*4.5. Assessment of Neuronal Death*

Three or seven days after GCI, we estimated hippocampal neuronal death using Fluoro-Jade B (FJB, Histo-Chem, Jefferson, AR) staining method (as previously described in detail [12]). Approximately nine to ten coronal brain sections (based on 30-μm thickness, 270- to 300-μm intervals) were obtained

from 3.00 to 4.68 mm caudal to bregma. These brain sections were mounted on gelatin-coated slides, and photographed using a fluorescence microscope (SZ61, Olympus, Shinjuku, Japan, FITC green fluorescence excitation-emission wavelength: 460–490 nm). To avoid experimenter bias, a blind observer counted the whole number of FJB-positive neurons in the hippocampal subiculum, cornu ammonis 1 (CA1), and CA2 regions. Collected data were used for statistical analysis.
