RNA extraction and reverse transcription-quantitative PCR (RT-qPCR) analysis

Total RNA was extracted from the hippocampal tissue using the RNeasy mini kit (Qiagen GmbH) following the manufacturer's protocol. RT to generate cDNA was performed using the ReverTra Ace qPCR RT Master Mix with gDNA Remover kit (Toyobo Co., Ltd.) according to the manufacturer's protocol. qPCR analysis was performed using the FastStart Essential DNA Green Master kit (Roche) according to the manufacturer's protocol. The primer sequences of the target genes were as follows: Interleukin-6 (IL-6), 5'-TGCAAGAGACTTCCATCCAGTT-3' (forward) and 5'-GAAGTAGGGAAGGCCGTGG-3' (reverse); tumor necrosis factor-α (TNF-α), 5'-GCACCACCATCAAGGACTC-3' (forward) and 5'-TGAGACAGAGGCAACCTGAC-3' (reverse); inducible nitric oxide synthase (iNOS), 5'-GGCAGCCTGTGAGACCTTTG-3' (forward) and 5'-GCATTGGAAGTGAAGCGTTTC-3' (reverse); chitinase-3 like protein (Ym1/2), 5'-CAGGGTAATGAGTGGGTTGG-3' (forward) and 5'-CACGGCACCTCCTAAATTGT-3' (reverse); GAPDH, 5'-ACTCCACTCACGGCAAATTC-3' (forward) and 5'-TCTCCATGGTGGTGAAGACA-3' (reverse). The PCR amplification conditions were adjusted based on the manufacturer's protocol and previous research (15): 95˚C for 10 min and 45 cycles of 95˚C for 10 sec and 60˚C for 1 min. The expression levels of these genes were normalized to the levels of GAPDH and presented as 2^-∆∆Cq with CB2R WT+O₂ as the control (27).