| BackgroundThe incidence of acute sleep deprivation(ASD)is increasing in the population.The most important impact of ASD on the brain is the impairment of daytime function and learning and memory process.Currently,the therapeutic methods and measures that can improve learning and memory are very limited.The fundamental reason is that the mechanisms about the impairment of sleep deprivation in learning and memory and intervention targets have not been identified.In recent years,more and more researches have shown that Orexin-A and its receptors not only play an important role in the regulation of sleep/wakefulness,but also related to the learning and memory closely.Studies have also shown that SD activates Orexin neurons.The hippocampus is a major part of the brain for learning and memory and is rich in orexin receptors.Studies on intracellular signaling pathways have shown that after Orexin-A binds to its receptor,extracellular signal-regulated kinase 1/2(ERK1/2)is activated mainly through Gq/PLC/PKC pathway,and ERK1/2 signaling pathway also plays an important role in hippocampal neurogenesis,which is closely related to the learning and memory.Based on this,whether it is possible to reduce the damage of learning and memory related to ASD by intervening the Orexin system is the starting point of this study.ObjectiveFirstly,Acute sleep deprivation(ASD)rat model was established to observe the expression of hypothalamus orexin-A after sleep deprivation.Adeno-associated virus(AAV)was used to down-regulate the Orexin receptor in the hippocampus of rats.The effects of ASD on learning,memory and neurogenesis after ASD,14 days after sleep recovery and 28 days after sleep recovery were observed,and the expression of PLCβ1 and phosphorylated ERK1/2 protein in the hippocampus were observed to further explore the possible pathway mechanism.Methods1.Establish ASD rat model and observe the expression of Orexin-A in the hypothalamus.8-10 weeks old Wistar male rats were firstly screened by Y maze forced alternation experiment and randomly enrolled into the group,then put the rats in a deprivation box,and stimulated the rat sleep by tactile stimulation by simulating gentle handing for 72 hours.In the acute sleep deprivation model,only 2 rats are placed in each deprivation box,while reducing the interference of the external experimental environment.Set the movement cycle of the metal rod as 10s(run)/110s(stop).Before the formal sleep deprivation,the rats need to be placed in the box to adapt to the environment for about 3 days.The expression of Orexin-A in the lateral hypothalamus of rats after 72h acute sleep deprivation was observed by immunohistochemical.2.Establish an adeno-associated virus intervention modelAdeno-associated virus(AAV)that carries the gene of interest was used to interfere with the expression of Orexin dual receptor(OXR)genes in the hippocampal CA1 area,and establish a rat model with low Orexin receptor expression in the hippocampus CA1 area.According to the receptor gene sequence,the virus siRNA gene vector is first designed,and the primary hippocampal cells are transfected,screened,and verified by PCR and Western blotting.Finally,stable protein and low gene expression sequences are selected for stereo localization in the hippocampal CA1 region injection.After 2 weeks of postoperative recovery,the follow-up experiment was performed.3.Mark newborn nerve cellsWe use 5-bromodeoxyuridine(BrdU)instead of thymine nucleoside to enter the process of cell proliferation to mark new neurons in the hippocampus.To observe the proliferation of nerve cells during ASD,BrdU was injected intraperitoneally at a fixed time point every day during ASD modeling,and the labeled BrdU nerve cells were observed by immunofluorescence.4.Evaluation of learning and memory abilityMorris water maze and Y maze were used to comprehensively evaluate the spatial learning and memory of rats.The evaluation contents mainly included spontaneous alternating times,escape latency,platform crossing times,swimming and movement speed.The evaluation time are after ASD,14 days and 28 days after sleep recovery.5.Immunofluorescence staining and molecular biological index detectionAfter the intervention,immunofluorescence co-labeling was used to observe the BrdU expression(0 time point),BrdU and DCX co-labeled expression(14 day point),and BrdU and NeuN co-labeled expression(28 day point)in the hippocampus of each group of rats.Confocal microscopy and semi-quantitative statistical analysis were used in this part.Western blotting and RT-PCR were used to determine the expression of OXR protein,pERK1/2 and PLCβ1 in the hippocampus of rats after ASD.Results1.The 72 hours sleep deprivation process was stable in rats using a sleep deprivation box to simulate artificial stroking through tactile stimulation,and no obvious irritating events such as fighting were observed.After ASD,the rat’s"revenge" sleep increased,indicating that the experimental ASD model was constructed.In addition,it was observed that the positive expression of Orexin-A in the lateral hypothalamus after ASD was significantly increased.2.Behavioral experiments of rats in each group showed that the number of spontaneous alternations of rats decreased in ASD groups,the duration of escape latency prolonged,and the number of platform crossings decreased in space exploration tasks.Compared with rats in the ASD group,the rats in the OXR-KD ASD group had stronger spontaneous alternation ability,shortened the escape latency,increased the number of platform crossings.And after 14 days sleep recovery,the escape latency of ASD rats was still prolonged,but there was no significant difference in the ability of spontaneous alternation and space exploration compared with the control group.There was no significant difference in the behavioral experiment of rats in each group after 28 days of sleep recovery.These results indicates that ASD impairs the short-term working memory and spatial learning and memory ability of rats,but this damage can be alleviated and recovered after sleep recovery,and downregulation of hippocampal OXR function can reduce this damage.3.After ASD,the immunofluorescence of the rats in each group showed that the BrdU-positive neurons in the rats decreased after ASD,and after 14 days of sleep recovery,the number of neuronal migration and differentiation produced during ASD was reduced by the co-labeling of BrdU and DCX,and after 28 days sleep recovery,the BrdU and NeuN co-labeling showed that the number of maturing neurons produced during the ASD period was reduced.Compared with the ASD group,the number of neurons in the OXR-KD ASD group increased at the above three time points.This results indicate that ASD damages the neurogenesis process in rats,and downregulation of hippocampal OXR function may alleviate these damage.4.After ASD the pERK1/2 and PLCβ1 proteins in the hippocampus of each group of rats were detected by Western blotting.It was observed that the expression of the above proteins increased after ASD,and the expression trend of the rats in OXR-KD ASD group was consistent with the control group.These results suggest that 72 hours ASD can cause the activation of the above-mentioned proteins,and down-regulation of hippocampal OXR function can alleviate this overactivation.ConclusionIn this study,we try to established an ASD rat model,our results showed the expression of Orexin-A in the lateral hypothalamus increased after ASD.72 hours ASD can impair short-term and long-term spatial learning and memory in wistar rats,but the impairment process can be improved after 14 days of sleep recovery.72 hours ASD can reduce the generation,migration,differentiation and maturation of new neurons in the hippocampal CA1 region.Down-regulation of hippocampal orexin receptor can alleviate the cognitive impairment caused by ASD and may protect the neurogenesis process of the hippocampus.This process may be associated with the Orexin-A-PLCβ1-pERK1/2 pathway. |
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