Mouse Brain Tissue Collection Methods

The mice brains were collected in a separate suite at the same time of the day during their active cycle following four different anesthesia and euthanasia methods: (1) Ketamine/Xylazine: mice were decapitated 45 min following an intraperitoneal injection of 100 mg/kg (#VINB-KET0-7021, Henry Schein Animal Health, Dublin, OH, USA) with 10 mg/kg xylazine hydrochloride supplement (#X1251-1G, Sigma-Aldrich, St. Louis, MO, USA). Serum ketamine levels are highest at 10–20 min post-injection (Ganguly et al., 2018 (link)); therefore to reduce the impact of ketamine on MAPK activity we chose the 45 min time-point, also because that is around the time a perfused brain would be collected. The anesthetic effect of a mixture of 100/10 mg/kg ketamine/xylazine is known to last up to 80 min with reflex suppressions and produces stable heart rates 40 min post-injection in mice (Erhardt et al., 1984 (link); Xu et al., 2007 (link)); thus the mouse is still sedated at our chosen time point; (2) Isoflurane: mice were placed in a plexiglass chamber with 5% isoflurane, USP (#NDC 13985-046-60, VetOne, Boise, ID, USA) for 5 min, and decapitated when fully sedated, as measured by a lack of active paw reflex; (3) Carbon Dioxide Asphyxiation: mice were placed in a new cage with corn cob bedding, and immediately euthanized by displacement of air with 100% carbon dioxide, within 5 min and decapitated for tissue collection; and (4) Decapitation: mice were gently restrained and decapitated in a new cage to minimize the exposure to blood from conspecific mice.