Prenatal disruption of blood-brain barrier formation via cyclooxygenase activation leads to lifelong brain inflammation

Abstract

Gestational maternal immune activation (MIA) in mice induces persistent brain microglial activation and a range of neuropathologies in the adult offspring. Although long-term phenotypes are well documented, how MIA in utero leads to persistent brain inflammation is not well understood. Here, we found that offspring of mothers treated with polyriboinosinic–polyribocytidylic acid [poly(I:C)] to induce MIA at gestational day 13 exhibit blood–brain barrier (BBB) dysfunction throughout life. Live MRI in utero revealed fetal BBB hyperpermeability 2 d after MIA. Decreased pericyte–endothelium coupling in cerebral blood vessels and increased microglial activation were found in fetal and 1- and 6-mo-old offspring brains. The long-lasting disruptions result from abnormal prenatal BBB formation, driven by increased proliferation of cyclooxygenase-2 (COX2; Ptgs2)-expressing microglia in fetal brain parenchyma and perivascular spaces. Targeted deletion of the Ptgs2 gene in fetal myeloid cells or treatment with the inhibitor celecoxib 24 h after immune activation prevented microglial proliferation and disruption of BBB formation and function, showing that prenatal COX2 activation is a causal pathway of MIA effects. Thus, gestational MIA disrupts fetal BBB formation, inducing persistent BBB dysfunction, which promotes microglial overactivation and behavioral alterations across the offspring life span. Taken together, the data suggest that gestational MIA disruption of BBB formation could be an etiological contributor to neuropsychiatric disorders.

Keywords: blood–brain barrier; cyclooxygenase; fetal programing; inflammation; pregnancy.

Fig. 1.

Gestational MIA induces long-term BBB hyperpermeability in adult offspring. (A) Timed pregnant mice were injected with poly(I:C) (“gestational MIA”) at GD13; outcomes were studied in PD30 and PD180 offspring. (B) Microglial cells markers Iba1 (red) and TMEM119 (green) and nuclear stain (DAPI, blue) in the cortex of the PD30 brain. Yellow arrows indicate Iba1+TMEM119+DAPI+ cells. (Scale bar, 50 µm.) (Insets) High-magnification views of Iba1+TMEM119+ microglial cells (white arrows). (Scale bar, 10 µm.) Quantification of Iba1+ cells (C), Iba1+TMEM119+ cells (D), and average branch number per Iba1+ cell (E) in saline and MIA cortex. (F and G) DCE MRI images and quantification of cortical BBB K_trans permeability in saline and MIA PD30 and PD180 offspring. (H and I) Molecular MRI of cerebrovascular inflammation using intravenous injections of MPIOs targeting VCAM1 shown by 3D GRE minimum intensity projection (mip) and quantification of the percentage of signal voids within the cortex, thalamus, and striatum of saline and MIA PD30 offspring. (J) Vascular inflammation marker VCAM1 (red) colocalization with endothelial cells (lectin+). Arrowheads point to VCAM1+lectin+ cells observed mainly in the MIA group. (Scale bar, 20 µm.) (Inset) Cortical location imaged. (K) Percentage of VCAM1/lectin colocalization in saline and MIA cortex. n = 4 mice per group. Colocalization of pericyte marker CD13 (red; L) and PDGFRβ (red; N) with endothelial cells (lectin+; green) in PD30 cortex of saline and MIA offspring (cortical region depicted in L, Inset). Arrowheads point to CD13/lectin and PDGFRβ/lectin staining overlap. Percentage of CD13/lectin (M) and PDGFRβ/lectin (O) signal overlap in saline and MIA cortex. (C, E, I, and M) n = 6 mice per group. (G) n = 12 mice per group. (D and O) n = 3 mice per group. Data represent mean ± SEM. (Scale bar, 20 µm.) **P < 0.01, two-tailed t test; ***P < 0.001, two-tailed t test.

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 PDGFRβ (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 PDGFRβ/PeCAM staining overlap. (Scale bars, 20 µm.) Quantification of SMA/PeCAM (D) and PDGFRβ/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.

The COX2 pathway is critical for gestational MIA effects on fetal BBB formation and function. Measures of (A) maternal serum and (B) fetal brain protein concentrations of proinflammatory cytokines IL-6, TNFα, and MCP-1 chemokine 6, 28, and 48 h after GD13 saline (dashed lines) or poly(I:C) injection (MIA; solid lines). (C) Measure of PGE2 tissue concentration in the whole fetal brain and maternal serum 28 h after maternal poly(I:C) injection. (D) Experimental scheme of COX2 blocking experiments. (E) Measure of total COX, COX1, and COX2 enzymatic activity in fetal brain 28 h after GD13 saline or poly(I:C) injection. (F and G) Representative images (F) and quantification (G) of endothelial (PeCAM+) and pericyte-like (SMA) colocalization in GD15 fetal cortex (nuclear marker DAPI, blue); dams received CX or vehicle (dimethylsulfoxide [DMSO]) 24 h after saline or MIA. Cortical region depicted in F, Inset. (Scale bar, 20 µm.) (H) Quantification (T1w SI) of tracer (Gd-DTPA) distribution into the fetal brain at GD15 in saline/MIA + vehicle (DMSO) and saline/MIA + CX groups. (A–G) n = 3 to 4 dams per group (one fetus per dam). (H) DMSO, n = 3 dams per group; CX, n = 4 dams per group. Data are presented as mean ± SEM. ns, not significant. *P < 0.05, one-way ANOVA with Tukey’s multiple comparison test (H); **P < 0.01, two-way ANOVA with Bonferroni’s multiple comparison test (E) and one-way ANOVA with Tukey’s multiple comparison test (G); ***P < 0.001, two-way ANOVA with Bonferroni’s multiple comparison test (A and E) and two-tailed t test (C).

Fig. 4.

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 K_trans images in PD30 offspring born from mothers treated with saline + CX and MIA + CX. (F) Quantification of cortical BBB K_trans 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. 5.

COX2 pathway activation by gestational MIA regulates microglial proliferation and localization to perivascular spaces in the fetal cortex. Representative images (A) of COX2-negative (red arrows) and COX2-positive (white arrows) parenchymal microglial cells (Iba1+) in saline and MIA fetal cortex at GD15. In the MIA group, most COX2+Iba1+ microglia are attached to vessels (yellow arrowheads). Nuclear marker DAPI, blue. Cortical region depicted in A, Inset. (Scale bar, 20 µm.) (Insets) High-magnification views of parenchymal and perivascular COX2+ microglial cells surrounded by dashed boxes. (Scale bar, 5 µm.) (B–D) Quantification of the portion of Iba1+ microglia (B), the fraction of microglia coexpressing Iba1 and COX2 (C), and the vessel-associated COX2+ microglia (D) in GD15 fetal cortex in saline and MIA groups, with (CX) and without (DMSO) CX treatment at GD14. (E) Experimental scheme of the BrdU experiment. (F, Left) Representative images of Iba1+BrdU+ in saline (+DMSO) and MIA (+DMSO) GD15 cortex. Nuclear marker DAPI, blue. Cortical region depicted in F, Inset. (Scale bar, 50 µm.) (F, Right) High-magnification views of Iba1+BrdU+ microglial cells (red arrowheads). (Scale bar, 10 µm.) (G) Quantification of Iba1+BrdU+ microglial cells density in saline (+DMSO), MIA (+DMSO), and MIA + CX at GD15. (H) Experimental scheme for the generation of fetus littermates with normal expression (COX2-FLOX) and myeloid cell–specific COX2 (Ptgs2) gene deletion (COX2-MKO). Quantification of Iba1+ microglial cells (I), vessel-associated Iba1+ cells (J), Iba1+Ki67+ microglia (K), and pericyte coverage of endothelial cells (L). (B–D and G) n = 3 dams per group (one fetus per dam). (I–L) n = 3 dams (two fetuses per dam). Data are presented as mean ± SEM. ns, not significant. (B–D and G) **P < 0.01, one-way ANOVA with Tukey’s multiple comparison test; ***P < 0.001, one-way ANOVA with Tukey’s multiple comparison test. (I–L) **P < 0.01, two-tailed t test; ***P < 0.001, two-tailed t test.