3.1.Long-term sevoflurane suppressed SEFL in the PTSD mice.
In test 1 on day 3, PTSD mice showed significantly enhanced freezing time. (68.12 ± 16.2%, p < 0.001 vs. Ctrl and Sevo (untreated), Fig. 1A, 1B) which indicated successful modeling of mice shocked six times seriously in context A on day 1. After exposed to sevoflurane for 6 h, mice in the PTSD + Sevo group exhibited decreased freezing time compared with PTSD (32.59 ± 8.35% vs. 60.74 ± 20.84, p < 0.001, Fig. 1A, 1C) in test 2 which designed for evaluating therapeutic effects of sevoflurane on PTSD mice and our results give a positive answer on it. To further explore the effective time of sevoflurane, we repeated test 2 after 7 days and the result was the same as before (28.03 ± 10.08% vs. 55.62 ± 14.33, p < 0.01 vs. PTSD, Fig. 1A, 1D). In addition, we performed test 3 in order to exclude the influence of memory generalization in this PTSD model. It can be observed that all groups showed a high degree of activity without fear in the context c (p > 0.05, Fig. 1A, 1E).
3.2.Long-term sevoflurane ameliorated anxiety in PTSD mice.
To further investigate the effects of sevoflurane on anxiety in PTSD mice, we conducted the open field test on days 12 (7 days after anesthesia). PTSD mice showed reduced time spent in the center of the arena compared with other groups (70.58 ± 16.28, p < 0.001 vs. Ctrl, Sevo and PTSD + Sevo, Fig. 2A). Similar data could be seen in the total distance of PTSD mice (37.72 ± 2.98, p < 0.001 vs. Ctrl, Sevo and PTSD + Sevo, Fig. 2B). In the forced swimming test, the freezing time was similar among all groups (p > 0.05, Fig. 2C).
3.3.Short-term sevoflurane improved SEFL transiently in the PTSD mice
For further investigating whether short-term sevoflurane inhaled could induce SELF either, we repeated experiment 1 expects that the duration of anesthesia was shortened to 2h. In test 1, the data showed obviously enhanced freezing time in the PTSD group. (66.93 ± 19.78%, p < 0.001 vs. Ctrl and Sevo (untreated), Fig. 3A, 3B). After exposed to sevoflurane for 2 h, mice in the PTSD + Sevo group exhibited decreased freezing time compared with PTSD (35.24 ± 11.12% vs. 62.41 ± 18.28, p < 0.001, Fig. 3A, 3C) in test 2. We repeated test 2 after 7 days and the result was modified. The PTSD mice still showed higher freeing time than the control group (54.50 ± 14.34% vs. 23.21 ± 7.30, p < 0.01, Fig. 3A, 3D), while mice exposure to sevoflurane after 7days could not revise the effect (45.34 ± 11.75%, p > 0.05 vs. PTSD, p < 0.05 vs. Ctrl and Sevo, Fig. 3A, 3D).
3.4.GR and CB1R expressions were increased in brain areas in PTSD mice and sevoflurane normalized this effect.
We explored the level of relative GR and CB1R expressions in the hippocampus, amygdala and prefrontal cortex (PFC) in all groups. In the PTSD group, the expression of GR was upregulated in the hippocampus (2.02 ± 0.30), amygdala (2.20 ± 0.35) and PFC (2.11 ± 0.26) compared with the control and Sevo group (p < 0.01 vs. Ctrl and Sevo). Sevoflurane administered for 6h modified these alterations in the three brain areas (hippocampus: 1.36 ± 0.19, amygdala1.23 ± 0.22, PFC: 1.22 ± 0.30, p < 0.05 vs. PTSD, Fig. 4A-B). Similar changes were seen in CB1R relative expression. Higher expression of CB1R was founded in the PTSD group in the hippocampus (2.22 ± 0.22), amygdala (3.03 ± 0.63) and PFC (2.33 ± 0.56) compared with the control and Sevo group (p < 0.01 vs. Ctrl and Sevo). 6h of sevoflurane treated normalized the alterations in the three brain areas (hippocampus: 1.41 ± 0.15, amygdala1.60 ± 0.47, PFC: 1.16 ± 0.39, p < 0.05 vs. PTSD, Fig. 4A, C).
3.5.The expression of D1/2 dopamine receptors decreased after modeling and sevoflurane normalized this change partly in the PFC.
We next investigated the expression of D1R and D2R in the hippocampus, amygdala and PFC in all groups. No alterations in D1R were observed in the hippocampus (1.11 ± 0.25) or amygdala (1.05 ± 0.16) of PTSD mice (p > 0.05 vs. all groups, Fig. 5A, B). In the PFC, a significant group effect was found for D1R in the PTSD group (0.55 ± 0.15, p < 0.05 vs. Ctrl, Sevo) and the sevoflurane inhaled increased the level (0.94 ± 0.13, p < 0.05 vs. PTSD, Fig. 5A, B). While when it came to D2R, it changed obviously. The level of D2R was decreased in the hippocampus (0.52 ± 0.14), amygdala (0.42 ± 0.10) and PFC (0.44 ± 0.14) in the PTSD mice (p < 0.01 vs. Ctrl and Sevo). Sevoflurane did not reverse the downregulation very well. Only in the PFC could sevoflurane improve the level of D2R (1.13 ± 0.27, p < 0.05 vs. PTSD, Fig. 5A, C). No differences were observed in the hippocampus (0.56 ± 0.10), amygdala (0.50 ± 0.15) compared with the PTSD group (p > 0.05, Fig. 5A, C).