| BackgroundPropofol is one of the new general anesthesia drugs,characterized with short-acting.Propofol is widely used in clinical anesthesia and ICU sedation because of its rapid onset,short duration,rapid recovery,low incidence of postoperative nausea and vomiting.In recent years,the impact of anesthetics on the developing brain cognition,learning and memory is a hot topic.Clinical studies showed that infants before the age of two exposure to anesthesia drug could occur personality and behavioral changes.However,the mechanisms of the neurotoxicity of propofol remain unclear.The piriform cortex located outside the ventral forebrain,originated from ancient cortex,belonged to the limbic system,is the largest olfactory cortex of mammalian.The piriform cortex accepts single synaptic input from olfactory bulb and there are complex projection fibers between piriform cortex and other related cortices as well as hippocampus.Piriform cortex consists of three layers.The first layer of piriform cortex named plexiform layer receives projections form olfactory bulb through the lateral olfactory tract(LOT),while the second and the third layers named cell layers receive the excitatory input from LOT and adjacent cortex and also receive the inhibitory input from local interneurons.Neural precursor cells in piriform cortex of the development brain have the properties to proliferate and differentiate into mature neurons.The piriform cortex,like the hippocampus and the subventricular zone possesses the potential of neurogenesis.In addition,the cell layers of piriform cortex mainly contain glutamater-releasing principal neurons and a much smaller number of GABA-releasing inhibitory interneurons.Interneurons in piriform cortex are commonly classified by their expression of two types of molecular markers: calcium-binding proteins and neuropeptides.The calcium-binding proteins interneurons consist of parvalbumin(PV),calbindin(CB)and calretinin(CR)and the neuropeptides interneurons consist of somatostatin(SOM),neuropeptide Y(NPY),cholecystokinin(CCK).The role of interneurons in piriform cortex is quite complex,involving dendritic inputs,axonal outputs and long-range connections between interneuron assemblies.It is precisely because of the regulatory role of interneurons in pirifrom cortex,the interneurons play an important role in olfactory learning and memory processes.Previous studies found that olfactory damage symptoms occurred before cognitive impairment for Alzheimer’s disease patients.Furthermore,there were common binding sites between the SOM interneurons,CR interneurons and pathologic markers of AD such as Aβ and Tau protein in piriform cortex,suggesting the vulnerability of the interneurons in piriform cortex in the progression of AD and cognitive impairment.Additionally,several studies have confirmed that urethane anesthesia could result in selective damage of interneurons in piriform cortex,which might be associated with cognitive impairment caused by anesthetics.In other words,the piriform cortex may be a new target site that anesthetics exert their neurotoxic effects.Neurobehavioral disorders consist of a variety of behavioral deficits caused by brain disease or brain injury.Neuroethology is a method to study the lesions of central nervous system(CNS)through a special behavioral change.Previous studies showed that neonatal exposure to inhaled anesthetic sevoflurane not only caused learning and cognitive impairment,but also resulted in social behavior abnormalities like autism.Combined with the previous study in our research group that propofol suppressed proliferation of neural stem cells in the dentate gyrus of developing brain,to investigate the effect of propofol on cognition-related behavior and the underlying mechanisms.MethodIn this study,we adopted the newborn mice at postnatal day(P)7 treated with 30 or 60 mg/kg of propofol or vehicle of equal volume to set up propofol model.c-Fos Immunohistochemistry was used to locate the target regios and vulnerable neurons of propofol on developing brain.Similarly,using immunohistochemistry to detect the neural precursor cell proliferation to clear the effect of propofol on piriform cortex neurogenesis of developing brain.In addition,newborn mice received i.p.injections of propofol at a dosage of 30 or 60 mg/kg on three subsequent days beginning on P7,an equivalent volume of i.p.administered intralipid was used as a vehicle control for propofol,and then reared to adulthood to observe the impact of propofol on long-term neurobehavioral.Buried food test was used to detect olfactory identification and olfactory learning and memory function of the propofol model mice and control mice.Using social experiment to detect social behavior and social preference of the propofol model mice and control mice.Using Novel object recognition test to detect cognitive memory function of the propofol model mice and control mice.At last,Immunohistochemistry was used to investigate the effect of propofol on vulnerable neurons to explore the possible mechanisms of propofol on neurobehavior of developing brain.The main results were as follows:1.Propofol activated interneurons in development piriform cortex.(1)c-Fos immunohistochemistry results demonstrated that compared with the control group of mice,the mice following propofol treatment at a dose of 30mg/kg(p<0.05)and 60mg/kg(p<0.05)showed significantly increased c-Fos+ cells in piriform cortex.(2)The immunofluorescence showed that there were no differences among the three groups in the number of Neu N labeled neurons.In addition,the mice following propofol treatment at a dose of 30mg/kg and 60mg/kg showed significantly increased c-Fos-NeuN double-labeled cells compared with the control group.However,there were no co-labeled between c-Fos and GFAP.(3)The immunofluorescence showed that the mice following propofol treatment at a dose of 30mg/kg and 60mg/kg showed significantly increased c-Fos-Calbindin double-labeled cells compared with the control group(P<0.01).However,there were no difference among the three groups in the co-labeled cells between c-Fos and other interneurons.2.The effect of propofol on neural precursor cell proliferation in development piriform cortex.(1)BrdU immunohistochemistry results demonstrated that compared with the control group of mice,the mice following propofol treatment at a dose of 30mg/kg(p<0.05)and 60mg/kg(p<0.01)showed decreased BrdU+ cells in piriform cortex.Furthermore,compared with the propofol 30mg/kg group,the mice treated with 60mg/kg propofol showed decreased BrdU+ cells significantly in piriform cortex(p<0.05).(2)Sox2 immunohistochemistry results demonstrated that the mice following propofol treatment at a dose of 30mg/kg showed no significant difference in piriform cortex,while the mice treated with 60mg/kg propofol showed decreased Sox2+ cells in piriform cortex.Similarly,the BrdU-Sox2 immunofluorescence results showed that there were no significant difference between control group and propofol 30mg/kg group,while the number of Brd U-Sox2 double labeled positive cells reduced significantly in piriform cortex in the mice following propofol treatment at a dose of 60mg/kg.(3)Nestin immunofluorescence results demonstrated that compared with the control group of mice,the mice following propofol treatment at a dose of 30mg/kg and 60mg/kg showed decreased nestin optical density(p<0.01).3.Neonatal exposure to propofol induced neurobehavior change in adult and its possible mechanism.(1)The buried food test results showed that there were no differ between the control group and the propofol 30mg/kg group,while the mice following propofol treatment at a dose of 60mg/kg showed significantly increased latency of extracting embedded cookies(P<0.01).(2)The social test results showed that the mice from the control group preferred to stay in the side contained a mouse(P<0.05),while the mice following propofol treatment at a dose of 30mg/kg and 60mg/kg showed no difference.In the second stage of the experiment,the mice from control group showed preference to the social novelty(P<0.01),while the mice following propofol treatment at a dose of 30mg/kg and 60mg/kg showed no such preference.(3)The novel object recognition test results showed that the mice from the control group preferred to explore the new object(P<0.001),while the mice following propofol treatment at a dose of 30mg/kg and 60mg/kg showed no such preference.In other words,the mouse treated with propofol showed decreased discrimination ratio of novel object as compared to the control group.(4)The immunohistochemistry results showed that compared with the control group,the mice following propofol treatment at a dose of 30mg/kg and 60mg/kg showed significantly reduced Calbindin interneurons in piriform cortex,suggesting the Calbindin interneurons showed specifically vulnerability in propofol anesthesia.ConclusionsThese results suggested that propofol affect the long-term cognitive,learning,and memory by inhibiting the neural precursor cells proliferation and activating the Calbindin interneurons in piriform cortex. |
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