Juvenile peripheral LPS exposure overrides female resilience to prenatal VPA effects on adult sociability in mice

Abstract

Autism spectrum disorder (ASD) exhibits a gender bias, with boys more frequently affected than girls. Similarly, in mouse models induced by prenatal exposure to valproic acid (VPA), males typically display reduced sociability, while females are less affected. Although both males and females exhibit VPA effects on neuroinflammatory parameters, these effects are sex-specific. Notably, females exposed to VPA show increased microglia and astrocyte density during the juvenile period. We hypothesized that these distinct neuroinflammatory patterns contribute to the resilience of females to VPA. To investigate this hypothesis, we treated juvenile animals with intraperitoneal bacterial lipopolysaccharides (LPS), a treatment known to elicit brain neuroinflammation. We thus evaluated the impact of juvenile LPS-induced inflammation on adult sociability and neuroinflammation in female mice prenatally exposed to VPA. Our results demonstrate that VPA-LPS females exhibit social deficits in adulthood, overriding the resilience observed in VPA-saline littermates. Repetitive behavior and anxiety levels were not affected by either treatment. We also evaluated whether the effect on sociability was accompanied by heightened neuroinflammation in the cerebellum and hippocampus. Surprisingly, we observed reduced astrocyte and microglia density in the cerebellum of VPA-LPS animals. These findings shed light on the complex interactions between prenatal insults, juvenile inflammatory stimuli, and sex-specific vulnerability in ASD-related social deficits, providing insights into potential therapeutic interventions for ASD.

Keywords: Autism spectrum disorder; Inflammation.

Figure 1

Intraperitoneal LPS injection induces an inflammatory response in young animals, and the every-other-day protocol of chronic inflammation allows animals to gain weight normally. (A) Animals prenatally exposed to the vehicle exhibit an increase in IL-1 expression 2 h after ip LPS injection, both in the spleen and in the hippocampus. ANOVA, Treatment effect: ***p < 0.001. n = 3 animals/group. (B) Throughout the juvenile chronic LPS injections, all animals exhibited similar weight gain patterns. n = 12–13 animals/group.

Figure 2

Juvenile LPS-exposure results in reduced sociability but normal repetitive behavior, exploration, and anxiety. (A) Animals were prenatally exposed to VPA or vehicle (VEH), and then injected every other day with LPS or saline (SAL) between PD22 and PD34. Behavior was evaluated in adulthood (> PD60). Animals were tested in the social interaction test, self-grooming test, and open field test, with one-week inter-test intervals. (B–D) Social interaction: (B) Time spent sniffing left (L) and right (R) cylinders during a 5-min habituation stage; (C) Time spent sniffing the cylinder containing the social stimulus (S) or the novel object (nonsocial, NS) during the 10-min test. ANOVA, Stimulus effect: ^###p < 0.001. (D) Sociability index was calculated as (S-NS)/(S + NS). ANOVA followed by Tukey’s posthoc analysis: *p < 0.05, **p < 0.01. (E) Time spent grooming in the 10-min self-grooming test. (F,G) Open field: (F) Total distance walked during a 10-min open field exposure; (G) Time spent in the center of the open field. Individual data are depicted by black dots, and group information is represented by the mean ± s.e.m. (B), (C) and (E–G)) or a boxplot (D). n = 10–13 animals/group.

Figure 3

Juvenile LPS exposure reverses VPA effects on cerebellar astrocytes and microglia. GFAP-positive area was determined in the molecular layer (A) and granular cell layer (B) of the lobule 7 of the cerebellum. Microglial (Iba1-positive) cell density was quantified in the molecular layer (C) and granular cell layer (D) of the lobule 7 of the cerebellum. In each region, total cells, ramified cells, and hypertrophic cells are reported. (E,F) Sholl analysis of Iba1-positive cells is reported for both regions. (G,H) Soma size was estimated for Iba1-positive cells. (I) Representative images of anti-GFAP immunofluorescence. Scale bar, 50 μm. (J) Representative images of anti-Iba1 immunofluorescence. Scale bar, 50 μm. (K) Linear density of calbindin-positive Purkinje cells in the lobule 7 of the cerebellum is shown. (L) Representative images of anti-calbindin immunohistochemistry. Scale bar, 50 μm. n = 4–5 animals/group. ANOVA followed by Tukey’s posthoc analysis: *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

Acute juvenile LPS effects on cerebellar lobule 7 astrocytes and microglia. (A–D) Two hours after SAL or LPS injection at PD22, GFAP and Iba1-positive cells were analyzed in the lobule 7 of the cerebellum. GFAP-positive area was determined in the molecular layer (A) and granular cell layer (B) of the lobule 7 of the cerebellum. Microglial (Iba1-positive) cell density was quantified in the molecular layer (C) and granular cell layer (D) of the lobule 7 of the cerebellum. Total cells, ramified cells, and hypertrophic cells are reported for each region. (E–H) At PD36 -two days after the last SAL or LPS injection-, GFAP and Iba1-positive cells were analyzed in the lobule 7 of the cerebellum. GFAP-positive area was determined in the molecular layer (E) and granular cell layer (F) of the lobule 7 of the cerebellum. Microglial (Iba1-positive) cell density was quantified in the molecular layer (G) and granular cell layer (H) of the lobule 7 of the cerebellum. Total cells, ramified cells, and hypertrophic cells are reported for each region. n = 4–5 animals/group. Two-way ANOVA main effects: *p < 0.05.