Figure 4: Effect of enriched environment on the proliferation of hippocampal neural stem cells of offspring exposed to sevoflurane inhalation in the second trimester of pregnancy. (A) Nestin/Ki67 immunofluorescence assay was used to detect proliferation in the hippocampus of offspring. Immunoreactivity of nestin and Ki67 were downregulated in the Sev group compared with the control group, and were restored in the Sev + EE group. Cells expressing nestin (red, stained by TRITC), Ki67 (green, stained by FITC) and DAPI (blue) were identified. Scale bars: 50 μm. (B, C) Protein and mRNA expression of nestin and Ki67 in hippocampus of offspring rats by western blot and real-time polymerase chain reaction, respectively. Data are shown as mean ± SD (eight litters/group, three offspring within each litter as replicates). *P < 0.05, vs. control (C) group; #P < 0.05, vs. sevoflurane (Sev) group (one-way analysis of variance followed by least significant difference post hoc test). DAPI: 4,6-Diamino-2-phenyl indole means nucleus; EE: environmental enrichment group; FITC: fluoresceine isothiocyanate.