Fig 1.
Early and progressive loss of pericyte coverage and capillary reductions in the somatosensory cortex of adult PdgfrβF7/F7 (F7/F7) mice.
(A) Representative confocal microscopy analysis of coronal sections showing CD13-positive pericyte coverage (magenta, left panels), lectin-positive endothelial vascular profiles (blue, middle panels) and merged (right panels) in the S1 region of the somatosensory cortex (S1Cx) layers II-IV of 6-, 12- and 38-week old F7/F7 and 38-week old control (+/+ 38w) mice. Bar = 40 μm. (B-C) Quantification of pericyte coverage (B) and total capillary length (C) in 4–6, 12–16, and 36-48-week old F7/F7 mice compared to age-matched littermate controls (+/+). Pericyte coverage was determined as a percentage (%) of CD13-positive pericyte surface area covering lectin-positive endothelial surface (See Methods and S1 Fig). Total capillary length was determined in mm of lectin-positive endothelial profiles of vessels ≤ 6 μm in diameter, and expressed per mm3 of cortical tissue. In each animal, 4–6 randomly selected fields were analyzed in 4 non-adjacent sections (~100 μm apart), and averaged per mouse to obtain individual values that were taken for statistical comparisons. Mean ± S.E.M., n = 5 animals per group. In B and C, one-way ANOVA and Bonferroni’s post hoc tests were used to compare data in F7/F7 mutants versus age-matched littermate controls and/or between different age groups of F7/F7 mutants only. P < 0.05 indicates statistically significant differences between groups.
Fig 2.
Early and progressive loss of pericyte coverage and capillary reductions in the hippocampus of adult PdgfrβF7/F7 (F7/F7) mice.
(A) Representative confocal microscopy analysis of coronal sections showing CD13-positive pericyte coverage (magenta, left panels), lectin-positive endothelial vascular profiles (blue, middle panels) and merged (right panels) in the CA1 subfield stratum pyramidale of the hippocampus (Hp) of 6-, 12- and 38-week old F7/F7 and 38-week old control mice (+/+ 38w). Bar = 40 μm. (B-C) Quantification of pericyte coverage (B) and total capillary length (C) in 4–6, 12–16, and 36-48-week old F7/F7 mice compared to age-matched littermate controls (+/+) determined as in Fig 1. Mean ± S.E.M., n = 5 animals per group. In B and C, one-way ANOVA and Bonferroni’s post hoc tests were used to compare data in F7/F7 mutants versus age-matched littermate controls, and/or between different age groups of F7/F7 mutants only. P < 0.05 indicates statistically significant differences between groups.
Fig 3.
Loss of pericyte coverage and capillary reductions in the posterior thalamus of adult PdgfrβF7/F7 (F7/F7) mice.
(A) Representative confocal microscopy analysis of coronal sections showing CD13-positive pericyte coverage (magenta, left panels), lectin-positive endothelial vascular profiles (blue, middle panels) and merged (right panels) in the posterior thalamus of 6-, 12- and 36-week old F7/F7 and 36-week old control mice (+/+ 36w). Bar = 40 μm. (B-C) Quantification of pericyte coverage (B) and total capillary length (C) in 4–6, 12–16, and 36-48-week old F7/F7 mice compared to age-matched littermate controls (+/+) determined as in Fig 1. Mean ± S.E.M., n = 5 animals per group. In B and C, one-way ANOVA and Bonferroni’s post hoc tests were used to compare data in F7/F7 mutants versus age-matched littermate controls, and/or between different age groups of F7/F7 mutants only. P < 0.05 indicates statistically significant differences between groups.
Fig 4.
Loss of pericyte coverage correlates with capillary reductions in the cortex and hippocampus of adult PdgfrβF7/F7 (F7/F7) mice.
(A-C) Correlations between age-dependent reduction in capillary length and loss of pericyte coverage in the somatosensory cortex S1 region (S1Cx) (A), the CA1 region of the hippocampus (Hp) (B), and posterior thalamus (Thal) (C) in F7/F7 mice. Single data points were from 4-6- (grey), 12–16 (red), and 36–48 (blue) week-old F7/F7 mice (n = 15 individual points; each point represents the mean value per mouse calculated as explained in Fig 1 legend and Methods). r = Pearson’s coefficient. P, significance.
Fig 5.
Loss of brain pericytes in adult PdgfrβF7/F7 (F7/F7) mice.
(A, C, E) Representative confocal images of CD13-positive pericyte cell bodies co-localized with nuclear 4,6-Diamidino-2-phenylindole, dihydrochloride (DAPI) staining indicated by yellow arrows in the somatosensory cortex S1 region layers II-IV (S1Cx) (A), the CA1 subfield of the hippocampus (Hp) (C), and posterior thalamus (Thal) (E). Bar = 40 μm. (B, D, F) Quantification of CD13-positive cell bodies per mm2 of tissue sections in S1Cx (B), Hp (D) and Thal (F). In each animal, 4–6 randomly selected fields in the cortex, hippocampus and thalamus were analyzed in 4 non-adjacent sections (~100 μm apart) and data were averaged per mouse. Mean ± S.E.M., n = 5 animals per group. In B, D, and F one-way ANOVA and Bonferroni’s post hoc tests were used to compare data in mutants versus age-matched littermate controls, and/or between different age groups of F7/F7 mutants. P < 0.05 indicates statistically significant differences between groups.
Fig 6.
Accumulation of fibrinogen and fibrin perivascular deposits in the brain of adult PdgfrβF7/F7 (F7/F7) mice.
(A-C) Representative confocal images of lectin-positive endothelial profiles and extravascular fibrinogen and fibrin deposits in the somatosensory cortex S1 region (S1Cx) layer IV (A), the CA1 region stratum pyrmidale of the hippocampus (Hp) (B) and posterior thalamus (C) of 6-, 16- and 46-week old F7/F7 mice and 16-week old control (+/+) mice. Bar = 20 μm. (D-F) Quantification of fibrinogen and fibrin-positive extravascular deposits in the S1Cx (D), Hp (E) and Thal (F) of 4–6, 12–16, and 36-48-week old F7/F7 mice and age-matched littermate controls (+/+). Mean ± SEM, n = 5 mice per group. In each animal, 4–6 randomly selected fields in the cortex, hippocampus and thalamus were analyzed in 4 non-adjacent sections (~100 μm apart) and averaged to calculate individual values per mouse. In D-F, one-way ANOVA and Bonferroni’s post hoc tests were used to compare data in mutants versus age-matched littermate controls, and/or between different age groups of F7/F7 mutants. P < 0.05 indicates statistically significant differences between groups. (G-I) Correlations between age-dependent fibrinogen and fibrin perivascular accumulation and loss of pericyte coverage in the S1Cx (G), Hp (H), and Thal (I) regions in F7/F7 mice. Single data points were from 4-6- (grey), 12-16- (red), and 36-48- (blue) week old F7/F7 mice (n = 15 individual points per mouse; r = Pearson’s coefficient; P, significance.
Fig 7.
Normal thickness of vascular smooth muscle cells (VSMCs)-covered arteriolar wall, VSMCs cell numbers and functional response to adenosine in adult PdgfrβF7/F7 (F7/F7) mice.
(A) Representative high magnification confocal images (see Methods for details) of α-smooth muscle actin (SMA)-positive VSMCs (green, left panels) and lectin-positive endothelial profiles (red, middle panels), and merged (left panels) in penetrating arterioles of the somatosensory cortex S1 layer 1 cortex (S1Cx) of 12-week old F7/F7 and age-matched littermate control mice (+/+). Interrupted lines indicate the edge of fluorescent signal. Da, SMA-positive arteriolar diameter; Dv, lectin-positive endothelial diameter. Scale bar = 50 μm. (B-C) Quantification of the arteriolar wall thickness (B) and VSMCs numbers (C) in 4-6-, 12-16- and 36-48-week old F7/F7 mice compared to the respective age-matched littermate controls (+/+). In B and C, individual points represent 10 vessels per animal from 5 animals per group. Mean ± S.E.M. from 50 arterioles per group. ns, not significant by one-way ANOVA. See Methods for details. (D) Laser Doppler flowmetry measurements of cerebral blood flow (CBF) response to endothelium-independent VSMCs relaxant adenosine (400 μM) in 4-6-, 12-16-, and 36-48-week old F7/F7 mice compared to age-matched littermate controls (+/+). Mean ± S.E.M.; n = 3 mice per group; ns, not significant by one-way ANOVA.