Intrauterine inflammation, insufficient to induce parturition, still evokes fetal and neonatal brain injury

Abstract

Exposure to prenatal inflammation is a known risk factor for long term neurobehavioral disorders including cerebral palsy, schizophrenia, and autism. Models of systemic inflammation during pregnancy have demonstrated an association with an immune response an adverse neurobehavioral outcomes for the exposed fetus. Yet, the most common route for an inflammatory exposure to a fetus is from intrauterine inflammation as occurs with chorioamnionitis. The aims of this study were to assess the effect of intrauterine inflammation on fetal and neonatal brain development and to determine if the gestational age of exposure altered the maternal or fetal response to inflammation. CD-1 timed pregnant mice on embryonic day 15 (E15) and E18.5 were utilized for this study. Dams were randomized to receive intrauterine infusion of lipopolysaccharide (LPS, 50 μg/dam) or normal saline. Different experimental groups were used to assess both acute and long-term outcomes. For each gestational age and each treatment group, fetal brains, amniotic fluid, maternal serum and placentas were collected 6h after intrauterine infusion. Rates of preterm birth, maternal morbidity and litter size were assessed. IL6 levels were assayed in maternal serum and amniotic fluid. An immune response was determined in the fetal brains and placentas by QPCR. Cortical cultures were performed to assess for fetal neuronal injury. Gene expression changes in postnatal day 7 brains from exposed and unexposed pups were determined. In the preterm period, low dose LPS resulted in a 30% preterm birth rate. Litter sizes were not different between the groups at either gestational age. IL6 levels were not significantly increased in maternal serum at either gestational time period. Low dose LPS increased IL6 levels in the amniotic fluid from exposed dams in the term but not preterm period. Regardless of gestational age of exposure, low dose intrauterine LPS activated an immune response in the placenta and fetal brain. Exposure to intrauterine LPS significantly decreased dendritic counts in cortical cultures from both the preterm and term period. Exposure to intrauterine inflammation altered gene expression patterns in the postnatal brain; this effect was dependent on gestational age of exposure. In conclusion, intrauterine inflammation, even in the absence of preterm parturition, can evoke fetal brain injury as evidence by alterations in cytokine expression and neuronal injury. Despite an absent or limited maternal immune response in low dose intrauterine inflammation, the immune system in the placenta is activated which is likely sufficient to induce a fetal immune response and subsequent brain injury. Changes in the fetal brain lead to changes in gene expression patterns into the neonatal period. Subclinical intrauterine inflammation can lead to fetal brain injury and is likely to be mechanistically associated with long term adverse outcomes for exposed offspring.

Figure 1

IL6 levels in maternal serum. Bar graph depicting means and standard deviations of IL6 levels in maternal serum from preterm (E15) and term (E18.5) exposure to low dose intrauterine LPS. Low dose intrauterine LPS did not significantly alter maternal IL6 levels in the preterm period. At term (E18.5), IL6 levels increased 3-fold but this was not statistically significant (P=0.09, T-test).

Figure 2

IL6 levels in amniotic fluid. Bar graph depicting means and standard deviations of IL6 levels in amniotic fluid from preterm (E15) and term (E18.5) exposure to low dose intrauterine LPS. There was wide variation in IL6 levels between the dams; the data were not normally distributed. IL6 levels were increased in the amniotic fluid in the preterm period but this did not reach statistical significance (Mann Whitney Rank Sum). In the term period, intrauterine inflammation significantly increased IL6 levels 27-fold compared to saline-exposed (P=0.004, Mann Whitney Rank Sum)

Figure 3

Cytokine expression in the placenta. Bar graph representing mean mRNA expression normalized to the amount of 18s rRNA in placentas exposed to saline or low dose LPS at both gestational time points (E15 and E18.5). Exposure to low dose LPS increased placental IL1β 12-fold (*P=0.008) on E15 and 5.8-fold on E18.5 (*P=0.009). Low dose LPS increased IL6 mRNA expression 8.6-fold (P=0.008) on E15 and 87-fold (P=0.06) on E18.5. Low dose LPS increased TNF 7-fold (P=0.002) on E15 and 5-fold (P=0.04) on E18.5. MRNA expression was compared using T-test when data was normally distributed and Mann Whitney Rank Sum test when the data was non-parametric.

Figure 4

Immune response in the fetal brain. Bar graph representing mean mRNA expression normalized to the amount of 18s rRNA in fetal brains exposed to saline or low dose LPS at both gestational time points (E15 and E18.5). In the preterm (E15) period, compared to saline, exposure to intrauterine low dose LPS resulted in an 2.8-fold increase in IL1 beta (P=0.007), a 1.4-fold increase in TNF (P=0.018) and 1.6-fold increase in COX-2 (P=0.02) mRNA expression. On E18.5, compared to saline, exposure to intrauterine low dose LPS increased IL1 beta 1.9-fold (P=0.13), TNF 1.8-fold (P=0.04) and COX2 (1.6-fold, P=0.04). IL6 mRNA expression was not significantly different in brains exposed to saline compared to LPS. Mean mRNA expression was compared using T-test for each gestational time period.

Figure 5

Fetal neuronal injury. Bar graph representing means and standard deviations of dendritic counts in the preterm and term period after exposure to low dose intrauterine inflammation or saline. Exposure to low dose LPS significantly decreased dendritic counts on E15 and E18.5 compared to saline-exposed (P<0.001 for both gestational time periods).