Maternal Sevoflurane Exposure Causes Abnormal Development of Fetal Prefrontal Cortex and Induces Cognitive Dysfunction in Offspring

Figures

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  Figure 1. Maternal sevoflurane exposure impaired learning and memory ability in offspring. (a) No significant differences in swimming speed were found between the control group and the sevoflurane group. (b) The escape latency of MWM in the sevoflurane group was longer than that in the control group. (c) No significant differences in platform crossing times were found between the control group and the sevoflurane group. (d) The %time in the opposite quadrant in the sevoflurane group was longer than that in the control group. Data are expressed as the mean ± SEM. ∗P < 0.05 and ∗∗P < 0.01.

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  Figure 2. Maternal sevoflurane exposure decreased the production of deep-layer immature neurons. (a) Schematic diagram of the timing of sevoflurane exposure and sacrifice to assess the differentiation in the fetal PFC. (b, c) Tbr1 (green) and Satb2 (red) immunofluorescence, combined with DAPI staining (blue) in the cortical plate at G18.5. Scale bars, 20 μm. (d) Quantification of the Tbr1+ and Satb2+ cells of the control and sevoflurane groups. (e, f) NeuN (red) immunofluorescence and DAPI staining (blue) in the cortical plate at G18.5. Scale bars, 20 μm. (g) Quantification of the NeuN+ cells of the control and sevoflurane groups. (h, i) GFAP (red) immunofluorescence and DAPI staining (blue) in the cortical plate at G18.5. Scale bars, 20 μm. (j) Quantification of the GFAP+ cells of the control and sevoflurane groups. Data are expressed as the mean ± SEM. ∗∗∗P < 0.001.

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  Figure 3. Maternal sevoflurane exposure suppressed the proliferation of the fetal PFC. (a) Schematic diagram of the timing of sevoflurane exposure, BrdU injection, and sacrifice to assess the proliferation and apotosis of the fetal PFC. (b, c) Ki67 (green) immunofluorescence and DAPI staining (blue) in the cortical plate at G15.5. Scale bars, 20 μm. (d) Quantification of the Ki67+ cells of the control and sevoflurane groups. (e, f) BrdU (red) immunofluorescence and DAPI staining (blue) in the cortical plate at G15.5. Scale bars, 20 μm. (g) Quantification of the BrdU+ cells of the control and sevoflurane groups. (h, i) Caspase-3 (green) immunofluorescence and DAPI staining (blue) in the cortical plate at G15.5. Scale bars, 20 μm. (j) Quantification of the GFAP+ cells of the control and sevoflurane groups. Data are expressed as the mean ± SEM. ∗∗∗P < 0.001.

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  Figure 4. Maternal sevoflurane exposure inhibited the expansion of neural progenitors in the fetal PFC. (a) Schematic diagram of the timing of sevoflurane exposure and sacrifice to assess the abundance of neural progenitors. (b, c) Nestin (red) immunofluorescence and DAPI staining (blue) in the cortical plate at G15.5. Scale bars, 20 μm. (d) Quantification of the nestin+ cells of the control and sevoflurane groups. (e, f) Pax6 (green) immunofluorescence and DAPI staining (blue) in the cortical plate at G15.5. Scale bars, 20 μm. (g) Quantification of the Pax6+ cells of the control and sevoflurane groups. (h, i) Tbr2 (green) immunofluorescence and DAPI staining (blue) in the cortical plate at G15.5. Scale bars, 20 μm. (j) Quantification of the Tbr2+ cells of the control and sevoflurane groups. Data are expressed as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

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  Figure 5. Maternal sevoflurane exposure decreased cell cycle exit and increased S-phase duration of neural progenitors in the fetal PFC. (a) Schematic diagram of the timing of sevoflurane exposure, BrdU injection, and sacrifice to assess the proportion of the cell cycle exit. (b, c) Coronal sections of the PFC from G16.5 mice were immunostained for BrdU (red) and Ki67 (green) at G16.5. Scale bars, 20 μm. (d) Numbers of BrdU+Ki67− cells are expressed as the numbers of BrdU+ cells. (e) Schematic diagram of the timing of sevoflurane exposure, IdU injection, and BrdU administration to assess the S-phase duration. Scells = cells labeled with BrdU; Lcells = cells labeled with IdU but not BrdU. (f, g) Coronal section through the cortex of the G15.5 fetal brain immunostained with antibodies specific for both BrdU and IdU (green) and BrdU alone (red) to identify Lcells (green-only cells) and Scells (red and green double-labeled cells). Arrowheads indicate Lcells. Scale bars, 20 μm. (h) Quantification of the length of S-phase in G15.5 embryos. Data are expressed as the mean ± SEM. ∗∗P < 0.01 and ∗∗∗P < 0.001.

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  Figure 6. Maternal sevoflurane exposure did not influence the duration of G1-, M-, and G2-phases of neural progenitors in the fetal PFC. (a) Schematic diagram of the timing of sevoflurane exposure and sacrifice to assess the expression of Ccnd1 and PH3. (b, c) Ccnd1 (green) immunofluorescence and DAPI staining (blue) in the cortical plate at G15.5. Scale bars, 20 μm. (d) Quantification of the Ccnd1+ cells of the control and sevoflurane groups. (e) PH3 (green) immunofluorescence and DAPI staining (blue) in the cortical plate at G15.5. PH3-positive cells were counted in radially arranged 200 mm-wide boxes, as illustrated. Scale bars, 20 μm. (f) Quantification of the PH3+ cells of the control and sevoflurane groups. Data are expressed as the mean ± SEM.