2.5. ELISA Analysis

For quantitative analysis of cytokines, an ELISA was used to measure the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), or transforming growth factor-β (TGF-β) in brain tissue [19]. Cytokines were extracted from mouse brains as follows: frozen hemibrains were placed in tissue homogenization buffer containing protease inhibitor cocktail (Sigma, St Louis, MO, USA) 1:1000 dilution immediately before use, and homogenized using polytron. Tissue sample suspensions were distributed in aliquots and snap frozen in liquid nitrogen for later measurements. Invitrogen ELISA kits were then used, according to manufacturer directions (Carlsbad, CA, USA).