3.1 The effect of LPS-induced intrauterine infection on survival of female and neonatal rats
In order to explore the appropriate dose of LPS for establishing an animal model of intrauterine infection, we observed maternal mortality, abnormal pregnancy rates, normal delivery rates and neonatal mortality by intraperitoneal injection of different doses of LPS in pregnant rats(Fig. 1A,n = 40 pregnant rats). We found that the increased doses of LPS did not cause premature delivery, but the fetus died in utero and were absorbed by the mothers or were directly responsible for the mother's death. All newborns were delivered at term. The normal delivery rates decreased with the increased LPS doses of 0,0.3,0.5,0.7,0.9 mg/kg,and the normal delivery rates were 100%, 50%, 33%, 25%and 0%. The abnormal pregnancy rates, which were the rates at which the fetus died in utero and were absorbed by the mothers, also varied with the dose of LPS. When LPS was administered at 0 ,0.3, 0.5, 0.7 mg/kg, the abnormal pregnancy rates were 0%, 50%, 67%, 75%, and the maternal mortality were all at 0%.However, when the dose was raised to 0.9 mg/kg, the maternal mortality rate rose by 25% with an abnormal pregnancy rate of 75%. As a result, we chosed a dose of 0.7 mg/kg LPS for subsequent experimental studies to maximize intestinal changes and adequate litters while minimizing the risk of maternal mortality. In order to ensure that pregnant rats have a similar response to chorioamnionitis by intraperitoneal injection of LPS at a dose of 0.7 mg/kg, we collected uterus specimens for pathological examination. Pregnant rats exposed to LPS had obvious inflammatory cell infiltration in the uterus(Fig. 1B).
During the experimental period of 7 days,we occasionally found the phenomenon that mothers ate their pups༌and apart from that, none of the pups die. The activities, feeding and hair color of all pups included in the LPS group and the sham group were normal after birth. There was no significant difference of birth weight and body weight gains between the LPS group and the sham group (Fig. 1C,n = 136 pups).
3.2 Histological analyses of intestinal ileum indicated that prenatal LPS exposure induced intestinal injury in neonatal rats
To determine whether prenatal LPS exposure would induce neonatal intestinal injury and has an effect on intestinal development, we collected distal ileum from different ages of pups for pathological histological analyses.We found that the overall structure of intestinal mucous from the sham groups were normal at any age,the intestinal villis were well-shaped and neatly arranged, the intestinal mucosal epithelial cells were not necrotic and exfoliated, and no inflammatory cells infiltration were observed in the intestinal mucosal layer(Fig. 2A). However, the intestinal structure of pups exposed to LPS at first days of life were disordered. Compared with the sham group, we could find shortened and irregular villis, edema of the submucosa, and the infiltration of a large number of inflammatory cells in the submucosa. At 3 days after birth, a large number of mucosal epithelial cells were denatured and swollen, submucosal edema was further aggravated and a large number of inflammatory cells were infiltrated. However, on the 7th day, the intestinal mucosa structure was better than before, villis structure were orderly arranged, no inflammatory cell infiltration was observed, but edema of some mucosal epithelial cells was still visible(Fig. 2A).
The results indicated that prenatal LPS exposure definitely induced fetal intestinal injury. Subsequently,we further assessed the injury using the intestinal injury score as previously described. The injury scores of the pups exposed to LPS were significantly higher than that of the sham groups at d0 and d3(Fig. 2B)( P = 0 .045, n = 9 pups in the sham group and 7 pups in the LPS group at d0; P = 0.038, n = 7 pups in the sham group and 9 pups in the LPS group at d3).Although the difference did not reach significance, the injury scores of LPS group was still higher than the sham group at 7 days after birth (P = 0.909; LPS n = 7, Sham n = 6).
3.3 Immunohistochemical analysis suggested that prenatal LPS exposure caused the loss of intestinal wall integrity in neonatal rats
We further evaluated intestinal wall integrity by staining epithelial tight junction protein ZO-1, which plays an important role in maintaining intestinal wall barrier integrity by connecting intestinal epithelial cells[23, 24].As shown in Fig. 2C, the intestinal epithelium ZO-1 protein in the sham groups showed a strong brown staining and was evenly distributed on the top of the junction of ileal epithelial cells. However, the ZO-1 protein staining in the ileum tissue of the LPS group was weaker and unevenly distributed, showing discontinuous spots or short bands.
Compared to controls,intestinal ZO-1 expression in pups with prenatal LPS exposure decreased significantly at d0 and d3 (Fig. 2D)( P = 0 .005 n = 10 pups in the sham group and 7 pups in the LPS group at d0; P = 0.003, n = 8 pups in the sham group and 10 pups in the LPS group at d3). At 7 days, we observed that the expression level of ZO-1 in the LPS group was slightly lower than that in the sham group, but there was no statistical difference(P = 0.327; Sham n = 8, LPS n = 7).
3.4 LPS-induced intestinal injury changes the composition of the intestinal microbiome
We next investigated the fecal microbiota to determine if prenatal LPS exposure would affect gut colonization in rats. As shown in Fig. 3A, the Shannon diversity index in two groups gradually increased with the increase of age, and the diversity index of the sham group at d7 was significantly higher than that of pups at 3 days after birth(P < 0.05), however, no significant difference was found in the diversity index between different ages in the LPS groups. Compared to sham controls,there were no significant differences in diversity index at any ages.
We further investigated the microbiota composition in different groups at different ages at the phyla and genus levels. At the phylum level, the composition of the microbiota in 20 fecal specimens of the two groups of pups revealed that the phyla of Firmicutes,Proteobacteria were the most abundant(Fig. 3B). The relative abundance of Bacteroidetes in the LPS group(0.045%) was significantly higher as compared to the sham group(0.006%) at 3 days of life (P = 0.045)( Fig. 3C). Interestingly, we found that the relative abundance of the Firmicutes in the sham group increased significantly from 49.52–91.61% (P = 0.012) and the relative abundance of the proteobacteria decreased significantly from 49.96–7.93% (P = 0.012) with the increasing age, however, no significant differences were observed in the composition of the microbiota at the phylum level among different ages in the LPS groups.Furthermore, we found that the proportion of Proteobacteria and Bacteroidetes in the LPS group significantly increased (P = 0.012 and 0.044)and the proportion of Firmicutes significantly decreased(P = 0.012) when compared with that in the Sham group at 7 days after birth (Fig. 3D).
As shown in Fig. 4, at the genus level, the population of Actinomyces and Enterococcus in the LPS group at 3 days of life was lower than the Sham group (P = 0.036 and 0.011), and the relative abundance of Bacteroides was higher in the LPS group compared to the Sham groups (P = 0.025)(Figure 4B).At 7 days of life, fecal samples from the LPS group had higher levels of Escherichia-Shigella(P = 0.012)and Bacteroides (P = 0.044)compared to Sham group. Meanwhile, the proportion of Lactobacillus, Rodentibacter and Veillonella significant decreased in LPS group as compared to Sham group(P = 0.012,0.036 and 0.021) (Figure 4C).