An investigation into the effects of antenatal stressors on the postpartum neuroimmune profile and depressive-like behaviors

Abstract

Postpartum depression is a specific type of depression that affects approximately 10-15% of mothers [28]. While many have attributed the etiology of postpartum depression to the dramatic change in hormone levels that occurs immediately postpartum, the exact causes are not well-understood. It is well-known, however, that pregnancy induces a number of dramatic changes in the peripheral immune system that foster the development of the growing fetus. It is also well-known that changes in immune function, specifically within the brain, have been linked to several neuropsychiatric disorders including depression. Thus, we sought to determine whether pregnancy induces significant neuroimmune changes postpartum and whether stress or immune activation during pregnancy induce a unique neuroimmune profile that may be associated with depressive-like behaviors postpartum. We used late-gestation sub-chronic stress and late-gestation acute immune activation to examine the postpartum expression of depressive-like behaviors, microglial activation markers, and inflammatory cytokines within the medial prefrontal cortex (mPFC) and the hippocampus (HP). The expression of many immune molecules was significantly altered in the brain postpartum, and postpartum females also showed significant anhedonia, both independently of stress. Following late-gestation immune activation, we found a unique set of changes in neuroimmune gene expression immediately postpartum. Thus, our data indicate that even in the absence of additional stressors, postpartum females exhibit significant changes in the expression of cytokines within the brain that are associated with depressive-like behavior. Additionally, different forms of antenatal stress produce varying profiles of postpartum neuroimmune gene expression and associated depressive-like behaviors.

Keywords: Depression; Forced swim test; Immune activation; Inflammation; LPS; Microglia; Neuroimmune; Postpartum; Pregnancy; Stress.

Figure 1. Timeline of Experiments 1 and 2

The experimental manipulations for Experiments 1.1, 1.2, 1.3, 2.1, and 2.2 are depicted. Animals in Experiments 1.1 and 2.1 did not undergo behavioral analysis and remained undisturbed during the time-matched equivalent of pre-pregnancy sucrose preference testing for females in Experiments 1.2, 1.3, and 2.2. All animals were bred, and females that did not get pregnant were assigned to the “Non-Pregnant” group. Females in Experiment 1 were subjected to forced swim stress or remained undisturbed between Embryonic (E) days 16-22. Females in Experiment 2 were treated with an i.p. injection of lipopolysaccharide (100μg/kg) or saline on E21. All females gave birth, and females in Experiments 1.1 and 2.1 were euthanized on the day of birth to collect brain tissue for analysis. Females in Experiments 1.2, 1.3, and 2.2 underwent postpartum sucrose preference testing immediately postpartum on P0-1, and animals in Experiments 1.3 and 2.2 repeated this test one week later on P7-8.

Figure 2. Effects of Reproductive State and Antenatal Forced Swim Test on Relative Inflammatory Gene Expression in the Medial Prefrontal Cortex and Hippocampus Immediately Postpartum

Stressed rats received daily forced swim stress from Embryonic (E) day 16 through E21 while rats in the No Stress groups remained undisturbed. Rats were sacrificed on the day of birth (P0) or 24 hours after the last day of forced swim test for non-pregnant animals. A, C, D. In the mPFC, CD11b, IL-6, and BDNF showed main effects of reproductive state. B. Il-1β analysis showed significant main effects of both reproductive state and forced swim stress. There were no effects on IL-4 expression in mPFC. E. In the HP, analysis revealed a significant main effect of reproductive state on the expression of IL-4. There were no significant changes in relative gene expression of CD11b, IL-1β, IL-6, or BDNF. *: p < 0.05; **: p < 0.001

Figure 3. Effects of Reproductive State and Antenatal Forced Swim Test on Microglia Density in the Medial Prefrontal Cortex and CA1, CA3, and Dentate Gyrus Regions of the Hippocampus Immediately Postpartum

Stressed rats received daily forced swim test from Embryonic (E) day 16 through E21 while rats in the No Stress groups remained undisturbed. Rats were sacrificed on the day of birth (P0) or 24 hours after the last day of forced swim test for non-pregnant animals and the density of Iba1 stain was performed in the (A) Medial Prefrontal Cortex, (B) CA1 hippocampus, (C) CA3 hippocampus, and (D) Dentate Gyrus [DG] of the hippocampus. B. Analysis of CA1 Iba1 densitometry revealed a significant main effect of reproductive state (p < 0.001) such that only postpartum females had significantly lower integrated density of microglia compared to non-pregnant animals. D. The DG revealed both a significant main effect of reproductive state as well as a main effect of sub-chronic stress (p < 0.05). There were no significant effects on integrated density of microglia in the mPFC or CA3 regions. *: p < 0.05; #: p < 0.001

Figure 4. Effects of Reproductive State and Antenatal Forced Swim Stress on Sucrose Preference Test Immediately Postpartum

Stressed rats received daily forced swim test from Embryonic (E) day 16 through E21 while rats in the No Stress groups remained undisturbed. Pre-Pregnancy Baseline tests were completed prior to breeding the animals. Sucrose measurements were taken on Postpartum (P) day 1. Statistical analysis revealed an interaction of reproductive state and stress immediately postpartum (p < 0.05). Post hoc analysis showed that Postpartum, No Stress and Non-pregnant, Stressed groups had significantly lower sucrose preference than Non-pregnant, No Stress controls. *: p < 0.05

Figure 5. Effects of Reproductive State and Antenatal Forced Swim Stress on Long-Term Sucrose Preference Postpartum

Stressed rats received daily forced swim test from Embryonic (E) day 16 through E21 while rats in the No Stress groups remained undisturbed. Pre-Pregnancy Baseline tests were completed prior to breeding the animals. Postpartum sucrose preference scores were taken on Postpartum (P) day 1 and P8. Statistical analysis revealed an interaction of reproductive state and stress immediately postpartum (P1) (p < 0.05). Post hoc analysis revealed Postpartum, No Stress and Non-pregnant, Stressed females had significantly lower sucrose preference than Non-pregnant, No Stress controls. There were no significant differences one week later. *: p < 0.05

Figure 6. Effects of Reproductive State and Acute Antenatal Immune Activation on Inflammatory Gene Expression in the Medial Prefrontal Cortex and Hippocampus Immediately Postpartum

Between Embryonic (E) day 21 and 22, rats received an injection of either lipopolysaccharide (LPS) (100μg/kg) or its saline vehicle at equal volume. Animals were euthanized on day of birth (P0) or at least 24 hours after the injection for non-pregnant animals. A. In the mPFC, analysis of CD11b expression revealed an interaction of pregnancy and immune activation. Post hoc analysis indicated the Postpartum, LPS-treated group had significantly lower expression than the Non-pregnant, LPS-treated group. B. IL-1β analysis revealed a significant interaction of pregnancy and immune activation. Post hoc analysis revealed trends that the Pregnant, LPS-treated group had lower expression than the Non-pregnant, LPS-treated and Postpartum, Saline-treated groups (p < 0.08). C. Analysis of IL-6 showed a significant main effect of pregnancy and an interaction of pregnancy and immune activation such that the Postpartum, Saline-treated animals and the Non-pregnant, LPS-treated animals had significantly higher expression compared to Non-pregnant, Saline-treated controls. D, E. IL-4 and BDNF analysis revealed a significant main effect of reproductive state in that only postpartum females showed higher gene expression relative to non-pregnant rats. F. In the HP, analysis of IL-1β showed a main effect of treatment. Only the Non-pregnant, LPS-treated group showed significantly higher gene expression compared to the other three groups (p < 0.05). G, H. IL-6 and IL-4 showed a main effect of pregnancy such that postpartum rats showed higher gene expression relative to the non-pregnant groups. No significant differences were found for CD11b or BDNF in the HP (not shown). *: p < 0.05; **: p < 0.001, #: p < 0.08

Figure 7. Effects of Reproductive State and Antenatal Immune Activation on Integrated Microglia Density in the Medial Prefrontal Cortex and CA1, CA3, and Dentate Gyrus Regions of the Hippocampus Immediately Postpartum

Between Embryonic (E) day 21 and 22, rats received an injection of either lipopolysaccharide (LPS) (100μg/kg) or its saline vehicle at equal volume. Animals were euthanized on day of birth (P0) or at least 24 hours after the injection for non-pregnant animals and the density of Iba1 stain was performed in the (A) Medial Prefrontal Cortex, (B) CA1 hippocampus, (C) CA3 hippocampus, and (D) Dentate Gyrus [DG] of the hippocampus. C. Analysis of CA3 revealed a significant main effect of reproductive state (p < 0.001) such that only postpartum females had significantly higher integrated density of microglia compared to non-pregnant animals. There were no significant effects on integrated density of microglia in the mPFC, CA1, or DG regions. **: p < 0.001

Figure 8. Effects of Reproductive State and Acute Antenatal Immune Activation on Immediate and Long-Term Sucrose Preference Postpartum

Between Embryonic (E) day 21 and 22, rats received an injection of either lipopolysaccharide (LPS) (100μg/kg) or its saline vehicle at equal volume. Pre-Pregnancy Baseline tests were completed prior to breeding the animals. Sucrose preference scores were taken on Postnatal (P) day 1, and one week later on P8. Analysis of sucrose preference revealed a significant main effect of reproductive state immediately postpartum (P1) such that females that had just given birth showed lower preference for sucrose compared to non-pregnant rats. One week later, analysis showed a main effect of treatment such that LPS-treated rats had greater sucrose preference compared to their saline counterparts. *: p < 0.05