Exposure to Sevoflurane Affects the Development of Parvalbumin Interneurons in the Main Olfactory Bulb in Mice

Abstract

Sevoflurane is widely used in adult and pediatric patients during clinical surgeries. Although studies have shown that exposure to sevoflurane impairs solfactory memory after an operation, the neuropathological changes underlying this effect are not clear. This study detected the effect of sevoflurane exposure on the development of calcium-binding proteins-expressing interneurons in the main olfactory bulb (MOB). We exposed neonatal mice to 2% sevoflurane at two different developmental time points and found that exposing mice to sevoflurane at postnatal day (PD) 7 significantly decreased the expression of GAD67 and parvalbumin (PV) in the olfactory bulb (OB) but did not alter the expression of calretinin (CR) or calbindin D28k (CB). The number and dendritic morphology of PV-expressing interneurons in the MOB were impaired by exposure to sevoflurane at PD7. However, exposure to sevoflurane at PD10 had no effect on calcium-binding protein expression or the number and dendritic morphology of PV-expressing interneurons in the MOB. These results suggest that exposing neonatal mice to sevoflurane during a critical period of olfactory development affects the development of PV-expressing interneurons in the MOB.

Keywords: CB; CR; PV; olfactory bulb; sevoflurane.

Figure 1

The effect of exposure to sevoflurane at postnatal days 7 (PD7) and 10 (PD10) on the laminar organization of main olfactory bulb (MOB). No significant change in the laminar organization of the MOB was observed between control mice (A) and mice exposed to sevoflurane at PD7 (B). Compared to control MOBs (C), the laminar organization of the MOB in mice that were treated with sevoflurane at PD10 (D) was unchanged. (A1–D1) High magnification images showing the structure of the MOB. Scale bars = 500 μm in (D) (applies to A–D) and 100 μm in (D1) (applies to A1–D1).

Figure 2

The effect of exposure to sevoflurane at PD7 on GAD67 and calcium-binding protein expression. (A) Representative autoradiogram showing the expression of GAD67 and calcium-binding proteins in the MOB at PD14, 28 and 42. Normal: control group. SEV: sevoflurane-treated group. Quantitative analysis of the results of western blot analysis to determine the expression of GAD67 (B), parvalbumin (PV) (C), calretinin (CR) (D), and calbindin D28k (CB) (E) Values are expressed as the Mean ± SD. ***P < 0.001, **P < 0.01, and *P < 0.05.

Figure 3

The effect of exposure to sevoflurane at PD7 on the number of calcium-binding protein-expressing neurons in the MOB. Control mice (A) had more PV interneurons in the MOB than the sevoflurane-treated mice (B). (A1,B1) High magnification images of PV interneurons in the MOB. The density of CR interneurons in the MOB of mice exposed to sevoflurane at PD7 (D) was unaffected compared to the control mice (E). (D1,E1) High magnification images of CR interneurons in the MOB. The density of CB interneurons in the MOB of mice exposed to sevoflurane at PD7 (G) was unaffected compared to the control mice (H). (G1,H1) High magnification images of CB interneurons in the MOB. Normal: control group. SEV: sevoflurane-treated group. Quantification analysis of the number of PV-ir neurons (C), CR-ir neurons (F) and CB-ir neurons (I) in different layers of MOB. Scale bars = 100 μm (applies to A–H,A1–H1). Values are expressed as the Mean ± SD. ***P < 0.001.

Figure 4

The effect of exposure to sevoflurane at PD7 on the dendritic architecture of calcium-binding protein-expressing neurons in the MOB. Compared to the control group (A,B), the dendritic morphologies of PV interneurons were impaired by exposure to sevoflurane at PD7 (C,D). (B1,B2,D1,D2) High magnification images of PV interneurons in the MOB. Compared to the control group (E,F), the dendritic morphologies of CR interneurons were not impaired by exposure to sevoflurane at PD7 (G,H). (F1,F2,H1,H2) High magnification images of CR interneurons in the MOB. Compared to the control group (I,J), the dendritic morphologies of CB interneurons were not impaired by exposure to sevoflurane at PD7 (K,L). (J1,J2,L1,L2) High magnification images of CR interneurons in the MOB. Scale bars = 100 μm in (K) (applies to A,C,E,G,I,K), 50 μm in (L) (applies to B,D,F,H,J,L), and 5 μm in (L2) (applies to B1,B2,D1,D2,F1,F2,H1,H2,J1,J2,L1,L2).

Figure 5

The effect of exposure to sevoflurane at PD10 on GAD67 and calcium-binding protein expression. (A) Representative autoradiogram of GAD67 and calcium-binding protein expression in the MOB at PD14, 28 and 42. Normal: control group. SEV: sevoflurane-treated group. Quantitative analysis of western blot results showing the expression of GAD67 (B), PV (C), CB (D), and CR (E). Values are expressed as the Mean ± SD.

Figure 6

The effect of exposure to sevoflurane at PD10 on the number and dendritic morphology of PV interneurons. Compared to the control group (A–C), the number and dendritic morphologies of PV interneurons were not impaired by exposure to sevoflurane at PD10 (D–F). (C1,C2,F1,F2) High magnification images of PV interneurons in the MOB. (G) Quantification analysis of the number of PV-ir neurons in different layers of MOB. Scale bars = 200 μm in (D) (applies to A,D), 100 μm in (E) (applies to B,E), 50 μm in (F) (applies to C,F), and 5 μm in (F2) (applies to C1,C2,F1,F2). Values are expressed as the Mean ± SD.