Assessing Fetal Brain Development

On GD 21, a day before parturition, animals—were deeply anesthetized with isoflurane in the anesthesia chamber and sacrificed by decapitation and fetuses were carefully separated. Two male fetuses from each dam were randomly selected from a similar position in the uterine horn. One pup per dam was used for immunoblotting while the other pup was used for immunohistochemistry. During normal development, male and female hippocampal development differ significantly and brain development in humans and animal is directly regulated by sex hormones (Yagi and Galea, 2019 (link); Mu et al., 2020 (link)). To accurately identify hippocampal-specific alterations induced by prenatal e-cig aerosol exposure without the confounding factor of sex, only male offspring were assessed in these experiments. Fetal whole brains from the male fetuses were extracted under a dissection microscope via craniotomy and serially washed in cold phosphate buffered saline (PBS). One of the fetal brains was dissected, meninges were removed, and hippocampi were micro-dissected in ice-cold HEPES buffer. Individual samples were then flash frozen and stored at −80°C until immunoblot analyses. For immunohistochemistry, the other whole fetal brain was embedded in optimal cutting temperature compound (OCT) (Sakura Finetek, Torrance, CA) and cryopreserved for immunofluorescence.