Abstract
<p style="text-align: justify;">This manuscript presents a protocol designed for the comprehensive investigation of early life stress (ELS) outcomes and a feasibility study conducted with this protocol. ELS alters normal development by interfering at various levels: hormonal changes, brain cellular architecture, epigenome, and chromosomal structural elements. The protocol combines classic behavioral tests with advanced molecular techniques to obtain comprehensive data and thus uncover the underlying mechanisms of ELS. In this protocol, the main source of stress is maternal separation. Briefly, a group of C57Bl/6 mice undergoes maternal separation; then, mice perform the radial maze test and the resident-intruder test. As a control, another group of mice stays undisturbed and performs the same behavioral tests in the same timeframe. After the behavioral tests, biosamples are collected, including urine for corticosterone measurements, peripheral blood, hippocampus, amygdala, and prefrontal cortex tissues for DNA isolation and its downstream analyses (DNA methylation profiling and telomere length measuring), and whole brains for immunohistochemistry analysis of the glucocorticoid receptor density. This protocol was successfully tested as a feasibility study for a large-scale investigation that addresses potential flaws to establish a robust methodology. This paper reports on a comprehensive approach to examining multiple aspects of development that interrogates a holistic analysis of multilayer and multidimensional data and may contribute valuable insights for both animal and human studies.</p>
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