Fig. 5. Neuronal differentiation and new neuronal density are not affected by isoflurane (iso) in aged rats. Stack of microscopic images acquired 1 mm apart in the Z-plane (A ) showing co-staining of the S-phase marker bromodeoxyuridine (BrdU) with the immature neuronal marker NeuroD 12 h after the last of two BrdU injections given intraperitoneally to aged rats on the fourth day after 4 h of isoflurane anesthesia. Neither the proportion of BrdU+ cells that coexpress NeuroD on day 5 after anesthesia (B ) nor the density of BrdU+/NeuroD+ cells (C ) is affected by isoflurane. New neuronal survival 28 days after a 4-day pulse of bromodeoxyuridine (50 mg/kg × 8 at 12-hourly intervals) starting 4 days after 4 h of isoflurane anesthesia (D , G ). Immunofluorescent image of a bromodeoxyuridine+/NeuN− cell (D , arrow ) and an image stack acquired on a confocal microscope showing colocalization of two bromodeoxyuridine+/NeuN+ cells (E ). The proportion of bromodeoxyuridine+ cells that coexpress the mature neuronal marker NeuN 35 days after isoflurane (n = 5) or no isoflurane (control, n = 6) does not differ between groups (F ). The density of bromodeoxyuridine+/NeuN+ cells in the DG is not different between groups either (G ). DAPI = 4′,6-diamidino-2-phenylindole.