mdivi compounds protect primary neurons against glutamate-induced excitotoxicity and OGD in vitro and attenuate ischemic brain damage in vivo. Primary rat cortical neurons (DIV 9) were exposed 4 h to OGD. After 4 h of OGD, the 1 × EBSS±glucose was replaced by NB+-medium containing glucose to all dishes and the cells were incubated for another 18–24 h in the regular oxygen-containing incubator. (a) Fluorescence microscope images ( × 40 objective) of DAPI-stained embryonic cortical neurons were obtained after 18 h of exposure to OGD. (b) Counting of ∼500 DAPI-stained neurons with a total n=5 per group displaying pycnotic nuclei after 4 h of OGD revealed that 25 μM Drp1 inhibitor mdiviA and B significantly protect neurons from hypoxic-hypoglycemic cell death. ***P<0.001 versus untreated neurons subjected to OGD (n=5, ANOVA, Scheffé's-test). Cultures treated with 25 μM mdiviA or B contained >60% healthy nuclei, whereas all other OGD-treated groups show 75% pycnotic and/or fragmented nuclei, indicating apoptotic damage (OGD, right white column). The respective control cultures with 1 × EBSS medium + glucose contained only very few apoptotic nuclei (Control, left white column). The experiments were repeated three times and the results are presented as mean±S.D. (c) Quantification of pycnotic nuclei in percentage of four independent experiments with a total n=5 per group. Primary rat cortical neurons (DIV 9) were treated for 18 h with glutamate (30 μM) and mdiviA and B (25 μM). After 18 h, cells were fixed in 4% PFA and stained with DAPI and ∼500 nuclei were counted per treatment condition. MdiviA and B significantly reduced glutamate-induced cell death. ***P<0.001 versus glutamate-treated neurons (n=5, ANOVA, Scheffé's-test). (d) Transient focal ischemia was induced for 45 min by a silicon-coated nylon filament that was introduced into the internal carotid artery to occlude the middle cerebral artery (MCA). Surgery was performed in deep isoflurane/N2O anesthesia with controlled ventilation, and ischemia and reperfusion were verified by Laser-Doppler flowmetry. The infarct volume was calculated on the basis of the histomorphometric data from 12 to 15 consecutive sections (10 μM, 500 μM apart) obtained 24 h after onset of ischemia. MdiviA (3 mg/kg) and mdiviB (1 and 3 mg/kg) reduced the mean infarct volume by 34 and 30% compared with vehicle (DMSO) controls, respectively. The photomicrographs show representative pictures of cresyl-violet-stained brain slices from animals, which received vehicle or mdiviB (1 and 3 mg/kg). The infarct area remained unstained and is highlighted by dashed lines