Figure 1 Prenatal LPS reduces focal demyelination lesion. Panels in (A) show Luxol fast blue staining of the EB‐injected corpus callosum of adult rats born to dams given either saline (pSal) or LPS (pLPS) at mid‐gestation. Demyelination lesion in pLPS rats is smaller than that seen in pSal. The bar graph in (B) shows that the size of the demyelination lesion in pLPS was significantly reduced in pLPS rats compared to pSal rats (P<.05). Micrographs in (C) show a double immunodetection of the axonal marker neurofilament (NF, green) and the myelin basic protein (MBP, red) in the vicinity of demyelination region within the corpus callosum of pSal and pLPS rats. Note the growth of myelinated axons within the lesioned area of the corpus callosum in pLPS rats. The dashed line indicates the border of the demyelination lesion. The dark bars in pLPS micrograph in (A) are the result of the merging of two pictures to show the whole area of the lesion. *P<.05, Scale bar in A)=400 μm, Scale bar in C)=50 μm

Figure 2 Prenatal LPS enhances the density of oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes. Panel in (A) shows an immunofluorescent detection of NG2+ cells (red) in the EB‐injected corpus callosum of adult rats born to dams given either saline (pSal) or LPS (pLPS) at mid‐pregnancy. A counter stain with DAPI was used for nuclear detection (blue). Bar graph in (B) shows the density of NG2+ cells in the corpus callosum in both basal condition (−EB) or in response to a focal demyelination (+EB). Prenatal LPS led to a significant increase in NG2+ cell density in the corpus callosum in the absence of focal demyelination. There was also a significant increase in the NG2+ cell density in the vicinity of the EB‐induced demyelination lesion in the corpus callosum of pLPS rats when compared to those of pSal rats (P<.05). Panel in (C) shows immunodetection of mature oligodendrocytes (CC1) in EB‐injected corpus callosum of pSal and pLPS rat groups. The bar graph in (D) shows that the cell density of CC1+ in the corpus callosum of adult rats was not affected by prenatal immune challenge (−EB). However, focal demyelination (+EB) led to a significant decrease in the density of CC1+ cells in the vicinity of the demyelination lesion of pSal rats group (P<.01). Such decrease was significantly dampened in pLPS rats (*P<.05, **P<.01). The schematic drawing shown on the top of the micrograph illustrates the site of demyelination lesion in the corpus callosum (red spot). The squares on both sides of the lesion illustrate the areas used for cell count. CC, corpus callosum; LV, lateral ventricle. Scale bars=50 μm

Figure 3 Impact of prenatal LPS on the expression of myelin basic protein isoforms during adulthood. Panel in (A) shows immunoblot detection of myelin basic protein (MBP) isoforms in the corpus callosum of adult rats born to dams given either saline (pSal) or LPS (pLPS) at mid‐gestation. Four major MBP‐immunoreactive bands were detected at an apparent molecular weight of (in KDa) 21.5, 18.5, 17, and 14. The densitometric analysis of both 17KDa and 18.5KDa were grouped due to the fusion of these two immunoreactive bands. Among these MBP bands, the expression level of the MBP 21.5 KDa (graph bars in B) was significantly enhanced in pLPS group when compared to that seen in pSal rat group (*P<.05)

Figure 4 Prenatal LPS dampens the inflammatory response to focal demyelination. Panel in (A) shows immunodetection of microglial cells (Iba1+) in the vicinity of demyelination lesion within the corpus callosum of pSal and pLPS rats. The bar graph (right) shows that, in the absence of focal demyelination (−EB), the density of microglial cells was significantly low and was not affected by prenatal LPS treatment. The density of microglial cells was significantly enhanced in the EB‐injected corpus callosum of pSal rat group. Such increase in microglial density was reduced in pLPS rat group. The protein levels of NFκB (B1) in the vicinity of EB‐injected corpus callosum of adult offspring was not affected by prenatal treatment with LPS. In contrast, the levels of IκB (B2) were significantly reduced in pLPS rat group when compared to that of pSal rat group (*P<.05, ***P<.001). Scale bar=50 μm

Figure 5 Impact of prenatal LPS on demyelination‐induced astrocytosis. Micrographs in (A) show that activated microglia (Iba1, green) were largely present at the center of the EB‐induced demyelination lesion in both pSal and pLPS rat groups. Astrocytes (GFAP, red) were mainly present at the edge of the demyelination lesion. DAPI (blue) was used to detect nuclear DNA. Micrographs in (B) show GFAP immunoreactivity at the edge of the lesion (red). The micrographs in black and white are a binarization rendering of the GFAP staining (binarized). The area fraction occupied by GFAP staining in the binarized pictures was measured and presented in the right upper bar graph while the integrated intensity of the GFAP immunofluorescence is presented in the right lower bar graph. EB injection resulted in a significant increase in the integrated intensity of GFAP in both saline‐ and LPS‐treated offspring. Prenatal LPS affected neither the fraction area occupied by GFAP immunoreactivity nor the overall integrated intensity of the GFAP immunofluorescence. Scale bar=50 μm

Figure 6 Long‐lasting impact of prenatal LPS on pIκB expression in microglia. Micrographs in (A and B) show pIκB (green) and Iba1 (red) immunodetection in the demyelinated area of the corpus callosum of rats born to dams given either saline (pSal) or LPS (pLPS) during pregnancy. Yellow arrowheads in the merged channels point to microglial cells containing pIκB while white arrows point to pIκB proteins, which are not localized in microglial cells. The white squares in the micrographs delineate the areas that are presented with a higher magnification below these micrographs. The bar graph in (C, upper panel) shows that prenatal LPS exposure resulted in a significant decrease in the density of cells containing pIκB. The bar graph in (C, lower panel) shows that prenatal LPS induced a significant reduction in the fraction of pIκB+ cells colabeled with the microglial marker Iba1. *P<.05. Scale bar=50 μm