| Postoperative cognitive dysfunction (POCD) is a clinical syndrome characterized by cognitive declines in patients after surgery. The pathogenic factors are not single, multi-factor induc postoperative cognitive dysfunction. Previous studies have suggested that surgery contributed to such impairment. It has been proven that neuroinflammation may exacerbate surgery-induced cognitive impairment in aged rats. Moreover, researches have suggested that superabundant oxygen free radical induced by operation would destroy cell membrane function, break the balance of homeostasis, make cell abnormal dead, impair some organ, lead cognitive decline finally.The free radical scavenger edaravone has high blood brain barrier permeability, and was demonstrated to effectively remove free radicals from the brain and alleviate the development of POCD in patients undergoing carotid endarterectomy, suggesting its potential role in preventing POCD.For this reason, this study was designed to determine whether edaravone is protective against POCD through its inhibitory effects on inflammatory cytokines and oxidative stress.Part Ⅰ Establishment of a rat model of postoperative cognitive impairmentObjective:To investigate the effect of surgery plus LPS administration to rats, establish a rat model of POCD.Methods:60 adult male SD rats were randomly divided into 6 groups,10 rats were in every group. Control group C (intraperitoneal inject placebo at date of surgery), LPS intraperitoneal injection group L (intraperitoneal inject LPS100μg/kg at date of surgery), surgery plus 50μg/kg LPS administration group SL50 ((intraperitoneal inject LPS50μg/kg 1h before surgery and underwent a left nephrectomy), surgery plus 100μg/kg LPS administration group SL100 ((intraperitoneal inject LPS100μg/kg 1h before surgery and underwent a left nephrectomy) and surgery plus 200μg/kg LPS administration group SL200 ((intraperitoneal inject LPS200μg/kg 1h before surgery and underwent a left nephrectomy). All of animals underwent MWM test training at 5 days before operation and were evaluated the cognition, meanwhile, they also accepted fear conditioning training at 1 day before operation. The MWM test and Fear conditioning test were respectively performed to assess learning, memory and cognitive flexibility at 3 days and 7 days after operation.Results:Every group could find platform quickly and accurately through 5 consecutive training days, there is no significant difference between groups(p>0.05). On postoperative day 3, compared to group C, the well time in the target quadrant of group SL100 and group SL200 decresed from29.14±6.88s to 19.64±8.28s and 19.38± 8.21s, the number of crossings of group SL100 and group SL200 decresed from3.44 ±1.51 to 2.40±1.58 and 2.22±1.64, the escape latency of group SL100 and group SL200 increased from 9.59±10.14s to 29.60±19.10s and 30.21±18.42s.The well time in the target quadrant in the first MWM probe trial in the SL100 group and SL200 group were decreased notably compared to C group(p<0.05), and the number of crossings also showed a decreasing tendency, although it did not reach significance(p>0.05). In the working memory test, the escape latency needed to reach the new platform was increased obviously (p<0.05) in the SL100 group and SL200 group compared to the C group. On postoperative day 7, there was no statistical difference between the SL100 group and SL200 group and other groups in dwelling time in the target quadrant, number of crossings, or escape latency(p>0.05). In the fear conditioning test, the tone-related fear conditioning test on postoperative day 3, compared to group C, the freezing time percentage of group SL100 and group SL200 decresed from 78.31±12.2 to 51.79±12.87 and45.00±15.90. The freezing time percentage was notably decreased in the SL100 group and SL200 group when compared to the C group (p<0.001),on postoperative day 7, freezing responses to the tone were not significantly different between any of the groups (p>0.05). In the fear conditioning test, the cortex-related fear conditioning test on postoperative day 3 the tone-related fear conditioning test on postoperative day 3, compared to group C, the freezing time percentage of group SL100 and group SL200 decresed from 70.38±19.60 to 29.60±9.98 and 21.26±8.72. On postoperative day 7, compared to group C, the freezing time percentage of group SL100 and group SL200 decresed from 77.43±14.20 to 56.02±16.27 and 56.02±16.27.In a novel context test, it revealed highly significant impairment in the SL100 group and SL200 group when compared to the C group on postoperative days 3 (p<0.01) and 7 (p <0.05).Conclusion:A left nephrectomy with 100μg/kg or 200μg/kg LPS administration could induce cognitive dysfunction in adult rats, and group SL100 and SL200 showed no difference in the above index,We chose surgery with 100μg/kg LPS administration as a rat model of POCD.Part II Effect of edaravone in adult rats with surgery and peripheral inflammation induced cognitive functionObjective:To investigate the effect of edaravone with surgery plus 100μg/kg LPS administration induced cognitive function in a rat model of POCD.Methods:80 adult male SD rats were randomly divided into 4 groups,20 rats were in every group. It were the control plus placebo group (C-P), control plus edaravone group (C-E), surgery plus placebo group (S-P), and surgery plus edaravone group (S-E). Each group was divided into two subgroups randomly (10 rats per group):the 3-day postoperative group and 7-day postoperative group. All of animals underwent MWM test training at 5 days before operation and fear conditioning training at 1day before operation. S-P group and S-E group were administrated LPS 100μg/kg i.p. Then they underwent a left nephrectomy 1h after injection. C-E group and S-E group were respectively administrated edaravone 3mg/kg until 3 days and 7 days after operation, and the animals were sacrificed at 3 days and 7 days postoperatively. We assessed rat’s cognition via MWM test and fear conditioning test. Superoxide dismutase activities and maiondialdehyde levels were measured in the hippocampus and prefrontal cortex on postoperative days 3 and 7, and microglial (Iba1) activation was also examined 3 and 7 days after surgery.Results:On postoperative day 3, compared to group C, the well time in the target quadrant of group SL100 and group SL200 decresed from 28.14±6.05s to 20.57± 4.69s and 27.32±6.19s (p<0.05),the number of crossings of group SL100 and group SL200 decresed from 3.20±1.62 to 2.22±0.98 and 2.71±1.70 (p<0.05),the escape latency of group SL100 and group SL200 decresed from 7.59±3.35s to 28.83±19.46s and 13.32±9.03s(p<0.05). In the fear conditioning test, the cortex-related fear conditioning test on postoperative day 3, compared to group C, the freezing time percentage of group SL100 and group SL200 decresed from73.78±20.44 to 39.95± 16.49 and 66.58±22.08. In the tone-related test, compared to group C, the freezing time percentage of group SL100 and group SL200 decresed from 73.16±22.58 to 44.31± 13.07 and 71.04±26.15. Rats that underwent surgery plus lipopolysaccharide administration showed significant impairments in spatial and working memory, accompanied by significant reductions in hippocampal-dependent and independent fear responses (p<0.05).All impairments were attenuated by treatment with edaravone. Moreover, compared to group C, superoxide dismutase activation in the hippocampi of group SL100 and group SL200 decresed from 102.8±38.31 U/mg protein to50.78± 20.34 U/mg protein and 71.37±20.23 U/mg protein (p<0.05), malondialdehyde levels incresed from 1.56±1.21nmol/mg protein to 3.65±1.25 nmol/mg protein and 1.89±0.83 nmol/mg protein (p<0.05); compared to group C, superoxide dismutase activation in the prefrontal cortices of group SL100 and group SL200 decresed from 129.7±44.73U/mg protein to54.65±12.00U/mg protein and 76.26±15.71U/mg protein (p<0.05), malondialdehyde levels incresed from 1.84±1.02nmol/mg protein to 3.30±1.04nmol/mg protein and 2.47±0.86 nmol/mg protein(p<0.05).Compared to group C, the number per area of microglial cell in the hippocampi of group SL100 and group SL200 incresed from 7.00±1.01 to 14.67±1.53 and 10.02±1.00 (p<0.05), the number per area of microglial cell in the prefrontal cortices of group SL100 and group SL200 incresed from 7.12±1.00 to 16.67±3.22 and 10.33±1.53 (p<0.05)。 All mentioned abnormal changes were totally or partially reversed by edaravone (p< 0.05)Conclusions:Edaravone could protect surgery plus lipopolysaccharide administration induced POCD. It might relate to its anti-oxidative stress and anti-inflammatory effects.Par tⅢ The protective mechanism of edaravone in adult rats with surgery and peripheral inflammation induced cognitive functionObjective:To investigate the protective mechanism of edaravone with surgery plus LPS administration induced cognitive function in rats.Methods:The hippocampi and prefrontal cortices of rats in every group of part Ⅱ were measured p-Akt, p-mTOR and EAAT3 protein expression with weston blot, They also were detected synaptophsin SYN-1 by immunofluorescence staining.Results:In the hippocampus, compared to group C, the gamma ratio of p-Akt of group SL100 and group SL200 decresed from 0.73±0.03 to 0.42±0.05 and 0.54±0.05, the gamma ratio of p-mTOR of group SL100 and group SL200 decresed from 0.71±0.04 to 0.49±0.03 and 0.59±0.03, the gamma ratio of EAAT3 of group SL100 and group SL200 decresed from 0.23±0.01 to 0.10±0.01 and 0.14±0.01. In the prefrontal cortex, compared to group C, the gamma ratio of p-Akt of group SL100 and group SL200 decresed from 0.97±0.18 to 0.51±0.03 and 0.88±0.09, the gamma ratio of p-mTOR of group SL100 and group SL200 decresed from 0.48±0.04 to 0.19±0.14 and 0.38± 0.10, the gamma ratio of EAAT3 of group SL100 and group SL200 decresed from 0.20 ±0.0 to 0.13±0.01 and 0.16±0.01. In the hippocampus, compared to group C, the synapsin-1 of group SL100 and group SL200 decresed from 1.68±0.11 to 0.74±0.13 and 1.28±0.07 (p<0.05).Down-regulation of p-Akt, p-mTOR and EAAT3 protein expression, and a statistically significant decrease in synapsin-1 were observed in the hippocampi and prefrontal cortices of rats after surgery with LPS injection (p<0.05) All mentioned abnormal changes were totally or partially reversed by edaravone (p< 0.05).Conclusions:The therapeutic effects of edaravone might be closely related to the maintenance of Akt/mTOR signal pathway activation, up-regulation of EAAT3 protein expression and protection of synaptic plasticity. |
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