Cytokine Measurements

Tissues were then processed for cytokine assessment using the Luminex‐200 multiplex analyzer (Luminex, Austin, TX) to simultaneously measure 10 cytokines (tumor necrosis factor‐α [TNF‐α], interleukin 1a [IL‐1a], interleukin 1b [IL‐1b], interleukin 2 [IL‐2], interleukin 4 [IL‐4], interleukin 6 [IL‐6], interleukin 10 [IL‐10], interleukin 12 [IL‐12], interferon‐γ, and granulocyte‐macrophage colony‐stimulating factor [GMCSF]) using a rat‐specific kit (Millipore, Billerica, MA). All values were corrected for protein concentration. The tissue was homogenized in PBS by applying a Dounce homogenizer for 20 strokes. The homogenate was then sonicated for 10 seconds for 3 times with an interval of 20 seconds, followed by centrifugation at 16,000g for 30 minutes. The supernatant was used for cytokine analysis. Protein levels in the supernatants were measured using the BCA (bicinchoninic acid) protein kit (Thermo Fisher Scientific Inc, Rockford, IL). The samples were run singly. Data for individual cytokines that were undetectable or out of range across multiple groups, suggesting a problem with the assay, are not shown (eg, interferon‐γ in serum or GMCSF levels in all individual brain regions). The values beyond the standard range were extrapolated automatically from the calibration curve by the Luminex software. Cytokine concentrations that were read as “out of range below” were nominally assigned a value at one hundredth of the lowest level on the calibration scale.