Fig 1.
Uterine and umbilical artery insufficiency and abnormal Tei index of the fetal heart exposed to intrauterine inflammation.
Doppler waveforms were interrogated six hours after phosphate-buffered saline (PBS) or lipopolysaccharide (LPS) exposure. Doppler was used to assess the uterine artery (UtA), placental side of the umbilical artery (UA) and fetal side of the UA in the PBS group, as well as the UtA, placental side of the UA and fetal side of the UA in the LPS group. (a) The systolic/diastolic (S/D) ratio, (b) resistance indices, (c) pulsatility indices and (d) rate of early diastolic notch in UtA were determined for the LPS group (n = 17 dams, 34 arteries) and the PBS group (n = 8 dams, 16 arteries). (e) Resistance indices, (f) pulsatility indices and (g) the rate of absent end-diastolic flow (AEDF) were found for both the placental (-P) and fetal (-F) sides of the UA in the LPS group (n = 17 dams, 38 pups) and the PBS group (n = 8 dams, 26 pups). Time intervals of Tissue Doppler for evaluating Tei indices are shown for the PBS group and the LPS group. (h) Tei indices, (i) the rate of abnormal Tei index (>0.44) and (j) fetal heart rates were compared for the LPS group (n = 17 dams, 40 pups) and the PBS group (n = 8 dams, 22 pups). *p<0.05, **p<0.01.
Fig 2.
MRI imaging and analysis, revealing decreased volume and thickness of placentas exposed to intrauterine inflammation.
(a) T2-weighted images of placentas in mice treated with phosphate-buffered saline (PBS) or lipopolysaccharide (LPS) were obtained. The blue and orange arrows note placental locations, while the dashed contours indicate examples of the manual delineation of the placentas (performed in 3D) for volumetric analysis. (b) 3D measurements shows the individual placenta volumes from the PBS (n = 15) and LPS (n = 14) groups. (c) Hematoxylin and eosin (H&E) stains were performed to compare LPS (n = 25) and PBS (n = 20) placental thicknesses at 5X magnification. The solid black line crossing the central cut surface of the placentas indicated the location of the measurements. The maternal segments (MS)–decidual zone–and fetal segments (FS) were identified as a compilation of junctional zone and labyrinth. (d) Statistical analysis shows the results of the H&E stain measuring maternal, fetal and total placental length at their thickest levels. (e) The images corresponded to the black boxes in (c) with high magnification. *p<0.05; ***p<0.001.
Fig 3.
Vimentin staining of placentas, indicating the endothelial cells.
(a) Hematoxylin and eosin (H&E) stains the neighbor sections indicates the central cut surface of the placentas. (b) Immunohistochemistry staining of vimentin (red) was shown for placentas exposed to phosphate-buffered saline (PBS, n = 5) or lipopolysaccharide (LPS, n = 5). DAPI (blue) stands for counter-staining of nuclei. The bottom panel is the magnification of the inserts in top panel. (c) Quantitative measurements show the percentage of vimentin expression in PBS and LPS placentas. *p<0.05.
Fig 4.
Virchow’s Triad, including hypercoagulability suggested by decreased fibrinogen in placentas exposed to intrauterine inflammation.
(a) This schematic illustrates the three primary factors that contribute to clot formation, according to Virchow’s triad, as well as the lab techniques used to verify their presence in our study mice. (b) The graph shows the fibrinogen levels detected by enzyme-linked immunosorbent assay (ELISA) in placentas exposed to phosphate-buffered saline (PBS, n = 7) or lipopolysaccharide (LPS, n = 8). (c) Histochemical staining of placentas for fibrin show abundant fibrin-rich thrombi (dark blue) in the villi of placenta exposed to LPS. The inserts were the high magnification of the black box in the same panel. *p<0.05.
Fig 5.
Iba 1 expression in fetal brain and correlation to placental thickness.
(a) Representative images of ionized calcium-binding adaptor molecule 1 (Iba1) expression in fetal brain of E17 mice six hours after lipopolysaccharide (LPS) intra-uterine exposure are presented, the studied areas were the cortical area, including the cortex plate, intermediate zone, and ventricular zone. (b) Negative control image using rabbit Ig G in place of primary antibody. (c) Iba1 positive expression in fetal brains was determined through quantitative analysis as the percentage of Iba1 expression area within the image using Image J (PBS: n = 7, LPS: n = 8). (d) Placental thickness was correlated with microglial activation (Pearson correlation analysis). A Panel: blue represents DAPI stain for nuclei, and red represents Iba1 stain. *** p<0.001; scale bar: 50μm. CP: cortical plate, IZ: intermediate zone, VZ: ventricular zone.