Early life stress and LPS interact to modify the mouse cortical transcriptome in the neonatal period

Abstract

Introduction Preterm birth (PTB) is closely associated with atypical cerebral cortical development and cognitive impairment. Early exposure to extrauterine life often results in atypical environmental and biological experiences that co-occur, including early life stress (ELS) and systemic inflammation. Understanding how these experiences interact to shape cortical development is an essential prerequisite to developing therapeutic interventions that will work in the complex postnatal environment of the preterm infant. Here, we studied the effects of a murine model of infection and ELS on the neonatal cortex transcriptome. Methods We used a mouse model of infection (1 ​mg/kg LPS at postnatal day (P)3) +/− ELS (modified maternal separation; MMS on days P4–P6) at timepoints with neurodevelopmental relevance to PTB. We used 4 groups: control, LPS, MMS and LPS ​+ ​MMS. Cortices were dissected at P6 for 3′RNA sequencing. Results LPS exposure resulted in reduced weight gain and increased expression of inflammation-associated genes in the brain. More genes were differentially expressed following LPS (15) and MMS (29) than with LPS ​+ ​MMS (8). There was significant overlap between the LPS and MMS datasets, particularly amongst upregulated genes, and when comparing LPS and MMS datasets with LPS ​+ ​MMS. Gene Ontology terms related to the extracellular matrix and cytokine response were enriched following MMS, but not following LPS or LPS ​+ ​MMS. 26 Reactome pathways were enriched in the LPS group, none of which were enriched in the LPS ​+ ​MMS group. Finally, a rank-rank hypergeometric overlap test showed similarities, particularly in upregulated genes, in the LPS and MMS conditions, indicating shared mechanisms. Conclusion LPS and MMS interact to modify the cortical transcriptome in the neonatal period. This has important implications for understanding the neural basis of atypical cortical development associated with early exposure to extrauterine life.

Altered hypothalamic DNA methylation and stress-induced hyperactivity following early life stress

“…In agreement with our previous report, there were no differences in weight gain (t-statistic = − 0.49, p = 0.62 for area under the curve, df = 18) (Fig. 1 A) [ 26 ]. MMS also had no effect on blood glucose (t-statistic = − 0.11, p = 0.91, df = 10) (Fig.…”

“…Animal models can be useful in furthering our understanding of the long-term effects of ELS and in facilitating the dissection of underlying mechanisms, however the large differences in experimental design and behavioural outcomes make interpretation difficult [ 47 ]; indeed, previous studies using maternal separation paradigms have described conflicting effects on behaviour [ 47 ]. We used a modified model of maternal stress [ 26 ] involving shorter periods of separation and frequent manipulation during the separation, with the lack of effects on weight gain and blood glucose levels supporting that MMS is a mild stressor and that maternal care is maintained. We speculate that the active manipulation component of MMS may lead to more consistent experience of the stress between pups and reduce heterogeneity in adult behavioural outcomes.…”

“…Within a litter, two males were randomly assigned to the control group and two to MMS. As previously described [ 26 ], MMS was performed between 1330 and 1500 daily from P4–P6 in a different room to their resident holding room. MMS pups were placed on a heating pad adjacent to the home-cage and for 1.5 h, pups were gently moved to the supine position whenever they returned to a prone position.…”

“…Sequencing was performed using the Ion PI Chip Kit v3 (Thermo Fisher Scientific, UK), 12 samples/chip with an average of 7,822,219 reads/sample generated. This depth of sequencing has previously been shown sufficient for differential expression analysis [ 26 , 30 , 31 ]. The Ion Hi-Q Sequencing 200 Kit (Thermo Fisher Scientific, UK) and the Ion Proton platform were used for analysis.…”

“…Maternal separation is the most common model of ELS which involves pup separation from the mother for ≥ 3 h/day for ≥ 10 consecutive days however considering the mother is the sole source of nutrition during this time, nutritional deficits may confound any findings in this model as hypoglycemia is independently associated with impaired neurodevelopment in humans [ 25 ]. To avoid this we used a modified maternal separation paradigm (MMS) to model ELS, involving short periods (1.5 h/day from postnatal day (P)4–P6 of brief maternal separation in combination with manual manipulation during the period of separation [ 26 ].…”