Consequences of Early Maternal Deprivation on Neuroinflammation and Mitochondrial Dynamics in the Central Nervous System of Male and Female Rats

Abstract

Early life stress (ELS) is associated with an increased risk for neuropsychiatric disorders, and both neuroinflammation and mitochondrial dysfunction seem to be central to mental health. Herein, using an animal model of ELS, a single episode of maternal deprivation (MD, 24 h on pnd 9) extensively documented to elicit behavioural anomalies in male and female Wistar rats, we investigated its consequences in terms of neuroinflammation and mitochondrial dynamics in the prefrontal cortex (PFC) and the hippocampal formation (HCF). MD differentially affected the brain content of cytokines: MD induced a transient increase in pro-inflammatory cytokines (IL-1β and IL-6) in the PFC, as well as in the levels of the anti-inflammatory cytokine IL-10 in the HCF. MD also induced a significant decrease mitochondria citrate synthase activity, but MD did not exert significant changes in mitochondria Complex IV activity, revealing a generalized decrease in mitochondrial density without any change in mitochondrial respiration. In the present study, we demonstrate that MD induces neuroinflammatory processes in specific brain regions. Additional research is needed to better understand the temporal pattern of such changes, their impact on the developing brain, and their participation in the already well-known behavioural consequences of MD.

Keywords: citrate synthase; complex IV; cytokines; early life stress (ELS); hippocampal formation; inflammatory processes; mitochondria; neonatal; prefrontal cortex; sex dimorphism.

Figure 1

Experimental design. The male and female offspring of Wistar rats were exposed to early life stress, maternal deprivation (MD, 24 h on pnd 9). During the study, animals’ body weight (BW) was recorded, animals’ performance in the righting reflex (RR) was evaluated, maternal behaviour was observed, and animals were then sacrificed at postnatal days 13 and 20 to obtain brain samples: prefrontal cortex (PFC) and hippocampal formation (HCF).

Figure 2

Effects of Maternal Deprivation (MD) on body weight (BW) gain from postnatal day (pnd) 9 until the end of the study. Rats were exposed to early life stress, MD (24 h on pnd 9, solid lines and symbols) or subjected to the same manipulation except for the separation period (CTRL, dashed line, and empty symbols). Pups were individually weighted before the procedure (pnd 9), immediately after (pnd 10), and on the day prior to their sacrifice (pnd 12 and 19); mean values are grouped by litter (12 individuals from three litters per experimental group). Data are presented as the mean body weight value (g) ± standard deviation (SD) of both male and female animals (Males: circles; Females: squares). As an insert: the percentage of BW changes during the deprivation period (mean ± SD). Repeated measures two−way ANOVA, followed by Tukey post-hoc comparisons: bbbb, p < 0.001, significant overall effect of neonatal condition.

Figure 3

Effects of maternal deprivation (MD) on the cytokine profile of the prefrontal cortex (PFC) of rats at pnd 13 (A,C,E,G) and at pnd 20 (B,D,F,H). Histograms represent the mean ± SD (five animals per experimental group). ANOVA and Tukey post-hoc comparisons or nonparametric analysis using the Kruskal–Wallis test followed by the Mann–Whitney test: a, p < 0.05 significant overall effect of sex; b, p < 0.05 significant overall effect of MD. Age differences are explained in the text.

Figure 4

Effects of maternal deprivation (MD) on the cytokine profile of the hippocampal formation (HCF) of rats at pnd 13 (A,C,E,G) and at pnd 20 (B,D,F,H). Histograms represent the mean ± SD (5 animals per experimental group). ANOVA and Tukey post-hoc comparisons or non-parametric analysis using the Kruskal–Wallis test followed by the Mann–Whitney test: b, p < 0.05 significant overall effect of MD. Age differences are explained in the text.

Figure 5

Effects of maternal deprivation (MD) on the activity of mitochondrial components of the respiratory chain, citrate synthase (CS), and complex IV (CIV) in the prefrontal cortex (PFC): CS activity at pnd 13 and 20 (A,B, respectively), CIV activity (C,D, respectively), and the activity ratio of CS relative to CIV (E,F, respectively). Histograms represent the mean ± SD (five animals per experimental group). ANOVA and Tukey post-hoc comparisons or nonparametric analysis using the Kruskal–Wallis test followed by the Mann–Whitney test: b, p < 0.05 significant overall effect of MD.

Figure 6

Effects of maternal deprivation (MD) on the activity of mitochondrial components of the respiratory chain, citrate synthase (CS), and complex IV (CIV) in the hippocampal formation (HF): CS activity at pnd 13 and 20 (A,B, respectively), CIV activity (C,D, respectively), and the activity ratio of CS relative to CIV (E,F, respectively). Histograms represent the mean ± SD (five animals per experimental group). ANOVA and Tukey post-hoc comparisons or nonparametric analysis using the Kruskal–Wallis test followed by the Mann–Whitney: a, p < 0.05 significant overall effect of sex; b, p < 0.05 significant overall effect of MD; c, p < 0.05 significant overall effect of age.

Figure 7

Effects of maternal deprivation (MD) on the expression levels of PINK and Parkin in the prefrontal cortex (PFC): PINK expression at pnd 13 and 20 (A,B, respectively) and Parkin expression (C,D, respectively) expressed as the change in expression relative to CTRL males. Representative images from the Western blots performed are presented under each histogram. Histograms represent the mean ± SD (four to five animals per experimental group). ANOVA and Tukey post-hoc comparisons or nonparametric analysis using the Kruskal–Wallis test followed by the Mann–Whitney: a, p < 0.05 significant overall effect of sex; * p < 0.05 vs. CTRL male; &&&, p < 0.005 vs. CTRL females. See “Supplementary Materials” for an example of the original Western blot images.

Figure 8

Effects of maternal deprivation (MD) on the expression levels of PINK and Parkin within the hippocampal formation (HCF): PINK expression at pnd 13 and 20 (A,B, respectively) and Parkin expression (C,D, respectively) expressed as the change in expression relative to CTRL males. Representative images from the Western blots performed are presented under each histogram. Histograms represent the mean ± SD (four to five animals per experimental group). ANOVA and Tukey post-hoc comparisons or nonparametric analysis using the Kruskal–Wallis test followed by the Mann–Whitney test: a, p < 0.05 significant overall effect of sex; b, p < 0.05 significant overall effect of MD; **** p < 0.001 vs. CTRL male; &&&, p < 0.005 vs. CTRL females. See “Supplementary Materials” for an example of the original Western blot images.