4.7. In Vitro Organotypic Slice Cultures and Oxygen-Glucose Deprivation

Hippocampal organotypic slice cultures were prepared from female neonatal (9–11 days old) Sprague–Dawley rats and the details of slice culture are as described previously [46,52,53,54]. Briefly, hippocampal slices were cultured for 14–20 days followed by exposure to nicotine (100 ng/mL) or saline (vehicle) for 14–16 days. At the end of the treatment period (~29–37 days), a subgroup of slices (either treated with nicotine or saline) were exposed to the inflammasome inhibitor isoliquiritigenin (ILG; Invivogen, San Diego, CA, USA) at different concentrations (1, 10, or 40 µM) dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) or vehicle control DMSO (1 µL/mL of medium) for 24 h. After inhibitor/control treatment, each subgroup was exposed to OGD (45 min) as described [46,52,53,54]. To determine the extent of neuronal damage following OGD, we used the propidium iodide (PI) method [46,52,53,54]. Briefly, slices were incubated in culture medium supplemented with 2 µg/mL PI (Sigma, St. Louis, MO, USA) for 1 h. Images were taken using an inverted fluorescence microscope. Images of the cultured slices were taken (1) at baseline prior to the ‘test’ ischemia procedure; (2) 24 h after the ‘test’ ischemic insult to assess ischemic damage; and (3) 24 h after N-methyl-d-aspartate (NMDA) treatment to assess maximum damage to neuronal cells. The hippocampal CA1 subfield was chosen as the region of interest, and quantification was performed using Scion Image software [46,52,53,54]. The percentage of relative optical intensity (ROI) served as an index of neuronal cell death [46,52,53,54].