Fig 2: Disruption of fetal BBB formation and cerebrovascular inflammation 48 h after MIA induction in utero. (A) Timed pregnant mice were injected with poly(I:C) (gestational MIA) at GD13; outcomes were studied in GD15 fetuses. Colocalization of pericyte markers SMA (red; B) and PDGFRß (red; C), endothelial cells marker PeCAM (green) and nuclear stain DAPI (blue) in saline and MIA GD15 cortex (cortical region depicted in B, Inset). Arrowheads indicate SMA/PeCAM and PDGFRß/PeCAM staining overlap. (Scale bars, 20 µm.) Quantification of SMA/PeCAM (D) and PDGFRß/PeCAM (E) signal colocalization in GD15 cortex. (F) Microglial cells markers Iba1 and P2ry12 (arrowheads) in GD15 cortex. (Scale bar, 50 µm.) (G and H) Iba1+ cells and Iba1+P2ry12+ cells in saline and MIA cortex. (I) Cerebrovascular inflammatory marker/VCAM1 (red) and endothelial cells (PeCAM+; green) and nuclear stain DAPI (blue). Arrowheads indicate signal overlap. Cortical region depicted in I, Inset. (Scale bar, 20 µm.) (J) Quantification of VCAM1/PeCAM signal colocalization in GD15 cortex. (D, G, and J) n = 6 dams per group. (E and H) n = 3 dams per group and one fetal brain per dam. (K) Anatomical T2*w image of a live GD15 fetus (dotted lines) in a saline-treated dam. (L) Example of T1w pre–Gd-DTPA (Upper) and post–Gd-DTPA contrast (Lower) images in a poly(I:C)-treated pregnant mouse at GD15 (asterisks indicate the fetal brain postcontrast). (M) T1w SI reflecting Gd-DTPA distribution and thus, BBB integrity in saline and MIA GD15 fetal brains. n = 10 saline-treated dams, and n = 9 MIA-treated dams (one to three embryos per dam). Data are presented as mean ± SEM. (D, E, G, H, and J) **P < 0.01, ***P < 0.001, two-tailed t test. (M) **P < 0.01, ****P < 0.001, one-way ANOVA with Tukey’s multiple comparison test.

Fig 3: Prenatal COX2 inhibition prevents MIA-induced BBB dysfunction in offspring. (A) Pericyte marker CD13 (red) overlaps with endothelial cell marker lectin (green) in the cortex of PD30 offspring born from mice treated prenatally with the COX2 inhibitor CX 24 h after saline or poly(I:C) (MIA) injection (blue, nuclear marker DAPI). Arrowheads point to areas of lectin/CD13 signal colocalization. Cortical region depicted in A, Inset. (Scale bar, 20 µm.) (B) Quantification of CD13/lectin signal overlap in the cortex PD30 offspring from mothers treated prenatally with saline, poly(I:C) (MIA), and vehicle (DMSO) or CX. (C) Vascular inflammation marker VCAM1 (red) and endothelial cell marker lectin (green) staining in the cortex of PD30 offspring born from mice treated prenatally with the COX2 inhibitor CX 24 h after saline or poly(I:C) (MIA) injection. Blue, nuclear marker DAPI. Cortical region depicted in C, Inset. (Scale bar, 20 µm.) (D) Quantification of VCAM1/lectin signal overlap in the cortex of PD30 offspring from mothers treated prenatally with saline, poly(I:C) (MIA), and vehicle (DMSO) or CX. (E) DCE MRI BBB Ktrans images in PD30 offspring born from mothers treated with saline + CX and MIA + CX. (F) Quantification of cortical BBB Ktrans permeability in PD30 offspring born from mice treated with saline or poly(I:C) (MIA) at GD13 with or without CX at GD14. DMSO, n = 3 mice per group; saline + CX, n = 4; MIA + CX, n = 6. (G) Iba1+ microglial cells (arrowheads) in the cortex of PD30 saline + CX and MIA + CX offspring (blue, nuclear marker DAPI). Cortical region depicted in G, Inset. (Scale bar, 20 µm.) (H) Iba1+ cells density in saline and MIA PD30 cortex with or without CX at GD14. (B, D, and H) n = 4 mice per group. Data are presented as mean ± SEM. ns, not significant. **P < 0.01, one-way ANOVA with Tukey’s multiple comparison test; ***P < 0.001, one-way ANOVA with Tukey’s multiple comparison test.

Fig 4: C5 blockade reduced renal IR-induced endothelial activation and inflammation. A, Representative images of VCAM-1 expression on kidney sections 24 h after reperfusion. Scale bar: 50 µm. B, Expression of VCAM-1 was significantly lower in BB5.1-treated mice compared to isotype-treated mice (##P < 0.01 for sham vs isotype; *P < 0.05, **P < 0.01 for isotype vs BB5.1). The data shown are mean ± SEM (n = 7–8). C, Plasma levels of HMGB1 measured by ELISA. Compared with sham, plasma HMGB1 levels were significantly higher 24 h after reperfusion in isotype-treated mice. Treatment with BB5.1 significantly reduced plasma HMGB1 levels. The data shown are mean ± SEM (n = 7–8). Statistical analysis was carried out using the Mann-Whitney U test (###P < 0.001 for sham vs isotype; **P < 0.01, ***P < 0.001 for isotype vs BB5.1). IR, ischemia-reperfusion; IRI, IR injury; SEM, standard error of the mean.