Maternal immune activation leads to defective brain blood vessels and intracerebral hemorrhages in male offspring (2022)
Figure 4

Figure 4. Maternal immune activation induces sex-specific alterations of the neurovascular unit changing the ration between ECs and PCs.

A. [left] Representative dot plots showing the gating strategy for E17 samples to distinguish NVU cells. The cells were pre-gated on single cells and live cells (Zombie Aqua negative). Live cells were pre-gated on CD45 (CD45-). [right] Brain Pericytes (PC) were defined as CD146+CD31- cells and brain Endothelial Cells (EC) as CD31+cells.

B. [left] Quantification of the ratio between PC number to EC numbers for male (blue) and female (purple) E17 offspring cortex samples. Male Ctrl = 1.10 ± 0.08, Male Poly I:C = 1.34 ± 0.09, Female Ctrl = 1.02 ± 0.08, Female Poly I:C = 1.12 ± 0.09. Mann-Whitney U test, *p < .05. Bars represent average ratio of individual embryos ± SEM, numbers in bars indicate the number of embryos. [right up] Percentages of ECs in Male Ctrl (0.79 ± 0.05), Male Poly I:C (0.90 ± 0.09), Female Ctrl (0.65 ± 0.07), Female Poly I:C (0.80 ± 0.07) samples and [right down] PCs in Male Ctrl (0.60 ± 0.05), Male Poly I:C (0.57 ± 0.06), Female Ctrl (0.53 ± 0.07), Female Poly I:C (0.62 ± 0.06) samples.

C. [left] Gating strategy used to analyze P30 samples: single cells, live (zombie Aqua negative), CD45- cells. [right] PCs are identified as CD146+CD13+CD31-; ECs are CD13-CD31+

D. [left] Quantification of EC/PC number ratio for male and female P30 offspring cortex samples, obtained by manual gating. Male Ctrl = 1.78 ± 0.29, Male Poly I:C = 1.74 ± 0.32, Female Ctrl = 1.49 ± 0.11, Female Poly I:C = 1.60 ± 0.19. [right up] Percentages of ECs in Male Ctrl (17.07 ± 1.88), Male Poly I:C (17.52 ± 1.90), Female Ctrl (14.58 ± 0.66), Female Poly I:C (15.73 ± 1.07) samples, and [right down] percentage of PCs in Male Ctrl (10.55 ± 1.89), Male Poly I:C (10.67 ± 1.04), Female Ctrl (10.07 ± 0.85), Female Poly I:C (10.20 ± 0.79).

E. Normalized percentage of live(CD45-) EdU-positive cells at E17 in Male Ctrl (1.00 ± 0.05), Male Poly I:C (0.62 ± 0.11), Female Ctrl (0.61 ± 0.04), Female Poly I:C (0.59 ± 0.11) samples. Mann-Whitney U test and Kruskal-Wallis test, *p < .05, **p < .001.

F. Percentage of PCs EdU-positive cells at E17 in Male Ctrl (7.88 ± 1.57), Male Poly I:C (4.55 ± 0.70), Female Ctrl (7.03 ± 1.04), Female Poly I:C (4.93 ± 0.83) samples. Student's t test, *p < .05.

G. Percentage of ECs EdU-positive cells at E17 in Male Ctrl (4.30 ± 0.71), Male Poly I:C (2.80 ± 0.53), Female Ctrl (2.84 ± 0.43), Female Poly I:C (2.29 ± 0.77) samples.

H. Normalized percentage of live(CD45-) EdU-positive cells at P30 in Male Ctrl (1.16 ± 0.14), Male Poly I:C (0.66 ± 0.16), Female Ctrl (1.00 ± 0.08), Female Poly I:C (0.91 ± 0.13) samples. Mann-Whitney U test, *p < .05.

I. Percentage of PCs EdU-positive cells at P30 in Male Ctrl (0.80 ± 0.24), Male Poly I:C (0.24 ± 0.06), Female Ctrl (0.26 ± 0.08), Female Poly I:C (0.38 ± 0.13) samples. Mann-Whitney U test, *p < .05.

J. Percentage of ECs EdU-positive cells at P30 in Male Ctrl (0.58 ± 0.10), Male Poly I:C (0.44 ± 0.19), Female Ctrl (0.38 ± 0.22), Female Poly I:C (0.25 ± 0.06) samples.

K. [left] CD146 normalized MFI of the EC cells at P30 in Male Ctrl (100.00 ± 8.27), Male Poly I:C (65.73 ± 3.63), Female Ctrl (57.08 ± 14.10), Female Poly I:C (43.07 ± 8.34). Mann-Whitney U test and Ordinary one-way ANOVA, **p < .01. [right] Representative histograms CD146 expression on ECs in Male Ctrl (blue hollow), Male Poly I:C (solid light blue), Female Ctrl (purple hollow), Female Poly I:C (solid purple) samples.

L. Percentages of PC CD146++, CD146+ and CD146- cells respectively at P30 in Male Ctrl (26.32/36.75/23.48 ± 2.84/3.31/4.62), Male Poly I:C (27.53/42.40/20.60 ± 2.25/0.58/1.94), Female Ctrl (29.05/40.98/2.19 ± 1.99/1.32/2.19), Female Poly I:C (27.17/46.60/17.10 ± 3.09/3.86/1.96) samples.

M. [left] CD105 normalized MFI of the EC cells at P30 in Male Ctrl (100.00 ± 7.39), Male Poly I:C (99.58 ± 6.23), Female Ctrl (100.30 ± 1.61), Female Poly I:C (96.67 ± 3.72). [right] Representative histograms indicating mean fluorescence intensity (MFI) of CD105 on EC in Male Ctrl (blue hollow), Male Poly I:C (solid light blue), Female Ctrl (purple hollow), Female Poly I:C (solid purple) samples.

N. [left] Quantitative data of the normalized MFI of CD105 in PCs at P30 in Male Ctrl (100.00 ± 3.43), Male Poly I:C (98.01 ± 5.14), Female Ctrl (98.57 ± 4.16), Female Poly I:C (87.99 ± 4.21). Mann-Whitney U test, *p < .05. [right] Representative histograms for CD105 expression on PCs in Male Ctrl (blue hollow), Male Poly I:C (solid light blue), Female Ctrl (purple hollow), Female Poly I:C (solid purple) samples.

O. [top] Representative images of CD13 (red) positive pericytes covering Lectin-positive vessels (green) in sections of P90 untreated male brains. [bottom] 3D rendering, Scale bar = 20 µm, enlargement scale bar = 10 µm.

P. Percentage of CD13-positive pericyte coverage at P90 in Male Ctrl (53.42 ± 9.22) and Poly I:C (27.73 ± 5.29). Student's t test, *p < .05.

Q. Percentage of PDGFrB-positive pericyte coverage at P90 in Male Ctrl (66.15 ± 3.09) and Poly I:C (55.08 ± 3.10). Student's t test, *p < .05.

R. Representative images of PDGFrB (red) and NG2 (white) positive pericytes covering Lectin-positive vessels (blu) in sections of P90 untreated male brains. Scale bar = 10 µm, enlargement scale bar = 5 µm.

S. Percentage of CD13-positive pericyte coverage at P90 in Female Ctrl (71.57 ± 8.37) and Poly I:C (69.13 ± 6.63).

T. Percentage of PDGFrB-positive pericyte coverage at P90 in Female Ctrl (66.77 ± 11.76) and Poly I:C (70.56 ± 8.32).

Data information: Embryos prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex single-cell suspension was analyzed by multi-color flow cytometry. Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. CD13 positive pericytes were represented ad CD146- (dark greed bars), CD146+ (green bars) and CD46++ (light green bars). Bars represent mean ± SEM, numbers in bars indicate the number of animals.