Inflammatory, synaptic, motor, and behavioral alterations induced by gestational sepsis on the offspring at different stages of life

Abstract

Background: The term sepsis is used to designate a systemic condition of infection and inflammation associated with hemodynamic changes that result in organic dysfunction. Gestational sepsis can impair the development of the central nervous system and may promote permanent behavior alterations in the offspring. The aim of our work was to evaluate the effects of maternal sepsis on inflammatory cytokine levels and synaptic proteins in the hippocampus, neocortex, frontal cortex, and cerebellum of neonatal, young, and adult mice. Additionally, we analyzed the motor development, behavioral features, and cognitive impairments in neonatal, young and adult offspring.

Methods: Pregnant mice at the 14^th embryonic day (E14) were intratracheally instilled with saline 0.9% solution (control group) or Klebsiella spp. (3 × 10⁸ CFU) (sepsis group) and started on meropenem after 5 h. The offspring was sacrificed at postnatal day (P) 2, P8, P30, and P60 and samples of liver, lung, and brain were collected for TNF-α, IL-1β, and IL-6 measurements by ELISA. Synaptophysin, PSD95, and β-tubulin levels were analyzed by Western blot. Motor tests were performed at all analyzed ages and behavioral assessments were performed in offspring at P30 and P60.

Results: Gestational sepsis induces a systemic pro-inflammatory response in neonates at P2 and P8 characterized by an increase in cytokine levels. Maternal sepsis induced systemic downregulation of pro-inflammatory cytokines, while in the hippocampus, neocortex, frontal cortex, and cerebellum an inflammatory response was detected. These changes in the brain immunity were accompanied by a reduction of synaptophysin and PSD95 levels in the hippocampus, neocortex, frontal cortex, and cerebellum, in all ages. Behavioral tests demonstrated motor impairment in neonates, and depressive-like behavior, fear-conditioned memory, and learning impairments in animals at P30 and P60, while spatial memory abilities were affected only at P60, indicating that gestational sepsis not only induces an inflammatory response in neonatal mouse brains, but also affects neurodevelopment, and leads to a plethora of behavioral alterations and cognitive impairments in the offspring.

Conclusion: These data suggest that maternal sepsis may be causatively related to the development of depression, learning, and memory impairments in the litter.

Keywords: Depression; Gestational sepsis; Memory; Motor damage; Synaptophysin.

Fig. 1

Gestational sepsis causes low-weight offspring. a Clinical score evaluation of pregnant mice over time after induction of sepsis at E14 with Klebsiella pneumoniae instillation in the sepsis group and instillation of saline solution in the control group. Each point is the mean +/− SEM from at least 12 pregnant mice. b LPS solution (3 mg/kg, 10 mg/kg, and 15 mg/kg) and saline solution were administered in pregnant mice at E14. Mice were monitored over 48 h for clinical scores. c TNF-α, IL-1β, and IL-6 levels measured by ELISA in the placenta 24 h after induction of sepsis (i.e., E15). Each bar is the mean +/− SEM from 12 placentas. d Representative pictures and their body-size of offspring measured at P2 and P8. e, f Body-weight of offspring measured at P2 (e) and P8 (f). Each bar is the mean +/− SEM from 20 animals. *p < 0.05 compared saline to sepsis. **p < 0.05 compared 10 mg/kg, and 15 mg/kg to saline. ***p < 0.05 compared 5 mg/kg, 10 mg/kg, and 15 mg/kg to saline. NS not significant

Fig. 2

Motor impairments in neonates submitted to gestational sepsis. a, d For the cliff aversion test, offspring at P2 (a) and P8 (d) were positioned on the edge of the box and the total time that it took mice to turn away from the cliff and move away from the edge was recorded. Each bar is the mean +/− SEM from 3 litters with 10–12 offspring each. b, e The negative geotaxis assesses the motor coordination of animals at P2 (b) and P8 (e) by comparing the time to complete the test. Each bar is the mean +/− SEM from 3 litters with 10–12 offspring each. c, f The hindlimb suspension assesses the animal strength by recording their latency to fall at P2 (c) and P8 (f). Each bar is the mean +/− SEM from 3 litters with 10–12 offspring each. g, h The animals were subjected to the Rota Rod test to evaluate the motor ability in young (g) and adult (h) offspring from mothers submitted to gestational sepsis. Each bar is the mean +/− SEM from 3 litters with 10–12 offspring each. *p < 0.05 compared saline to sepsis. NS not significant

Fig. 3

Gestational sepsis downregulates synaptophysin and PSD95 expression, and induces immune activation in neonates’ brains at P2. a–d Western Blot was performed to detect synaptophysin and β-tubulin in the hippocampus (a), neocortex (b), frontal cortex (c), and cerebellum (d). Each bar is the mean +/− SEM from 3 to 7 animals. e–h Western blot was performed to detect PSD95 and β-tubulin in the hippocampus (e), neocortex (f), frontal cortex (g), and cerebellum (h). Each bar is the mean +/− SEM from 3 to 4 animals. i–l The immune response at P2 was evaluated with TNF-α, IL-1β, and IL-6 levels analyzed by ELISA in the hippocampus (i), neocortex j), frontal cortex (k), and cerebellum (l). Each bar is the mean +/− SEM from 3 animals from different litter each one. *p < 0.05 compared saline to sepsis. NS not significant

Fig. 4

Gestational sepsis downregulates synaptophysin and PSD95 expression, and induces immune activation in neonates’ brains at P8. a–d Western Blot was performed to detect synaptophysin and β-tubulin in the hippocampus (a), neocortex (b), frontal cortex (c), and cerebellum (d). Each bar is the mean +/− SEM from 4 to 6 animals. e–h Western blot was performed to detect PSD95 and β-tubulin in the hippocampus (e), neocortex (f), frontal cortex (g), and cerebellum (h). Each bar is the mean +/− SEM from 3 to 4 animals. (I – L) The immune response at P8 was evaluated through TNF-α, IL-1β, and IL-6 levels analyzed by ELISA in the hippocampus (i), neocortex (j), frontal cortex (k), and cerebellum (l). Each bar is the mean +/− SEM from 3 animals from different litter each one. *p < 0.05 compared saline to sepsis. NS not significant

Fig. 5

Gestational sepsis downregulates synaptophysin and PSD95 expression, and induces immune activation in young offspring brains (P30). a–d Western blot was performed to detect synaptophysin and β-tubulin in the hippocampus (a), neocortex (b), frontal cortex (c), and cerebellum (d). Each bar is the mean +/− SEM from 3 to 7 animals. e–h Western blot was performed to detect PSD95 and β-tubulin in the hippocampus (e), neocortex (f), frontal cortex (g), and cerebellum (h). Each bar is the mean +/− SEM from 3 to 4 animals. i–l The immune response at P30 was evaluated through TNF-α, IL-1β, and IL-6 levels analyzed by ELISA in the hippocampus (i), neocortex (j), frontal cortex (k), and cerebellum (l). Each bar is the mean +/− SEM from 3 animals from different litter each one. *p < 0.05 compared saline to sepsis. NS not significant

Fig. 6

Gestational sepsis downregulates synaptophysin and PSD95 expression and induces immune activation in adult offspring brains (P60). a–d Western blot was performed to detect synaptophysin and β-tubulin in the hippocampus (a), neocortex (b), frontal cortex (c), and cerebellum (d). Each bar is the mean +/− SEM from 4 to 7 animals. e–h Western blot was performed to detect PSD95 and β-tubulin in the hippocampus (e), neocortex (f), frontal cortex (g), and cerebellum (h). Each bar is the mean +/− SEM from 3 to 4 animals. i–l The immune response at P60 was evaluated through TNF-α, IL-1β, and IL-6 levels analyzed by ELISA in the hippocampus (i), neocortex (j), frontal cortex (k), and cerebellum (l). Each bar is the mean +/− SEM from 5 to 9 animals. *p < 0.05 compared saline to sepsis. NS not significant

Fig. 7

Cognitive impairment of young (P30) and adult (P60) offspring from mice subjected to gestational sepsis. a–d Morris water maze test. a, b The amount of time spent by young (a) and adult (b) offspring to climb the hidden platform along 4 days of experiment was measured. Each point is the mean +/− SEM from 9 to 12 animals. c, d Evaluated the time on the hidden platform quadrant of young (c) and adult (d) offspring. Each bar is the mean +/− SEM from 9 to 12 animals. e, f Aversive memory was evaluated in the fear conditioning test at young e) and adult (f) ages evaluating the time spent in freezing position. Each group of saline or sepsis administration is represented by a square or triangle, and the mean +/− SEM from 9 to 12 animals are indicated by lines. *p < 0.05 compared saline to sepsis. NS not significant

Fig. 8

Depressive behavior of offspring from mice subjected to gestational sepsis. The forced swim test (a, d) and tail suspension test (b, e) evaluated depressive-like behavior of young (a–c) and adult (d, e) animals. Anhedonia was tested by the sucrose preference test (c, f). Each bar is the mean +/− SEM from 9 to 12 animals. *p < 0.05 compared saline to sepsis. NS not significant