Fig. 1. Density of activated caspase 3-stained neurons (profiles/mm3) in isoflurane-exposed versus control brains. Transverse sections (∼800) were cut serially across the entire forebrain and midbrain and counts were performed on sections selected at 2.24-mm intervals (every 32nd section). An outline of each section was entered into the computer and all stained neuronal profiles within the outlined space were counted and marked for location. The total number of stained neurons divided by the total tissue space (determined by the applied software) yielded a neuronal density count (number of activated caspase-3-stained profiles per cubic millimeter) for each brain. The median density (range) of activated caspase-3-positive neurons per cubic millimeter for the isoflurane-exposed brains was 32.5 (18.0-48.2) and for the control brains was 2.5 (1.1-5.2). This amounts to a 13-fold increase in the density of apoptotic neurons in the isoflurane-exposed brains. The difference in the median density of apoptotic neurons between the isoflurane and control groups was 30 (95% confidence interval, 15.5-45.7, P = 0.008).
