| Objective:The present study is to investigate the potential regulatory capacity of hypoxia exposure on emotions and spatial learning and memory in chronic fatigue syndrome mice induced by restraint and forced swimming. Furthermore, we explore the underlying mechanism via the varying of CREB activation and BDNF expression in hippocampus.Methods:1. AnimalsExperiment 1:Seventy-two male ICR mice (weighting 30-40g) were randomly divided into six groups:normal control group (C, n=12), chronic fatigue syndrome model group (CFS, n=12), seven days natural recovery normal control group (C1, n=12), seven days natural recovery chronic fatigue syndrome model group (CFS1, n=12), seven days 15%O2 exposure chronic fatigue syndrome model group (15%CFS1, n=12) and seven days 18%O2 exposure chronic fatigue syndrome model group (18%CFS1, n=12). The mice were induced chronic fatigue syndrome by five weeks restraint and forced swimming in group CFS, CFS1,15%CFS1 and 18%CFS1. The other two groups were normally fed in the same condition with chronic fatigue syndrome mice.Experiment 2:Forty-eight male ICR mice (weighting 30-40g) were randomly divided into four groups:ten days natural recovery normal control group (C2, n=12), ten days natural recovery chronic fatigue syndrome model group (CFS2, n=12), ten days 12%O2 exposure chronic fatigue syndrome model group (12%CFS2, n=12) and ten days 15%O2 exposure chronic fatigue syndrome model group (15%CFS2, n=12). The mice were induced chronic fatigue syndrome by five weeks restraint and forced swimming in group CFS2,15%CFS2 and 18%CFS2-Another group was normally fed in the same condition with chronic fatigue syndrome mice.2. Hypoxia exposure protocolExperiment 1:The mice were sacrificed in C and CFS after five weeks restraint and forced swimming. The other four groups naturally recovered for seven days. In addition, the mice were separately received 15%O2 and 18%O2 intervention in 15%CFS1 and 18%CFS1 for one hour every day during the recovery period.Experiment 2:All the four groups naturally recovered for ten days. In addition, the mice were separately received 12%O2 and 15%O2 intervention in 12%CFS2 and 15%CFS2 for four hours every day during the recovery period.3. Animal behavior testsBehavior tests for the mice in C and CFS:The morris water maze (MWM) was performed at the fifth week of restraint and forced swimming, and the tail suspension test (TST) was carried out following MWM. Then the open field test (OFT) and O maze test (OMT) were performed after twenty-four hours.Behavior tests for the mice in C1, CFS1,15% CFS1 and 18% CFS1:The MWM was performed at the last six days during 7-day natural recovery, and the tail suspension test (TST) was carried out following MWM. Then the open field test (OFT) and O maze test (OMT) were performed after twenty-four hours.Behavior tests for the mice in C2, CFS2,12% CFS2 and 15% CFS2:The MWM was performed at the last six days during 10-day natural recovery, and the tail suspension test (TST) was carried out following MWM. Then the open field test (OFT) and forced swimming test (FST) were performed after twenty-four hours.4. Sample collection and outcome measurementThe mice in C and CFS were sacrificed after thirty-six hours following the last restraint and forced swimming or hypoxia exposure, which was to avoid the effect of acute stress. After anesthetization, blood was drawn from the eye and serum prepared by centrifugation. Then the hippocampus was separated on the ice immediately. Elisa was used to detect serum pro-inflammation cytokines IL-6, TNF-α and IL-1β. Western blot was used to detect the protein expression of CREB, p-CREB and BDNF. The ratio of p-CREB to CREB (p-CREB/CREB) represented CREB activation.Results1. Effects of hypoxia exposure on learning, memory and emotion1) Compared to C, the mice took longer (P<0.05) to reach an invisible platform hidden under the water in CFS, and spent less time (P<0.05) in the target quadrant. However, there were no significant differences in OFT, TST and OMT between two groups (P>0.05).2) After 7-day hypoxia intervention, repeated measures ANOVA showed group effect F(5,54)=5.46, P<0.05, indicted that the escape latency was different among C1, CFS1,15% CFS1 and 18% CFS1. The escape latency in 15% CFS1 was shorter than in C1 at the second day of MWM (P<0.05), and the escape latency in 15% CFS1 was also shorter than in CFS1 at the fifth day of MWM (P<0.05). In contrast to C1, the mice in CFS1 and 18% CFS1 spent significantly less time in the target quadrant (P<0.05), but there was no significant difference (P >0.05) between C1 and 15% CFS1. No significant differences were observed in OFT, TST and OMT among all the groups (P>0.05).3) After 10-day hypoxia intervention, there were no significant change in learning and memory (P>0.05). In addition, the mice in 12% CFS2 and 15% CFS2 exhibited significant less (P<0.05) immobile time than in CFS2. No significant differences were found in FST and OFT (P> 0.05).2. Effects of hypoxia exposure on serum IL-6, TNF-α and IL-1β1) Compared to C, the serum IL-6, TNF-a and IL-1β significantly increased in CFS (P<0.05).2) After 7-day hypoxia intervention, the serum IL-6 and IL-1β levels in CFS1 and 18% CFS1 were significantly higher than in C1 (P<0.05), but it was not significantly different in IL-6 between 15% CFS1 and C1(P>0.05). No significant differences were observed in the serum TNF-α among all the groups (P>0.05).3) After 10-day hypoxia intervention, there were no significant differences in the serum IL-6 and IL-1β levels among C2,12% CFS2 and 15% CFS2 (P>0.05), but the serum IL-6 and IL-1β levels in CFS2 were still higher than in C1 (P<0.05). No significant differences were found in the serum TNF-α among all the groups (P>0.05).3. Effects of hypoxia exposure on hippocampus CREB activation and BDNF expression1) Compared to C, the mice demonstrated lower CREB activation in CFS (P<0.05), but no significant difference was observed in BDNF expression (P>0.05).2) After 7-day hypoxia intervention, the hippocampus CREB activation in CFSi was significant lower than C1 and 15% CFS1 (P<0.05), but no significant difference was observed between C1 and 15% CFS1 (P> 0.05). The hippocampus CREB activation in 18% CFS1 showed no significant differences with C1 and CFS1 (P>0.05). Compared to C1, the hippocampus BDNF expression exhibited significantly lower in CFS1,15% CFS1 and 18% CFS1 (P>0.05).3) After 10-day hypoxia intervention, it was not significantly different in the hippocampus CREB activation among C2,12% CFS2 and 15% CFS2 (P>0.05), but all the three groups were significant higher than CFS2 (P<0.05). There were no significant differences in the hippocampus BDNF expression among C2,12% CFS2 and 15% CFS2 (P >0.05), but it was significant lower in CFS2 than the other groups (P <0.05).Conclusions1. The spatial learning and memory were impaired in chronic fatigue syndrome mice induced by 5-week restraint and forced swimming, and the depression appeared later than the spatial learning and memory impairment.15% O2 exposure (1h/day) enhanced the spatial learning and memory in chronic fatigue syndrome mice during the 7-day natural recovery. In addition,12%O2 or 15%O2 exposure (4h/day) did not promote the spatial learning and memory in chronic fatigue syndrome mice during the 10-day natural recovery, but produced anti-depression effects.2. The serum IL-6, TNF-α and IL-1β persisted in high level for ten days in chronic fatigue syndrome mice induced by 5-week restraint and forced swimming.15%O2 exposure (1h/day) reduced the serum IL-6 to the normal level during 7-day natural recovery. In addition, the serum IL-6 and IL-1β decreased to the normal level following 12%O2 or 15%O2 exposure (4h/day) during 10-day natural recovery.3. The hippocampus CREB activation was inhibited in chronic fatigue syndrome mice induced by 5-week restraint and forced swimming, and the hippocampus BDNF expression decline was later than CREB activation inhibition.15%O2 exposure (1h/day) promoted hippocampus CREB activation during 7-day natural recovery, but showed no effect on the BDNF expression. However,12%O2 or 15%O2 exposure (4h/day) enhanced the hippocampus CREB activation and BDNF expression during 10-day natural recovery. |
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