zlokovic, B. V. Blood-spinal cord barrier pericyte reductions contribute to increased capillary permeability. J. Cereb. Blood Flow Metab. Off. J. Int. Soc. Cereb. Blood Flow Metab. 32, 1841 1852 (2012). 63. Winkler, E. A. et al. GLUT1 reductions exacerbate Alzheimer s disease vasculo-neuronal dysfunction and degeneration. Nat. Neurosci. 18, 521 530 (2015). 64. Li, T. et al. The neuritic plaque facilitates pathological conversion of tau in an Alzheimer s disease mouse model. Nat. Commun. 7, 12082 (2016).
65. Wang, Y. et al. 3K3A-activated protein C stimulates postischemic neuronal repair by human neural stem cells in mice. Nat. Med. 22, 1050 1055 (2016). 66. Goodpaster, T. & Randolph-Habecker, J. A flexible mouse-on-mouse immunohistochemical staining technique adaptable to biotin-free reagents, immunofluorescence, and multiple antibody staining. J. Histochem. Cytochem. Off. J. Histochem. Soc. 62, 197 204 (2014).