(A) Confocal microscopy analysis of PDGFRβ on perivascular pericytes (red), SMI-32 neurofilament marker (green, top panels), neuronal-specific marker NeuN (green, bottom panels) and lectin-positive brain capillaries (blue) in the layer II parietal cortex of a 2 month old Pdgfrβ^+/+ and Pdgfrβ^+/− mice showing no colocalization of PDGFRβ with neurons.
(B) Representative confocal microscopy analysis of desmin immunodetection showing pericyte coverage (red) of lectin-positive brain capillaries (green) in the parietal cortex of an 8 month old Pdgfrβ^+/+ mouse and 1, 8 and 16 month old Pdgfrβ^+/− mice.
(C) Age-dependent loss of pericyte coverage in the cortex and hippocampus of 1, 6–8, and 14–16 month old Pdgfrβ^+/− mice (black bars) and in 6–8 month old F7 mice (gray bars) compared to age-matched Pdgfrβ^+/+ controls (white bars). Pericyte coverage was determined as a percentage (%) of desmin-positive pericyte surface area covering lectin-positive capillary surface area that was arbitrarily taken as 100%. Total of 36 and 24 randomly chosen fields (420 × 420 μm) from 6 non-adjacent cortical and hippocampal sections, respectively, were analyzed per mouse. Mean ± s.e.m., n=6 mice per group; *p<0.05 or **p<0.01 by one-way ANOVA.
(D) Length of lectin-positive capillary profiles in the cortex and hippocampus of 1, 6–8, and 14–16 month old Pdgfrβ^+/+ mice (white bars) and Pdgfrβ^+/− mice (black bars), and 6–8 month old F7 mice (gray bars). Total of 36 and 24 observations in the cortex and hippocampus from the same fields as in C were analyzed per mouse, respectively. Mean ± s.e.m., n=6 mice per group; *p<0.05 or **p<0.01 by one-way ANOVA.
(E) Positive correlation between age-dependent reduction in capillary length and loss of pericyte coverage in the cortex of Pdgfrβ^+/− mice. Single data points were from 1(red), 6–8 (blue) and 14–16 (black) month old Pdgfrβ^+/− mice (n=18). r = Pearson’s coefficient.
(F) Confocal microscopy analysis of TUNEL (green, top panels), PDGFRβ immunodetection on pericytes (red) and lectin-positive capillaries (blue) in the cortex of 6 month old Pdgfrβ^+/+, 1 and 6 month old Pdgfrβ^+/− mouse and 6 month old F7 mouse. Closed arrow, TUNEL-positive attached pericyte; *, TUNEL-positive detaching pericyte; Open arrow, TUNEL-positive endothelial cell. (See Fig. S1)

(A) Confocal microscopy analysis of plasma-derived IgG (red) and fluorescein-conjugated lectin-positive microvessels (green) in the hippocampus of an 8 month old Pdgfrβ^+/+ and in 1, 8 and 16 month old Pdgfrβ^+/− mice. Arrows, extravascular IgG deposits.
(B–C) Quantification of extravascular IgG deposits in the hippocampus of 1 month old Pdgfrβ^+/− mice (black bar) and Pdgfrβ^+/+ controls (white bar) (B), and in 6–8 and 14–16 month old Pdgfrβ^+/− mice (black bars), and Pdgfrβ^+/+ controls (white bars) and 6–8 month old F7 mice (gray bar) (C).
In B and C, 24 randomly chosen fields (420 × 420 μm) within the hippocampus were analyzed from 6 non-adjacent sections per mouse. Values are mean ± s.e.m., n=6 mice per group; *p<0.05 or **p<0.01 by one-way ANOVA. (See Fig. S2).
(D) Negative correlation between an age-dependent IgG brain accumulation and loss of pericyte capillary coverage in the hippocampus of Pdgfrβ^+/− mice. Single data points were from 1 (red), 6–8 (blue) and 14–16 (black) month old Pdgfrβ^+/− mice (n=18). r = Pearson’s coefficient.
(E) Confocal microscopy imaging of IgG deposits (green) around lectin-positive capillaries (white) lacking pericyte coverage (red; negative desmin staining) in an 8 month old Pdgfrβ^+/− mouse. Orthogonal views suggest that microvessels lacking pericyte coverage have significant perivascular IgG accumulation.
(F) Confocal microscopy analysis of IgG (green) and a pericyte marker PDGFRβ (red) on lectin-positive (blue) pericyte-covered capillary in an 8 month old F7 mutant mouse. Orthogonal views show IgG accumulation in a PDGFRβ-positive pericyte in a F7 mouse.

(A) In vivo time-lapse multiphoton imaging of tetramethylrhodamine (TMR) dextran (MW = 40,000; red) leakage from cortical vessels (layer II and III, approximately 100 μm from the cortical surface) in an 8 month old Pdgfrβ^+/+ mouse, and 1, 8 and 16 month old Pdgfrβ^+/− mice within 30 min of TMR-dextran intravenous administration.
(B–C) The cerebrovascular permeability surface area product (PS) for TMR- dextran (40,000 Da) determined by multiphoton imaging as in A. in 1 month old Pdgfrβ^+/− mice (black bar) and control Pdgfrβ^+/+ (white bar) mice (B), and 6–8 and 14–16 month old Pdgfrβ^+/− (black bar) and control Pdgfrβ^+/+ (white bars) mice, and 6–8 month old F7 mice (gray bar) (C).
(D) Negative correlation between the PS product for TMR-dextran and age-dependent loss of pericyte coverage of cortical capillaries in Pdgfrβ^+/− mice. Single data points derived from 1 (red), 6–8 (blue) and 14–16 (black) month old Pdgfrβ^+/− mice (n=18). r = Pearson’s coefficient.
(E) The blood-brain barrier permeability PS product for TMR-dextran (40,000 Da), Cy-3-IgG (150,000 Da) FITC-Dextran (MW=500,000 Da) and fluorescein-conjugated mega-Dextran (MW=2,000,000 Da) in 6–8 month old Pdgfrβ^+/+ (white bar), Pdgfrβ^+/− (black bar) and F7 (gray bar) mice determined by a non-invasive fluorometric tissue analysis not requiring the cranial window surgical procedure. Mean ± s.e.m., n=4–6 animals per group. *p<0.05 or ** p<0.01 by one-way ANOVA. ND. Non-detectable.
(F) Immunoblotting of ZO-1 and occludin in isolated cortical and hippocampal capillaries from a 16 month old Pdgfrβ^+/+ control mouse and 1, 8 and 16 month old Pdgfrβ^+/− mice.
(G–H) Quantification of ZO-1 (G) and occludin (H) relative abundance by densitometry analysis of immunoblots in 1, 8 and 16 month old Pdgfrβ^+/+ and Pdgfrβ^+/− mice showing a progressive decrease in the tight junction proteins in the aging pericyte-deficient mice. β-actin was used as a loading control. In B, C, G and H, mean ± s.e.m., n=6 animals per group. *p<0.05 or ** p<0.01 by one-way ANOVA. (See Fig. S3–S5).

(A) Confocal analysis of hypoxyprobe-1 (pimonidazole)-positive hypoxic (O₂ <10 mm Hg) tissue in the hippocampus of 8 month old Pdgfrβ^+/+, Pdgfrβ^+/− and F7 mice.
(B) Quantification of hypoxyprobe-1-positive area expressed as a percentage of total tissue area in the cortex and hippocampus of 6–8 month old Pdgfrβ^+/+, Pdgfrβ^+/− and F7 mice. A total of 36 and 24 randomly chosen fields (420 × 420 μm) from 6 non-adjacent cortical and hippocampal sections, respectively, were analyzed per mouse. Mean ± s.e.m., n=6 mice per group; *p<0.05 or **p<0.01 by one-way ANOVA. (See Fig. S7).

(A) Confocal microscopy analysis of Iba1-positive microglia in the cerebral cortex of an 8 month old control Pdgfrβ^+/+ mouse and 1, 8 and 16 month old Pdgfrβ^+/− mice.
(B) The number of Iba1-positive microglia in the cortex of 1, 6–8 and 14–16 month old Pdgfrβ^+/− mice (black bars) and age-matched Pdgfrβ^+/+ controls (white bars). Total of 36 randomly chosen fields (420 × 420 μm) from 6 non-adjacent cortical sections were analyzed per mouse.
(C) Bright-field microscopy analysis for neutrophils (red) using dichloracetate esterase staining in the cerebral cortex of an 8 month old Pdgfrβ^+/+ mouse and 1, 8 and 16 month old Pdgfrβ^+/− mice. MCAO, a positive control showing neutrophils infiltration (arrows) of ischemic brain tissue of a Pdgfrβ^+/+mouse 24 h after permanent middle cerebral artery occlusion (MCAO).
(D) The number of neutrophils in the cortex of 1, 6–8 and 14–16 month old Pdgfrβ^+/− mice and mice receiving the MCAO compared to control 1 month old Pdgfrβ^+/+ mice.
(E–I) Real-time quantitative PCR for mouse interlukein-1 beta (Il-1β) (E) interlukein 6 (Il-6) (F) tumor necrosis factor alpha (Tnf-α) (G), chemokine C-C motif ligand 2 (Ccl2)(H) and intercellular adhesion molecule 1 (Icam-1) (I) mRNA in the cerebral cortex of 1, 6–8 and 14–16 month old Pdgfrβ^+/− (black bars) and age-matched control Pdgfrβ^+/+ (white bars) mice.
In B, D and E–I, mean ± s.e.m, n=4–6 mice per group; *p<0.05 by one-way ANOVA. NS, non-significant.

(A) Perfusion of cortical microvessels studied by in vivo multiphoton microscopy analysis of fluorescein-conjugated mega-dextran (MW=2,000,000; green) in 8 month old Pdgfrβ^+/+ mouse, and 1, 8 and 16 month old Pdgfrβ^+/− mice.
(B) Quantification of perfused capillary length from angiograms obtained as in A. in 1, 6–8, and 14–16 month old Pdgfrβ^+/+ mice (white bars) and Pdgfrβ^+/− mice (black bars), and in 6–8 month old F7 mice (gray bars). Two to three angiograms were analyzed per mouse.
(C) Positive correlation between age-dependent reductions of brain capillary perfusion and desmin-positive pericyte coverage of cortical capillaries in Pdgfrβ^+/− mice. Single data points were from 1 (red), 6–8 (blue) and 14–16 (black) month old Pdgfrβ^+/− mice (n=18). r = Pearson’s coefficient.
(D) ¹⁴C-iodoantipyrine (IAP) autoradiography of the cerebral blood flow (CBF) in an 8 month old Pdgfrβ^+/+ mouse, 1 and 8 month old Pdgfrβ^+/− mice and an 8 month old F7 mouse.
(E) Local CBF determined using ¹⁴C-IAP autoradiography as in D. in 1, 6–8, and 14–16 month old Pdgfrβ^+/+ mice (white bars) and Pdgfrβ^+/− mice (black bars), and in 6–8 month old F7 mice (gray bars). (See Table S1).
(F) CBF responses to whisker-barrel cortex vibrissal stimulus (% increase from basal values) in 1, 6–8 and 14–16 month old Pdgfrβ^+/+ mice (white bars) and Pdgfrβ^+/− mice (black bars).
(G) Positive correlation between diminished CBF responses to a stimulus and an age-dependent loss of pericyte coverage of cortical capillaries in Pdgfrβ^+/− mice. Single data points were from 1 (red), 6–8 (blue) and 14–16 (black) month old Pdgfrβ^+/− mice (n=18). r =Pearson’s coefficient.
In B, C, E and F, mean ± s.e.m., n=6 mice per group; *p<0.05 or **p<0.01 by one-way ANOVA.

(A) Confocal microscopy analysis of fibrin (green) and CD31-positive microvessels (red) in the cortex of an 8 month old Pdgfrβ^+/+ and 1, 8 and 16 month old Pdgfrβ^+/− mice. Arrows, extravascular fibrin deposits.
(B–C) Quantification of fibrin extravascular deposits in the cortex of 1 month old Pdgfrβ^+/+ (white bar) and Pdgfrβ^+/− mice (black bar) (B), and 6–8 and 14–16 month old Pdgfrβ^+/+ (white bars) and Pdgfrβ^+/− mice (black bar), and 6–8 month old F7 mice (gray bar) (C). In B– C, 36 randomly chosen fields (420 × 420 μm) in the cortex were analyzed from 6 non-adjacent sections per mouse. Values are mean ± s.e.m., n=6 mice per group; *p<0.05 or **p<0.01 by one-way ANOVA. (See Fig. S2).
(D) Negative correlation between an age-dependent fibrin extravascular accumulation and loss of pericyte coverage in the cortex of Pdgfrβ^+/− mice. Single data points derived from 1 (red), 6–8 (blue) and 14–16 (black) month old Pdgfrβ^+/− mice (n=18). r = Pearson’s coefficient.
(E) Confocal microscopy imaging of fibrin deposits (green) around lectin-positive capillary (white) lacking desmin-positive (red) pericyte coverage in an 8 month old Pdgfrβ^+/− mouse. The orthogonal views show a significant perivascular fibrin accumulation.
(F) Confocal microscopy imaging of fibrin (green) and a pericyte marker PDGFRβ (red) covering a lectin-positive capillary (blue) in an 8 month old F7 mutant mouse. Orthogonal views show fibrin accumulation in a PDGFRβ-positive pericyte in a F7 mouse.
(G) Quantification of Prussian blue-positive hemosiderin deposits in sagittal brain sections of cortex plus hippocampus of 8 month old Pdgfrβ^+/− and F7 mice compared to age-matched Pdgfrβ^+/+ controls. Inset, a hemosiderin deposit in the cortex of an 8 month old Pdgfrβ^+/− mouse. A total of 6 non-adjacent sections were analyzed per mouse.
(H) Immunoblotting of thrombin, plasmin and laminin in capillary-depleted brain tissue of 8 month old Pdgfrβ^+/+, Pdgfrβ^+/− and F7 mice. β-actin was used as a loading control. Graphs show quantification of relative protein abundance using densitometry analysis. Mean ± s.e.m., n=6 animals per group; *p<0.05 or **p<0.01 by one-way ANOVA.

(A–B) Low magnification bright-field microscopy analysis of Golgi-Cox staining of neurons (A) and high magnification bright-field microscopy images of dendritic spine density (B) in the CA1 region of the hippocampus of an 8 month old Pdgfrβ^+/+ control and 1, 8 and 16 month old Pdgfrβ^+/− mice.
(C–E) Dendritic length (C), dendritic spine density (D) and spine length (E) of Golgi-Cox stained neurons in the CA1 region of the hippocampus in 1, 6–8, and 14–16 month old Pdgfrβ^+/− mice (black bars) and control Pdgfrβ^+/+ (white bars) mice, and 6–8 month old F7 mice (gray bars). Total of 30 fields (500 × 375 μm) from 3 adjacent 100 μm thick sections were analyzed per mouse for dendritic length measurements. Total of 100 randomly selected dendrites from 3 adjacent 100 μm thick sections were analyzed per mouse for dendritic spine density measurements and for spine length quantification.
(F) Bright-field microscopy analysis of hemotoxylin and eosin staining of stratum pyramidale in the CA1 hippocampal region of 6 month old Pdgfrβ^+/+, Pdgfrβ^+/− and F7 mice.
(G) Quantification of NeuN-positive neurons in the stratum pyramidale of the CA1 hippocampal subfield in 1, 6–8, and 14–16 month old Pdgfrβ^+/− (black bars) and control Pdgfrβ^+/+ (white bars) mice, and 6–8 month old F7 mice (gray bar). Total of 24 randomly chosen CA1 fields (420 × 420 μm) in the hippocampus were analyzed from 6 non-adjacent sections per mouse.
(H) Confocal microscopy analysis of TUNEL (green), NeuN-positive neuronal nuclei (red) and thrombin (blue) in the cortex of an 8 month old Pdgfrβ^+/− and control Pdgfrβ^+/+ mouse.
(I–J) Novel object location (I) and novel object recognition (J) exploratory preference in 1, 6–8, and 14–16 month old Pdgfrβ^+/− mice (black bars) and age-matched Pdgfrβ^+/+ control mice (white bars) and in 6–8 month old F7 mice (gray bars). (See Fig. S6).
In C–E, G and I–J, mean ± s.e.m., n = 6–10 mice per group; *p<0.05 by one-way ANOVA. NS, non-significant.

(A) Confocal microscopy analysis of SMI-32 immunodetection of neurites (green) and Topro3 nuclear staining (blue) in the hippocampus of 8 month old Pdgfrβ^+/+ and F7, and Meox2^+/+ and Meox2^+/− mice. Pyr., Stratum Pyramidale and Rad., Stratum Radiatum.
(B) Quantification of SMI-32 positive neurite density in the cortex and hippocampus of 8 month old Pdgfrβ^+/+ and F7, and Meox2^+/+ and Meox2^+/− mice.
(C) Confocal microscopy analysis of NeuN-positive neurons (green) in the cortex in 8 month old Pdgfrβ^+/+ and F7, and Meox2^+/+ and Meox2^+/− mice.
(D) Quantification of NeuN-positive neurons in the cortex and hippocampus CA1 stratum pyramidale of 8 month old Pdgfrβ^+/+ and F7 mice, and Meox2^+/+ and Meox2^+/− mice.
In B and D, a total of 36 and 24 randomly chosen fields (420 × 420 μm) from 6 non-adjacent cortical and hippocampal sections, respectively, were analyzed per mouse. Mean ± s.e.m., n=6 mice per group; *p<0.05 or **p<0.01 by one-way ANOVA

PDGFRβ deficient signaling in brain pericytes leads to a progressive age-dependent pericyte loss resulting in (1) reductions in brain microcirculation with diminished cerebral blood flow (CBF) and CBF responses to a stimulus (CBF dysregulation) leading to a chronic perfusion stress and hypoxia, and (2) blood-brain barrier breakdown with accumulation of serum proteins and several cytotoxic and neurotoxic pathologic deposits in brain. Both, arms 1 and 2 may contribute to secondary neuronal injury and neurodegeneration mediated by a neurovascular insult.