Quantifying Neuroinflammatory Gene Expression

On day 7, a sub-set of animals (N=6 per group) was sacrificed by conscious decapitation and blood collection and cryostat tissue punches were taken from the PVN of fresh frozen brains (Kapusta et al., 2012 (link); Kapusta et al., 2013 (link)). Total RNA was isolated using RNeasy kits (Qiagen) according to the manufacturer’s instructions, and 200 ng of purified RNA was reverse transcribed with a high-capacity cDNA reverse transcription kit (Qiagen). The IL-1β, IL-6, tumor necrosis factor-α, and IL-10 mRNA levels were analyzed by quantitative real-time PCR using specific primers and probes in a PRISM 7000 sequence detection system (Applied Biosystems) (Shi et al., 2010 (link)). Data were normalized to 18s ribosomal RNA, and the 2^−ΔΔCT method was used to calculate relative changes in gene expression (Zuriaga et al., 2017 ).