Repeated Sevoflurane Exposures in Neonatal Rats Increased the NKCC1/KCC2 Ratio in the PFC at P14, Which was Alleviated by Pretreated with the NKCC1 Inhibitor Bumetanide
The authors investigated whether repeated sevoflurane exposures in neonatal rats altered the expression the NKCC1/KCC2 ratio in the PFC during the day of fear conditioning training at P14. The results showed repeated sevoflurane exposures in neonatal rats at P5-7 had no statistically difference on expression of NKCC1 (F(2, 15) = 1.21, p = 0.11; One-way ANOVA; Fig. 2A) and KCC2 levels in the PFC at P14 (F(2, 15) = 0.94, p = 0.08; One-way ANOVA; Fig. 2B). However, the resulting NKCC1/KCC2 ratio in the PFC at P14 was significantly increased for neonatal sevoflurane-exposed rats (F(2, 15) = 3.27, p < 0.05; Fig. 2C), which was alleviated by pretreated with the NKCC1 inhibitor bumetanide (F(2, 15) = 3.27, p < 0.05; Fig. 2C). These results suggested the possibility the brain depolarizing GABAAR activity might be altered when exposed to the electric foot shock stress in fear conditioning training at P14 for neonatal sevoflurane-exposed rats, which could be attenuated by pretreated with the NKCC1 inhibitor bumetanide.
Repeated Exposures to Sevoflurane in Neonatal Rather Than Juvenile Rats Increased the Stress Response to Electric Foot Shock in the Fear Conditioning Training
In order to assess whether neonatal exposure to sevoflurane alters the susceptibility to stress exposure, serum corticosterone levels under basal and stress conditions were measured in blood samples collected from P14 rats 60 min before the process of fear conditioning and 5 min after the last unconditioned stimulus (After-US). The results showed neonatal sevoflurane-exposed had unaltered secretion of corticosterone under the basal condition (Fig. 3A), however, the levels of corticosterone under stress condition for neonatal sevoflurane-exposed rats was significantly higher than CON + VEH group (F(3,21) = 18.17, p < 0.05, one-way ANOVA; Fig. 3A). Pretreatment of neonatal rats prior to each sevoflurane exposure with BUM significantly attenuated the heightened secretion of corticosterone after the stress exposure in fear conditioning training (F(3,21) = 18.17, p < 0.05; Fig. 3A).
To test whether sevoflurane exposure at different developmental brain stage affect the stress response, juvenile rats receiving identical sevoflurane exposures on P25, 26, 27 were explored. The results showed there were no statistical difference between SEV + VEH and CON + VEH groups in secretion of corticosterone under basal and stress conditions (Fig. 3B) at P34.
These results indicated repeated exposures to sevoflurane in neonatal rather than juvenile rats increased the neuroendocrine response to future stress exposure, which might be associated with the neonatal enhanced brain depolarizing GABAAR activity.
Repeated Sevoflurane Exposures in Neonatal Rats Increased the Brain Vulnerability to Future Stress Exposure
The authors tested whether repeated sevoflurane exposures in neonatal rats increased the brain vulnerability to the future stress exposure and whether the adverse results could be attenuated by bumetanide. The basal neuroapoptosis for neonatal sevoflurane-exposed rats were determined without receiving electric foot shock stress at P14. Other independent rat groups were used in determination of stress-induced neuroapoptosis in the PFC six hours after exposed to electric foot shock stress in fear conditioning training at P14. The results showed there was no significantly difference in the neuroapoptosis in the PFC for rats not receiving electric foot shock stress (p = 0.15; Fig. 4). However, repeated sevoflurane exposures in neonatal rats at P5, 6, 7 increased the neuroapoptosis in the PFC six hours after exposed to electric foot shock stress in fear conditioning training at P14 (F(2, 15) = 5.12, p < 0.01; Fig. 3C), which was alleviated by pretreated with the NKCC1 inhibitor bumetanide (Fig. 3C, F(2,15) = 5.12, p < 0.05; Fig. 3C). Repeated sevoflurane exposures for juvenile rats at P25-27 had no significant effects on neuroapoptosis in the PFC six hours after exposed to stress in fear conditioning training at P34 (Fig. 3D).
These results indicated repeated exposures to sevoflurane in neonatal rather than juvenile rats increased the brain vulnerability to adverse post-stressful factors, which might be associated with the neonatal enhanced brain depolarizing GABAAR activity.
Neonatal Rather Than Juvenile Sevoflurane-exposed Rats Exhibited Deficits in Fear Extinction Training and Recall
For neonatal sevoflurane-exposed rats, there was no significantly difference in the total distance travelled in Open-field test among CON + VEH, SEV + VEH and SEV + BUM groups at P18 (Fig. 5A). The fear conditioning acquisition was determined after the Open-field test. One-way ANOVA analysis revealed there were no group differences in pre-CS freezing prior to CS-presentation (F(2,33) = 0.63, p = 0.79; Fig. 5C). The results showed the rate of freezing under CS-presentation at P18 was significantly increased for CON + VEH, SEV + VEH and SEV + BUM groups (Fig. 5C). Although the SEV + VEH group exhibited higher rate of freezing than CON + VEH and SEV + BUM groups in fear conditioning acquisition, there was no statistical difference among CON + VEH, SEV + VEH and SEV + BUM groups (F(2,33) = 0.86, p = 0.13; Fig. 5C; One-way ANOVA). For juvenile sevoflurane-exposed rats, there was no significantly difference in the total distance travelled in Open-field test between CON and SEV groups at P38 (Fig. 5B). The fear conditioning acquisition at P38 was significantly increased for CON and SEV groups at P38 (Fig. 5D).
Following the determination of fear conditioning acquisition, the authors performed the fear extinction training for neonatal sevoflurane-exposed rats, which was the laboratory basis of exposure therapy for anxiety disorders. Regarding the rate of freezing change during extinction training, there was a significant effect of extinction trial (F(5,165) = 24.43, p < 0.01; Fig. 6A). Repeated measures two-way ANOVA showed there was significant effect of treatment (F(2,165) = 18.87, p < 0.01; Fig. 6A) on the rate of freezing response in fear-extinction training and an interaction of treatment-by-trial (F(10,165) = 29.65, p < 0.01; Fig. 6A). The post hoc Bonferroni test showed SEV + VEH rats had a higher rate of freezing response than the CON + VEH rats at CS3, CS4, CS5 and CS6. Pretreated with the NKCC1 inhibitor bumetanide before neonatal sevoflurane exposures lowered the rate of freezing response in fear-extinction training compared with SEV + VEH rats at CS5 and CS6.
The CON + VEH and SEV + BUM rats exhibited comparable, low CS-elicited freezing at CS5 and CS6 in fear-extinction training, which suggested the success in retention of fear extinction for both groups (Fig. 6A), however, SEV + VEH rats still exhibited high CS-elicited freezing compared with the CON + VEH and SEV + VEH groups at CS5 and CS6, suggesting the deficit in retention of fear extinction. Then rats were tested for extinction recall at P20 by receiving 2 min CS presentation. All groups exhibited comparable, low, pre-CS freezing during the adaptation period prior to extinction recall (F(2,33) = 1.28, p = 0.78; Fig. 6B ), however the groups differed significantly in CS-elicited freezing (F(2,33) = 16.21, p < 0.01; Fig. 6B). The post hoc Bonferroni test showed that the SEV + VEH rats exhibited significantly higher rate of freezing response compared with the CON + VEH group (F(2,33) = 16.21, p < 0.01; Fig. 6B) in extinction recall. Pretreated with the NKCC1 inhibitor bumetanide before neonatal sevoflurane exposures reduced the rate of freezing response in fear-extinction recall (F(2,33) = 16.21, p = 0.019; Fig. 6B).
For juvenile sevoflurane-exposed rats, there was no difference between the two groups in pre-CS freezing prior to extinction training (p = 0.78; Fig. 6C). Regarding the rate of freezing change during extinction training, there was significant effect of extinction trial (F(5,110) = 16.23, p < 0.01; Fig. 6C). However, repeated measures two-way ANOVA showed there was no effect of group (F(1,110) = 0.96, p = 0.67; Fig. 6C) and trial-by-group interaction (F(5,110) = 1.49, p = 0.39; Fig. 6C), indicating that both groups exhibited comparable rates of extinction (Fig. 6C). Both groups exhibited comparable, low, pre-CS freezing during the adaptation period prior to extinction recall (p = 0.57; Fig. 6D). There was also no difference between the two groups in CS-elicited freezing (Fig. 6D).