Environmental influences on placental programming and offspring outcomes following maternal immune activation

Abstract

Adverse experiences during pregnancy induce placental programming, affecting the fetus and its developmental trajectory. However, the influence of 'positive' maternal experiences on the placenta and fetus remain unclear. In animal models of early life stress, environmental enrichment (EE) has ameliorated and even prevented associated impairments in brain and behavior. Here, using a maternal immune activation (MIA) model in rats, we test whether EE attenuates maternal, placental and/or fetal responses to an inflammatory challenge, thereby offering a mechanism by which fetal programming may be prevented. Moreover, we evaluate life-long EE exposure on offspring development and examine a constellation of genes and epigenetic writers that may protect against MIA challenges. In our model, maternal plasma corticosterone and interleukin-1β were elevated 3 h after MIA, validating the maternal inflammatory response. Evidence for developmental programming was demonstrated by a simultaneous decrease in the placental enzymes Hsd11b2 and Hsd11b2/Hsd11b1, suggesting disturbances in glucocorticoid metabolism. Reductions of Hsd11b2 in response to challenge is thought to result in excess glucocorticoid exposure to the fetus and altered glucocorticoid receptor expression, increasing susceptibility to behavioral impairments later in life. The placental, but not maternal, glucocorticoid implications of MIA were attenuated by EE. There were also sustained changes in epigenetic writers in both placenta and fetal brain as a consequence of environmental experience and sex. Following MIA, both male and female juvenile animals were impaired in social discrimination ability. Life-long EE mitigated these impairments, in addition to the sex specific MIA associated disruptions in central Fkbp5 and Oprm1. These data provide the first evidence that EE protects placental functioning during stressor exposure, underscoring the importance of addressing maternal health and well-being throughout pregnancy. Future work must evaluate critical periods of EE use to determine if postnatal EE experience is necessary, or if prenatal exposure alone is sufficient to confer protection.

Keywords: 11-β hydroxysteroid dehydrogenase; Corticotropin releasing hormone; Early life stress; Environmental enrichment; Fkpb5; Glucocorticoid receptor; Intervention; Maternal health; Maternal immune activation; Neurodevelopment; Opioid; Placenta; Programming; Stress.

Figure 1.

A) Schematic representation of timeline and experimental design. Experiment 1. Female rats habituated to either standard (SD) or environmentally enriched (EE) housing and bred. Pregnant rats received an injection of LPS (100 μg/kg) or equivolume of saline on gestational day 15. 3 h and 24 h after injection, plasma and tissue samples were collected from placenta, fetus and pregnant rats to analyze corticosterone, Hsd11b1, Hsd11b2, Nr3c1, Nr3c2, and epigenetic writers. Experiment 2. Pregnant SD and EE rats received an injection of LPS (100 μg/kg) or equivolume of saline on gestational Day 15. After birth on postnatal day (P)22, offspring were weaned. Between P25-P28, offspring were habituated to an open arena where social behavior tests would take place. On P29 and P30 the social preference and social discrimination tests were conducted, respectively. On the afternoon following behavioral testing on P29, offspring plasma, hippocampus and hypothalamus samples were collected for future analyses. B) Example set-ups of the multi-level environmental enrichment caging system. C) Representative photographs of the standard cages used in the present study.

Figure 2.

A) Illustration of maternal blood collection, embryonic tissue collection and PCR to determine fetal sex. Lane (L) 1; molecular weight ladder. L2 is the ‘known’ male (M) control sample, confirmed by the presence of the top band (Sry gene). L3 is the ‘known’ female (F) control, as depicted by an absence of Sry. L4 = female; L5–7 = males. Maternal plasma levels of B) corticosterone (pg/mL) and C) IL-1β (pg/mL) were elevated 3 h post LPS, validating maternal immune activation. Placental levels of D,G) Hsd11b1, E,H) Hsd11b2, F,I) Hsd11b2/Hsd11b1, J,N) OGT, K,O) DNMT1, L,P) DNMT3a, M,Q) MECP2, 3 h and 24 h post LPS. Group designations: SD-Saline (open squares), SD-LPS (closed squares), EE-Saline (open circles), EE-LPS (closed circles). Error bars represent mean ± SEM, n = 7 per sex/housing/MIA group. If there were no significant effects of sex, male and female offspring data were collapsed together for visualization. *p < 0.05, ** p < 0.01, *** p < 0.001, ****p <0.0001, LPS vs saline; ^ap < 0.05, ^aap < 0.01, ^aaap < 0.001, ^aaaap <0.0001, male vs female; ^bp < 0.05, ^bbp < 0.01, ^bbbp < 0.001, ^bbbbp <0.0001, standard housing vs environmental enrichment; ; ^&p < 0.05, ^&&p < 0.01, ^&&&p < 0.001, ^&&&&p <0.0001, SD-Saline vs SD-LPS; ^#p < 0.05, ^##p < 0.01, SD-LPS vs EE-LPS. Fetal brain data are presented in Supplementary Table 2.

Figure 3.

A) Illustration of juvenile locomotor activity, social preference (Trial 1), and social discrimination (Trial 2) tests using automated behavioral tracking software. B) Total distance traveled (mm), C) velocity (mm/sec), D) duration spent in center (sec), total time investigating (seconds) E) both the novel object and the novel conspecific, F) the novel conspecific, and G) the social preference score for Trial 1. H) Total distance traveled (mm), I) velocity (mm/sec), J) duration spent in center (sec), total time investigating (seconds) K) both the novel and familiar conspecifisc, L) the novel conspecific, and M) the social discrimination score for Trial 2. Group designations: SD-Saline (open squares), SD-LPS (closed squares), EE-Saline (open circles), EE-LPS (closed circles). Error bars represent mean ± SEM, n = 8–10 per sex/housing/MIA group. If there were no significant effects of sex, male and female offspring data were collapsed together for visualization. *p < 0.05, ** p < 0.01, *** p < 0.001, ****p <0.0001, LPS vs saline; ^ap < 0.05, ^aap < 0.01, ^aaap < 0.001, ^aaaap <0.0001, male vs female; ^bp < 0.05, ^bbp < 0.01, ^bbbp < 0.001, ^bbbbp <0.0001, standard housing vs environmental enrichment; ^&&p < 0.01, SD-Saline vs SD-LPS ^{; #}p < 0.05, SD-LPS vs EE-LPS.

Figure 4.

Juvenile brain mRNA expression of hippocampal (left) and hypothalamic (right) AB) Crh, CD) Crhr1, EF) Crhr2, GH) Crhr2/Crhr1 normalized to Gapdh. Group designations: SD-Saline (open squares), SD-LPS (closed squares), EE-Saline (open circles), EE-LPS (closed circles). Error bars represent mean ± SEM, n = 7 per sex/housing/MIA group. If there were no significant effects of sex, male and female offspring data were collapsed together for visualization. *p < 0.05, ** p < 0.01, *** p < 0.001, ****p <0.0001, LPS vs saline; ^ap < 0.05, ^aap < 0.01, ^aaap < 0.001, ^aaaap <0.0001, male vs female; ^bp < 0.05, ^bbp < 0.01, ^bbbp < 0.001, ^bbbbp <0.0001, standard housing vs environmental enrichment.

Figure 5.

Juvenile brain mRNA expression of hippocampal (left) and hypothalamic (right) AB) Nr3c1, CD) Nr3c2, EF) Nr3c1/Nr3c2, GH) Fkbp5, and I) Oprm1 normalized to Gapdh. Group designations: SD-Saline (open squares), SD-LPS (closed squares), EE-Saline (open circles), EE-LPS (closed circles). Error bars represent mean ± SEM, n = 7 per sex/housing/MIA group. If there were no significant effects of sex, male and female offspring data were collapsed together for visualization. *p < 0.05, ** p < 0.01, *** p < 0.001, ****p <0.0001, LPS vs saline; ^ap < 0.05, ^aap < 0.01, ^aaap < 0.001, ^aaaap <0.0001, male vs female; ^bp < 0.05, ^bbp < 0.01, ^bbbp < 0.001, ^bbbbp <0.0001, standard housing vs environmental enrichment.