Prolonged maternal separation induces undernutrition and systemic inflammation with disrupted hippocampal development in mice

Abstract

Objective: Prolonged maternal separation (PMS) in the first 2 wk of life has been associated with poor growth with lasting effects in brain structure and function. This study aimed to investigate whether PMS-induced undernutrition could cause systemic inflammation and changes in nutrition-related hormonal levels, affecting hippocampal structure and neurotransmission in C57BL/6J suckling mice.

Methods: This study assessed mouse growth parameters coupled with insulin-like growth factor-1 (IGF-1) serum levels. In addition, leptin, adiponectin, and corticosterone serum levels were measured following PMS. Hippocampal stereology and the amino acid levels were also assessed. Furthermore, we measured myelin basic protein and synapthophysin (SYN) expression in the overall brain tissue and hippocampal SYN immunolabeling. For behavioral tests, we analyzed the ontogeny of selected neonatal reflexes. PMS was induced by separating half the pups in each litter from their lactating dams for defined periods each day (4 h on day 1, 8 h on day 2, and 12 h thereafter). A total of 67 suckling pups were used in this study.

Results: PMS induced significant slowdown in weight gain and growth impairment. Significant reductions in serum leptin and IGF-1 levels were found following PMS. Total CA3 area and volume were reduced, specifically affecting the pyramidal layer in PMS mice. CA1 pyramidal layer area was also reduced. Overall hippocampal SYN immunolabeling was lower, especially in CA3 field and dentate gyrus. Furthermore, PMS reduced hippocampal aspartate, glutamate, and gamma-aminobutyric acid levels, as compared with unseparated controls.

Conclusion: These findings suggest that PMS causes significant growth deficits and alterations in hippocampal morphology and neurotransmission.

Keywords: Hippocampus; IGF-1; Inflammation; Leptin; Malnutrition; Maternal separation; Stereology.

Fig. 1

Relative weight and tail length gain from experimental mice during timed-prolonged maternal separation (PMS) (N=16) and respective non-separated controls (N=10). Curves are presented as percentage of initial weight at post-natal day 1. Results are expressed in mean ± SEM and were analyzed by unpaired Student t test. * P <0.05 vs non-separated controls. Results are expressed in mean ± SEM and were analyzed by unpaired Student t test. * P <0.05 vs non-separated controls.

Fig. 2

Behavioral tests (A: surface righting) and (B: dorsal immobility) conducted in the first two weeks of life in the non-separated (N=6) and prolonged maternal separated (PMS) (N=8) groups. For the surface righting testing, results are shown in scores. The performed time was scored as follows: 2-righting ≤1 sec, 1-righting >1 and ≤2 sec, 0–>2 sec. For the dorsal immobility test, data are shown as latency mean time in seconds. The results are shown as mean ± SEM and were analyzed by unpaired Student t test. * P <0.05 vs non-separated controls.

Fig. 3

C-reactive protein serum levels of experimental mice following prolonged maternal separation (PMS) (N=10) and the respective unseparated-controls (N=10) (on day 14). Data are expressed in mg/L. Results are shown as mean ± SEM and were analyzed by unpaired Student t test. * P <0.05 vs non-separated controls.

Fig. 4

IGF-1 and leptin serum levels of experimental mice following prolonged maternal separation (PMS) and the respective unseparated-controls (on day 14). At least five mice were used for these analyses. Data are expressed in ng/mL. Results are shown as mean ± SEM and were analyzed by unpaired Student t test. * P <0.05 vs non-separated controls.

Fig 5

Hippocampal amino acid levels of experimental mice following prolonged maternal separation (PMS) (N=10) and the respective unseparated-controls (N=9), detect by high-performance liquid chromatography on day 14. Data are expressed in μmol/g of tissue. Results are shown as mean ± SEM and were analyzed by unpaired Student t test. * P <0.05 vs non-separated controls.

Fig. 6

Representative hippocampal synaptophysin (SYN) immunohistochemistry from experimental groups, 100X. N=5 for each group. Scale bar: 50 μm.