Long-Term Impact of Early-Life Stress on Hippocampal Apoptotic Gene Expression in BALB/c Mice

Abstract Early-life stress (ELS), such as maternal separation, has been associated with neuronal apoptosis and impaired hippocampal function in rodent models. This study investigated the long-term effects of two ELS paradigms—unpredictable maternal separation (MS) and MS combined with unpredictable maternal stress (MSUS)—on the hippocampal mRNA expression of proapoptotic ( Bax , Tp53 and Casp3 ) and prosurvival ( Bcl2 ) genes in offspring, both in vivo and in a complementary in vitro neuronal culture model. The study included three groups (Control, MS and MSUS groups), with ELS applied from postnatal days 1 to 14 to BALB/c mice. For the in vitro experiments, total RNA from the MSUS hippocampus was nucleofected at different concentrations into healthy mouse hippocampal cells, followed by 3D neuronal cell culture using N-heptyl-D-galactonamide (GalC7) hydrogels. Quantitative real-time PCR was used to assess gene expression, which was analyzed via the comparative Ct method (2 −ΔΔCt ). This study revealed that Bax mRNA expression was significantly lower in the MS group than in the control group, whereas both the MS and MSUS groups presented significant increases in Bcl2 mRNA expression. In addition, the expression ratio of Bcl2/Bax was significantly greater in the MS and MSUS groups. No significant differences in Tp53 or Casp3 mRNA expression levels were detected between the groups. Although the in vitro mRNA expression levels were not significantly different, the mRNA expression ratio of Bcl2/Bax reached equilibrium as the concentration of total RNA nucleofected increased. Our results suggested that, in response to ELS, hippocampal cells adapt to prioritize survival over apoptosis.