To determine the impact of RZ on the hippocampal BDNF, mice receiving ADR or RZ were euthanized for 4 weeks after the initiation of RZ treatment, and BDNF ELISA was performed as described [34 (link)]. Brains were immediately extracted from the skull (N = 8 to 9 mice per group). The hippocampus was microdissected from each cerebral hemisphere, flash-frozen by immersing the cryo-vials in the liquid nitrogen, and stored at − 80 °C until assayed. Each hippocampus was weighed and transferred into 500 μl ice-cold lysis buffer (NPER, Neuronal Protein Extraction Reagent, ThermoScientific) containing sodium orthovanadate (0.5 mM, Santa Cruz), phenyl-methylsulfonyl fluoride (PMSF, 1 mM, Santa Cruz), aprotinin (10 μg/ml, Santa Cruz), and leupeptin (1 μg/ml; Santa Cruz). Tissues were then sonicated individually and centrifuged at 4 °C, and the supernatants were collected and diluted at 1:5 or 1:10 with ice-cold Dulbecco’s PBS (Gibco). The supernatants were acidified to pH 2.6 and then neutralized to pH 7.6. The BDNF levels were assayed using a commercially available ELISA kit (E-EL-M0203, Elabscience Biotechnology) and uncoated ELISA plates (Nunc MaxiSorp, Biolegend). The colorimetric measurements were performed at 450 nm wavelength using a microplate reader (BioTek SynergyMx).
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