| Pediatric OSAHS can caused the multi-system damage,Cognitive impairment caused by OSAHS has become a hot topic in recent years,but the research conclusions were inconsistent.Cognitive function requires the collaborative participation of multiple brain regions to deliver and store information.The hippocampus is the most important structure in these brain regions.It has been found that the multi-system damage caused by OSAHS is closely related to oxidative stress,while the cAMP/ PKA-CREB signaling pathway are involved in cognitive function.Therefore,In this study,young rat model of intermittent hypoxia will be established to simulate the process of OSAHS in children,and give Edaravone,a free radical scavenger to treat,in order to explore the effects of oxidative stress on the cognitive function,hippocampus and cAMP/ PKA-CREB signaling pathway,as to provide theoretical evidence and drug treatment for OSAHS causes cognitive dysfunction.Methods:1.Eighty healthy 4-week-old male Wistar rats were randomly divided into four groups(20 rats per group).(NC group),rats were exposed to intermittent compressed air without any drug treatment;(IH group)rats were exposed to intermittent hypoxic conditions without any drug treatment;(IH + ED group)rats were intraperitoneally administered 5 mg/kg edaravone and were then exposed to intermittent hypoxic conditions;(IH + NS group)rats were intraperitoneally administered the same volume of normal saline as edaravone and were then exposed to intermittent hypoxic conditions.After total exposure was 4 weeks,the water maze experiment was conducted to determine the time of evading latency,the time of crossing the targetquadrant and the number of times crossing the platform.2.After the water maze experiment,decapitation and the brain was taken out.HE staining was used to determine the pathology of the hippocampus in each group,Nissl bodies in hippocampal neurons was detected by Nissl staining,the ultrastructure of hippocampal neurons was observed by transmission electron microscopy,and the expression of hippocampal neuron-specific marker(NeuN)was determined by immunofluorescence.3.The content of superoxide anions,the capacity of inhibiting hydroxyl radical,Mn-SOD,Cu/ Zn-SOD,CAT,MDA,8-OHdG and Protein carbonyl in the hippocampus of each group were detected by spectrophotometer or enzyme marker.4.cAMP content in hippocampal tissues of each group was detected by ELISA,The expression levels mRNA and protein of PKAc,CREB,p-CREB,bcl-2 and BDNF in hippocampal tissues of each group were detected by RT-PCR and Western blot.Results:1.Effects of intermittent hypoxia on cognitive function in young rats and the intervention effect of edaravoneEscape latencies significantly increased,the traveling time in the target quadrant and number of times crossing the platform were decreased in the IH and IH+NS groups as compared with those in the NC group(p < 0.05),but they did not differ between the NC and the IH+ED group(p > 0.05).however,Escape latencies significantly decreased,the traveling time in the target quadrant and number of times crossing the platform were increased in the IH and IH+NS groups as compared with those in the IH+ED group(p < 0.05).2.Effects of intermittent hypoxia on the general and subtle structures of hippocampal neurons in young rats(1)HE staining: The IH and IH+NS groups exhibited a loose intercellular matrix in the CA1 area with a widened pericellular space.Degenerative and necrotic neurons were also found.The karyon was shrunk and hyperchromatic,the nucleus was hyperchromatic and partially irregular,and the nucleolus was unclear.Notably,the morphological and structural abnormalities in the CA1 area was attenuated in the IH+ED group.The damage in CA1 area was significantly more than those in CA3 area.(2)Nissl staining: in the IH and IH+NS groups,Nissl bodies decreased.Notably,in the IH+ED group,Nissl bodies increased.In the IH,IH+NS,and IH+ED groups,Nissl bodies decreased more in the CA1 area than in the CA3 area.(3)NeuN immunofluorescence: compared with the NC group,the mean fluorescence intensity of the CA1 area in the IH group and the IH+NS group was significantly decreased(p < 0.05),and there was no significant difference between the IH+ED group and the NC group(p >0.05).The mean fluorescence intensity of IH+ED group was significantly higher than that of IH group and IH+NS group(p <0.05).There was no statistical significance in the mean fluorescence intensity of CA3 between NC group,IH group,IH+NS group and IH+ED group(p > 0.05).(4)Electron microscopy: Compared with the NC group,the neuronal chromatin was significantly reduced,the density was non uniform,the nuclear membrane was invaginated,some mitochondria had vacuolar degeneration,and the number of mitochondria decreased.Notably,in the IH+ED group,the neuronal chromatin decreased slightly,the chromatin density was uniform,and mitochondrial morphology and quantity were close to those in the NC group.3.Oxidative stress in the hippocampus(1)Changes in oxide content of hippocampus:Compared with the NC group,the capacity of inhibiting hydroxyl radical was significantly decreased(p < 0.05),the levels of superoxide anion was significantly increased in the IH and IH+NS groups(p< 0.05),but they did not differ between the NC and the IH+ED group(p >0.05).Compared with IH group and IH+ NS group,the capacity of inhibiting hydroxyl radical was significantly increased and the levels of superoxide anion was significantly decreased in IH+ED group(p < 0.05).(2)Changes in antioxidant content of hippocampus:The expression levels of Mn-SOD and CAT were significantly decreased in the IH and IH + NS groups compared with the NC group(P < 0.05)or the IH+ED group(P < 0.05),but did not differ between the NC group and the IH + ED group(P > 0.05).The expression levels of Cu/Zn-SOD slightly decreased in the IH,IH+ED,and IH + NS groups compared with the NC group,but no significant differences were found among the 4 groups(P >0.05).(3)Changes in oxidation products of hippocampal tissue: Compared with the NC group,significantly increased levels of MDA,8-OHdG and Protein carbonyl were observed in the IH and IH + NS groups(P < 0.05).Notably,no significant differenceswere found between the NC group and the IH+ED group(P > 0.05).Moreover,significantly decreased levels of MDA,8-OHdG and Protein carbonyl were detected in the IH+ED group compared with the IH and IH + NS groups(P < 0.05).4.The expression levels of the cAMP/ PKA-CREB signaling pathway-related proteins and mRNA in the hippocampusThe protein expression levels of cAMP,PKAc,p-CREB,bcl-2,and BDNF in the hippocampus were significantly downregulated in the IH and IH + NS groups compared with the NC group or the IH+ED group(P < 0.05),but did not differ between the NC group and the IH+ED group(P > 0.05).The expression levels of CREB did not differ among the four groups(P > 0.05);however,p-CREB levels were decreased in the IH and IH+NS groups compared with the NC group or the IH+ED group(P < 0.05),and no differences were detected between the NC group and the IH+ED group(P > 0.05).The findings suggest that intermittent hypoxia downregulate protein expressions of cAMP,PKAc,p-CREB,Bcl-2,and BDNF in the cAMP/PKA-CREB signaling pathway,and edaravone can up-regulate the expression of these proteins.The expression trend of mRNA corresponding to the protein was consistent with these proteins.Conclusion:(1)Intermittent hypoxia can lead to Cognitive impairment in young rats,mainly manifested by decreased learning and memory ability.Edaravone can alleviate IH-induced cognitive dysfunction.(2)Cognitive dysfunction caused by intermittent hypoxia is closely related to hippocampal oxidative stress injury.Compared with the CA3 area,the CA1 area is vulnerable to oxidative stress.Edaravone can reduce hippocampal damage by removing ROS and normalizing oxidative balance.(3)The cAMP/ PKA-CREB signal transduction pathway is closely related to cognitive dysfunction caused by intermittent hypoxia,edaravone attenuated IH-induced cognitive impairment and hippocampal damage by upregulating p-CREB in young rats. |
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