2.3. Cathepsin B Activity Assay

Brain cathepsin B activity was measured 2 h after trauma using a fluorometric assay kit, as described by the manufacturer (ab65300; Abcam, Cambridge, MA, USA). Briefly, tissues were washed twice in ice-cold phosphate-buffered saline and then homogenized in extraction buffer, as described by the manufacturer. After 10-min incubation on ice, the extract was centrifuged at 10,000 g for 5 min, and 50 μL of supernatant was mixed with an equal volume of 2 × reaction buffer and 2 μL of substrate in a 96-well microplate. Plates were kept in the dark at 37 °C for 1 h, and fluorescence was recorded using a FLUOstar Optima plate reader (BMG LABTECH GmbH, Ortenberg, Germany). Protein concentration was determined by the bicinchoninic acid assay method (Bio-Rad, Hercules, CA, USA). Cathepsin B activity was measured in triplicate and was expressed as fluorescent units/mg of protein. For the determination of enzyme activity, we isolated the region of trauma for analysis.