Effects Of Lentiviral Vector With SiRNA To Silence OXR Gene Of The Hippocampus On Dentate Gyrus Cell Proliferation In Rats Of Sleep Deprivation

Research BackgroundSleep deprivation(SD)refers to the condition that individuals can not satisfy normal sleep because of their own or environmental reasons,and their sleep time can not satisfy the sleep time needed by the body to keep awake and alert,which is manifested as sleep loss or sleep interruption.Sleep deprivation is divided into selective sleep deprivation(SSD)and total sleep deprivation(TSD),and the former is divided into rapid eye movement(REM)sleep deprivation and non-rapid eye movement(NREM)sleep deprivation.The hippocampal structure is an important part of the limbic-cognitive system,including the hippocampus,dentate gyrus(DG),endoolfactory cortex,inferior and anterior inferior receptacles.Its basic function is to receive sensory information from various cortex sources,and encode and store the information in a short time.It plays an important role in cognitive function.Sleep plays an important role in the formation of hippocampus-dependent memory.Short-term memory fragments are reactivated during sleep,and long-term memory is formed after analysis and combination.However,sleep deprivation can lead to disturbance of human or animal thinking and impairment of learning and memory.The main mechanisms are as follows:(1)Sleep deprivation interferes with the induction of long-term potentiation(LTP)in the dentate gyrus,and decreases the basic level of phosphorylated cAMP response element binding protein(CREB)and the expression of calcium/calmodulin kinase IV(CaMK IV)and brain-derived neurotrophic factor(BDNF)in the dentate gyrus of the hippocampus,and then destroys the plasticity of hippocampal synapses[1-2].(2)Long-term sleep deprivation can reduce the expression and function of glutamate receptors in the hippocampus,decrease the proportion of NMDA/AMPA receptors in the hippocampus,and cause changes in the structure of NMDA receptors and damage the formation of LTP[3-4].(3)After sleep deprivation the level of adenosine increases,activates adenosine A1 receptor,reversibly inhibits action potential produced by pyramidal cell layer stimulating hippocampal CA1 region and decreases the frequency of excitatory postsynaptic currents(EPSCs)[5],resulting in impairment of hippocampal function and learning and memory.Other studies have confirmed that long-term sleep deprivation or sleep interruption can reduce the activity of the hippocampus,inhibit the regeneration of hippocampal neurons,and ultimately damage the plasticity and function of the hippocampus,resulting in cognitive impairment[6].At present,effective solutions to sleep deprivation and learning and memory impairment caused by sleep deprivation are extremely limited.Studies have reported that caffeine can prevent learning and memory impairment caused by sleep deprivation[7],but the target of caffeine intervention on memory impairmenticaused by sleep deprivation is not clear,and it can cause REM and NREM sleep structural changes.If long term use of caffeine can lead to sleep disorders,it will aggravate memory damage.Therefore,further study of the molecular and cellular mechanisms and specific targets of sleep deprivation and learning and memory impairment is of great clinical significance for the development of effective drugs to treat the learning and memory impairment associated with sleep deprivation.Orexin,also known as hypocretin(Hcrt),was found independently in 1998 by two laboratories[8-9].Orexin is an excitatory hypothalamic neuropeptide with two subtypes,Orexin-A(also known as Hcrt-1)and Orexin-B(also known as Hcrt-2).Earlier studies focused on the role of the Orexin system in food intake and energy balance.With the further study of Orexin it was found that Orexin also participated in many physiological functions,such as sleep-wake,body temperature regulation,blood pressure and neuroendocrine regulation.There are many researches on sleep wakefulness,learning and memory.Orexin acts by activating two cell surface Orexin receptors(OXRs),also known as Hypocretin receptors(Hcrtrs),which are OX1R(Hcrtr 1)and OX2R(Hcrtr 2)that are coupled to G proteins.Orexin neurons originate mainly from the lateral hypothalamus and emit excitatory projections to the central nervous system except the cerebellum.They mainly project to awakening-promoting cell groups,such as histaminergic cells in the papillary body nucleus,dopaminergic and non-dopaminergic cells in the ventral tegmental area,serotoninergic cells in the dorsal raphe nucleus,noradrenergic cells in locus coeruleus and cholinergic cells in dorsolateral tegmental nucleus/tegmental nucleus of pons foot and basal forebrain.The expression of Orexin receptors in the brain is consistent with these projection areas,but the distribution of OX1R and OX2R in the brain is different.OX1R was mainly distributed in locus coeruleus,while OX2R was mainly distributed in the papillary body nucleus of nodules.OX1R and OX2R were co-located in the dorsolateral tegmental nucleus/tegmental nucleus of pontine foot,dorsal raphe nucleus and ventral tegmental area.Recent studies have shown that Orexin-A participates in hippocampal learning and memory by regulating acetylcholinergic,glutamatergic,adrenergic and gamma-aminobutyric acid(GABA)energy systems to induce synaptic plasticity[10-11]It has been further confirmed that Orexin-A can regulate learning and memory by regulating the activation of extracellular regulated protein kinases 1/2(ERK1/2)in the hippocampal dentate gyrus of epileptic rats[12].However,Orexin neurons are very sensitive to sleep deprivation.After sleep deprivation,the synthesis,secretion and receptor expression of Orexin-A are increased[13].Moreover,the increase of Orexin-A expression induced by sleep deprivation in REM phase can lead to bilateral hippocampal neuronal damage[14]These studies suggest that the molecular and cellular mechanisms of sleep deprivation impairing learning and memory can be explored through Orexin-A signaling pathway.Blocking a specific target in this signaling pathway can not only regulate sleep,but also improve the learning and memory impairment caused by sleep deprivation.Therefore,the study of this target has important application value.Most studies on cell proliferation have been based on the use of 5-bromo-2’-deoxyuridine(BrdU),a synthetic thymidine-uracil analogue that is ingested by dividing cells and can replace thymine(T)in the S phase of DNA synthesis by incorporating into replicating DNA molecules.BrdU,a marker of cell division,is considered to be an important marker for cell proliferation.BrdU,doublecortin(DCX)and neuronal nuclear antigen(NeuN)markers can well observe the dynamic development of neural stem cells in dentate gyrus of hippocampus after sleep deprivation.BrdU+ cells are considered as new cells,while BrdU+/DCX+ double labeled cells are considered to be new migratory immature neurons,and BrdU+/NeuN+ double labeled cells are new mature neurons.During adult nerve regeneration,DCX expression began at the time of neural stem cell formation,peaked at the second week,and decreased with the appearance of neuron marker NeuN[15-16].The differentiation and maturation of the neonatal cells were usually marked at 14 and 28 days after BrdU injection.ObjectiveThe aim of this study was to observe the changes of hypothalamic Orexin-A neurons and hippocampal dentate gyrus cells proliferation after sleep deprivation and the effect of lentivirus(LV)mediated small interfering RNA(siRNA)silencing of hippocampal Orexin receptor gene on the proliferation of hippocampal dentate gyrus cells in sleep deprived rats,and to investigate the molecular and cellular mechanisms underlying learning and memory impairment induced by sleep deprivation via Orexin-A related signaling pathways.MethodsThe experiment is divided into two parts:immunohistochemical part and immunofluorescence part.Immunohistochemistry was used to observe the changes of Orexin-A neurons in hypothalamus of rats after sleep deprivation.In the immunofluorescence part,the effects of sleep deprivation on the proliferation,differentiation and maturation of rat dentate gyrus cells were studied by lentivirus vector transfection,siRNA gene silencing,double immunofluorescence labeling and confocal technique.1.Immunohistochemical section Selected 24 healthy adult male Wistar rats were selected and randomly divided into three subgroups:cage control group(CC),treadmill control group(TC)and sleep deprivation group(SD),with 8 rats in each subgroup.A.Intermittent treadmill was used to carry out 72 hours total sleep deprivation in SD rats.The treadmill speed is set to 10cm/s,running for 3 seconds,stopping for 12 seconds;the TC group runs for 15 minutes,stopping for 60 minutes,running and stopping circulating.This setting did not restrict sleep in the TC group,and allowed a 60-minute break during the 75-minute cycle,but achieved the same amount of exercise as the SD group.Rats in group CC were fed normally.B.Specimens were extracted immediately after 72 hours sleep deprivation.The number of Orexin-A positive neurons in hypothalamus of rats was detected by immunohistochemistry.2.immunofluorescent section Selected 60 healthy adult male Wistar rats were randomly divided into five subgroups:cage control group(CC),treadmill control group(TC),sleep deprivation group(SD),Lentivirus group(LV)and lentivirus mediated siRNA group(LM),with 12 rats in each subgroup.A.Stereotactic injection was performed on LV and LM rats.The hippocampal coordinates were 3.6 mm in the anterior fontanelle,2 mm in the middle line,and 3 mm below the skull surface.LV group was injected with 1.6 uL Lentivirus and LM group was injected with 1.6 uL Hcrtr-siRNA(Hcrtr 1-siRNA:Hcrtr 2-siRNA 0.6:1).B.The rats in SD,LV and LM groups were deprived of sleep for 72 hours by intermittent treadmill.The speed of treadmill was set to 10cm/s,running for 3 seconds and stopping for 12 seconds.The rats in TC group ran for 15 minutes,stopping for 60 minutes,running and stopping circulating.This setting did not restrict sleep in the TC group.The TC group had a 60-minute break during the 75-minute cycle,but achieved the same amount of exercise as the SD,LV and LM groups.Rats in group CC were fed normally.C.During sleep deprivation,rats in each group were given intraperitoneal injection of BrdU 50 mg/kg twice a day for 3 days.D.The expression of BrdU+,BrdU+/DCX+ and BrdU+/NeuN+ cells in dentate gyrus of hippocampus was detected by immunofluorescence technique at 3 time points after 5,14 and 28 days of 72 hours sleep deprivation respectively,4 at each time point.Results1.The immunohistochemical results showed that the expression of Orexin-A positive cells in the hypothalamus of the SD group was higher than that of the TC group(P<0.05),but there was no statistical significance between CC and TC groups(P>0.05).2.The results of immunofluorescence showed that the expression of BrdU+,BrdU+/DCX+ and BrdU+/NeuN+ cells in the dentate gyrus of SD rats decreased compared with TC group(P<0.05),but there was no statistical significance between CC group and TC group(P>0.05),and the expression of BrdU+,BrdU+/DCX+ and BrdU+/NeuN+ cells in the hippocampal dentate gyrus were increased markedly in the LM rats when compared with the LV group(P<0.05),but there was no statistical significance between LV group and SD group(P>0.05).Conclusion1.Sleep deprivation can increase the expression of Orexin-A neurons in the hypothalamus of rats.2.Sleep deprivation can inhibit the proliferation,differentiation and maturation of hippocampal dentate gyrus cells in rats.3.Lentiviral Vector with siRNA silencing of Orexin receptor gene in hippocampus can promote cell proliferation,differentiation and maturation in dentate gyrus of sleep deprived rats.4.Overexpression of Orexin-A induced by sleep deprivation can inhibit the regeneration of hippocampal dentate gyrus cells,while blocking Orexin receptor gene can attenuate the inhibition of hippocampal dentate gyrus cell proliferation.Sleep deprivation may affect the proliferation of hippocampal dentate gyrus neurons and learning and memory through Orexin signaling pathway.