Study On The Mechanism Of Dual Orexin Receptor Antagonist Attenuates Learning And Memory Impairment In Chronic Sleep Deprivation Mice Model

ObjectiveSleep deprivation(SD)or lack of sleep is becoming more common among young people.Chronic sleep deprivation(SD)disturbances the daily sleep/wake cycle regulated by the orexin system and further increases the risk of insomnia or sleep disorders,which is an important risk factor for cognitive impairment is a significant risk factor for cognition impairment.Some studies believe that sleep disorders are the principal risk factors and characteristic manifestations of progressive mild cognitive impairment(MCI)and Alzheimer’s disease(AD).Recent research showed that sleep is necessary for the process of adequate metabolite clearance and protein,including beta-amyloid(Aβ)and phospho-tau protein from the central nervous system(CNS)of AD patients.SD will lead to the accumulation of metabolic waste in the CNS that increases the production of metabolic proteins and slows down the clearance speed,negatively affecting cognition.In recent years,the glymphatic system formed by the participation of astrocytes and worked during astrocytes,which opens the door to the study of metabolic waste clearance in the brain,which is active in sleep.Studies have also shown that astrocytes shrink in size during sleep,increasing the extracellular space volume,helping to remove metabolic waste.These studies showed that maintaining the normal morphology and function of astrocytes is very important.Furthermore,studies have found that the orexin system can enhance the migration of rat brain astrocytes through the OX1R-PLC-Ca2+-PKCa signaling pathway.At present,research on the orexin system focuses on the sleep/wake cycle mechanism,and there is little evidence on whether orexin is directly involved in cognitive memory.Moreover,whether the orexin system affects learning and memory by affecting the clearance function of the glymphatic system is worthy of further discussion.Methods1.Establish an animal model of chronic sleep deprivationFor SD studies,mice in SD and SD/ALM groups were placed in a sleep fragmentation chamber.Briefly,it employs intermittent tactile stimulation using a near-silent motorized horizontal bar sweeping just above the cage floor from one side to the other.The sweeping bar moved along the cage’s bottom every 2 minutes last 20 hours a day(11 am-7 am).Moreover,the sweep bar automatically shut off and was stationary during the set time(7 am-11 am)by a timing device.Mice in the Con and Con/Veh groups that received undisturbed sleep were placed in sleep fragmentation chambers with stationary sweep bars.SD exposure lasted for 6 weeks,during which mice with food and water available.After 6 weeks,the Y-maze and Morris water maze(MWM)tests were deployed to evaluate the mice’s short-and long-term spatial learning and memory.2.Establish a drug intervention modelMice in the SD/ALM group were treated with almorexant at 30 mg/kg/day..According to the instructions,almorexant is formulated in a vehicle containing 5%DMSO,40%PEG300,5%Tween-80,and 50%deionized water.As a control,the Con/Veh group mice were treated with vehicle alone(5%DMSO/40%PEG300/5%Tween-80/50%in deionized water).The almorexant was injected intraperitoneally every day for 6weeks after the sweeping bar stopped working.The almorexant and solutions were freshly prepared every day.3.Detection of molecular biological indicatorsPCR,immunohistochemistry,and western blot were used to detect the expression of Aβ protein,tau/phospho-tau protein,and GFAP in the hippocampus in each group of mice after the intervention.Results1.The sleep deprivation chamber that simulates artificial caressing by tactile stimulation is stable and feasible for the sleep deprivation process of mice has little effect on the stress state of mice,and has low mortality during the intervention period.Furthermore,it was observed that the learning and memory impairment of mice in the 20-hour sleep deprivation group was more significant than that in the 9-hour sleep deprivation group.2.We observed that the SD group mice swim more around the pool wall than the control group.The hidden platform test revealed that the groups all started similarly for the first 2 days but separated on days 3-5,and the escape latency of the hidden platform test was significantly longer after SD,which may indicate learning impairment.However,mice in the SD/ALM group found the platform faster than the SD group.In the Y-maze test,the percentage of spontaneous alternation was decreased in the SD and SD/ALM group mice.Importantly,mice treated with almorexant showed a significant increase in spontaneous alternation percentage than SD group mice.One-way ANOVA analysis also showed a significant difference between the Con and SD group mice in terms of time spent in the novel arm,and the SD/ALM group mice spent more time in the novel arm than the SD group.3.Immunohistochemistry results showed GFAP-positive astrocytes throughout all hippocampal.We also found GFAP expression highly enriched in the perivascular or the area close to the CSF compartment.Furthermore,compared with the control group,we observed the astrocytes in the SD group showed an activated state:enlarged cell bodies and increased synapses in the hippocampus,while almorexant treatment effectively decreased the immunoreactivity of GFAP during chronic SD in CA1 and hf area.4.Immunohistochemical staining showed that the phosphorylated tau protein was more strongly expressed in the cortex than the hippocampus,but there was no much difference in phosphorylated tau protein expression in the hippocampus of each group.Consistently,western blot quantitative analysis observed that total tau protein expression changes were not significant in the hippocampus.Moreover,we observed that phosphorylated tau protein expression was slightly increased after chronic SD.Compared with the SD group,phosphorylated tau protein expression in the SD/ALM group was slightly reduced,but the difference was not statistically significant.No expression of the Aβ protein was observed.These results show that sleep intervention in our modal has little effect on tau protein pathology.ConclusionIn conclusion,cognition impairment and astrocyte activation were observed in the chronic SD mice,and treatment with almorexant could prevent this impairment by suppressing astrocyte activation in the hippocampus in mice.