Effect of sustained postnatal systemic inflammation on hippocampal volume and function in mice

Abstract

Background: Premature infants are at risk for persistent neurodevelopmental impairment. Children born preterm often exhibit reduced hippocampal volumes that correlate with deficits in working memory. Perinatal inflammation is associated with preterm birth and brain abnormalities. Here we examine the effects of postnatal systemic inflammation on the developing hippocampus in mice.

Methods: Pups received daily intraperitoneal injections of lipopolysaccharide (LPS) or saline between days 3 and 13. Ex vivo magnetic resonance imaging (MRI) and microscopic analysis of brain tissue was performed on day 14. Behavioral testing was conducted at 8-9 wk of age.

Results: MR and microscopic analysis revealed a 15-20% reduction in hippocampal volume in LPS-treated mice compared with controls. Behavioral testing revealed deficits in hippocampal-related tasks in LPS-treated animals. Adult mice exposed to LPS during the postnatal period were unable to select a novel environment when re-placed within a 1-min delay, were less able to remember a familiar object after a 1-h delay, and had impaired retention of associative fear learning after 24 h.

Conclusion: Systemic inflammation sustained during the postnatal period contributes to reduced hippocampal volume and deficits in hippocampus-dependent working memory. These findings support the novel and emerging concept that sustained systemic inflammation contributes to neurodevelopmental impairment among preterm infants.

Figure 1

Kaplan-Meier estimator of survival as a function of time for mice injected daily with either saline or LPS between postnatal days 3 and 13. Approximately 50% of newborn mice survived to day 14 when injected daily with 0.3 mg/kg of LPS (dotted line) compared to 83% of mice injected with saline (solid line; p < 0.05). The death rate was comparable among the litters in the LPS group.

Figure 2

(A) Representative MRI image illustrating a non-enhanced coronal T 2-weighted 3D RARE sequence reconstruction of a 512×512×512 matrix with in plane resolution of 27 µm and (B) a light microscopy image of a 30 µm Crysel Violet-stained tissue section showing the hippocampal formation as the region of interest manually demarcated and its area measured using Image J software (NIH).

Figure 3

(A) Average volumes of the hippocampal formation calculated as the summation of both right and left hippocampal regions in all of the sequential images from each brain multiplied by the section thickness (27 µm). (B) Average areas of the hippocampal formation calculated by adding the areas of both right and left hippocampal regions in rostral and caudal sections at matching levels from each brain. The hippocampal volume was reduced by 20% and its area reduced by 25% in the LPS compared to the control group on postnatal day 14 (M±SEM; *p < 0.01 vs. control; LPS: solid bars, control: open bars).

Figure 4

Cognitive assessment of mice between 8 and 9 weeks of life with a battery of tests of hippocampus-dependent working memory tasks that include spontaneous alternation on a T- maze (panel A), novel object recognition (panel B), and contextual fear conditioning (panels C and D; LPS: solid line; control: dotted line). Mice that were injected daily with LPS during the postnatal period were not able as adults to register and select a novel environment when re-placed within a 1-minute delay on a T-maze, were less able to remember a familiar object after a 1-hour delay and showed impaired retrieval and retention of associative learning after 24 hours (panel D), despite adequate initial acquisition of associative learning to the fearful stimulus (panel C; M±SEM; ^†p < 0.01 vs. 1-minute on day 1; *p < 0.05 vs. control).

Figure 5

Proposed mechanisms that may have resulted in the observed association between postnatal exposure to LPS and subsequent impairment of hippocampal development in mice. (WM= white matter; GM=germinal matrix).