Prenatal stress causes alterations in the morphology of microglia and the inflammatory response of the hippocampus of adult female mice

Abstract

Background: Stress during fetal life increases the risk of affective and immune disorders later in life. The altered peripheral immune response caused by prenatal stress may impact on brain function by the modification of local inflammation. In this study we have explored whether prenatal stress results in alterations in the immune response in the hippocampus of female mice during adult life.

Methods: Pregnant C57BL/6 mice were subjected three times/day during 45 minutes to restraint stress from gestational Day 12 to delivery. Control non-stressed pregnant mice remained undisturbed. At four months of age, non-stressed and prenatally stressed females were ovariectomized. Fifteen days after surgery, mice received an i.p. injection of vehicle or of 5 mg/kg of lipopolysaccharide (LPS). Mice were sacrificed 20 hours later by decapitation and the brains were removed. Levels of interleukin-1β (IL1β), interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), interferon γ-inducible protein 10 (IP10), and toll-like receptor 4 mRNA were assessed in the hippocampus by quantitative real-time polymerase chain reaction. Iba1 immunoreactivity was assessed by immunocytochemistry. Statistical significance was determined by one-way or two-way analysis of variance.

Results: Prenatal stress, per se, increased IL1β mRNA levels in the hippocampus, increased the total number of Iba1-immunoreactive microglial cells and increased the proportion of microglial cells with large somas and retracted cellular processes. In addition, prenatally stressed and non-stressed animals showed different responses to peripheral inflammation induced by systemic administration of LPS. LPS induced a significant increase in mRNA levels of IL-6, TNF-α and IP10 in the hippocampus of prenatally stressed mice but not of non-stressed animals. In addition, after LPS treatment, prenatally stressed animals showed a higher proportion of Iba1-immunoreactive cells in the hippocampus with morphological characteristics of activated microglia compared to non-stressed animals. In contrast, LPS induced similar increases in expression of IL1β and toll-like receptor 4 in both prenatally stressed and non-stressed animals.

Conclusion: These findings indicate that prenatal stress induces long-lasting modifications in the inflammatory status of the hippocampus of female mice under basal conditions and alters the immune response of the hippocampus to peripheral inflammation.

Figure 1

A) Representative images of the dentate gyrus of hippocampus showing immunoreactivity for Iba1. A) Non-stressed female treated with vehicle; B) Prenatally stressed female treated with vehicle; C) Non-stressed female treated with LPS and D) Prenatally stressed female treated with LPS. Inserts show details of the morphology of Iba1-immunoreactive cells at high magnification. Scale bar, 50 μm. In the inserts, the scale bar represents 7.5 μm. B) Number of Iba1-immunoreactive cells/mm³ in the hilus of dentate gyrus of hippocampus. NS, non-stressed female mice; PS, prenatally stressed females. Filled bars, animals treated with vehicle (VEH). Empty bars, animals treated with LPS. Data are mean ± SEM. *, Significant differences (P <0.05) versus NS-VEH. ^aSignificant difference (P <0.05) versus NS-VEH and PS-VEH mice. ^bbSignificant difference (P <0.01) versus NS-VEH and PS-LPS.

Figure 2

Morphological changes of Iba1-immunoreactive cells in the hilus of dentate gyrus of hippocampus. The upper panels show examples of the five morphological types in which Iba1-immunoreactive cells were classified: Type I, cells with few cellular processes (two or less); Type II, cells showing four short branches; Type III, cells with numerous cell processes and a small cell body; Type IV, cells with large somas and retracted and thicker processes and Type V, cells with amoeboid cell body, numerous short processes and intense Iba1 immunostaining. The graph shows the proportion of each morphological type in non-stressed (NS) and prenatally stressed (PS) animals treated with vehicle (VEH) or LPS. aaa, Significant difference (P <0.001) of II type cells versus PS-VEH, NS-LPS and PS-LPS mice. bbb, Significant difference (P <0.001) of III type cells between NS-VEH, NS-LPS and PS-LPS mice. ccc, Significant difference (P <0.001) of type IV cells between NS-VEH mice. ddd, Significant difference (P <0.001) of type IV cells versus PS-LPS mice. eee, Significant difference (P <0.01) of type IV cells versus PS-VEH mice. ff, Significant difference versus NS-VEH and PS-VEH mice.

Figure 3

mRNA levels of inflammatory markers in the hippocampus. A) Interleukin-1β (IL1β); B) Interleukin-6 (IL6); C) Tumor necrosis factor α (TNF-α) and D) IFN-inducible protein 10 (IP10). NS, non-stressed female mice; PS, prenatally stressed females. Filled bars, animals treated with vehicle (VEH). Empty bars, animals treated with LPS. Data are mean ± SEM. *,**,** Significant differences (*P <0.05, **P <0.01; ***P <0.001) of LPS groups versus their respective VEH groups. a, significant differences (P <0.05) versus NS-LPS values. #, Significant difference (P <0.05) versus NS-VEH group.

Figure 4

Toll-like receptor 4 (TLR4) mRNA levels in the hippocampus. NS, non-stressed female mice; PS, prenatally stressed females. Filled bars, animals treated with vehicle (VEH). Empty bars, animals treated with LPS. Data are mean ± SEM. *, Significant differences (P <0.05) of LPS groups versus their respective VEH groups.