Effects Of PM2.5 On Learning And Memory Behaviors And Brain Pathological Changes In Young,Aged And APP/PS Transgenic Mice

Objective:PM2.5 is the main composition of granule contamination in the polluted air.Many studies have shown that long-term exposure to PM2.5 may jeopardize human health.At present,studies have found that PM2.5 across blood-brain barrier and cause nervous system damage.However,the pathogenic factors and microglia-related mechanisms of it have not been reported.The purpose of this study is to observe the effects of PM2.5 on learning and memory behaviors,the degeneration of hippocampal and cortical neurons,the Aβ plaques and the activation and release of microglia in young,aged and APP/PS transgenic mice,which may help to clarify the role of PM2.5 in learning memory defects,(even in Alzheimer’s disease(AD))and the microglia related mechanisms.In addition,whether the aging of the population and susceptibility genes of AD involve in this process were compared and analyzed as well.Methods:1.Animal grouping,behavioral testing and tissue sampling: Twelve mice of young(3.5months old),aged(12 months old)and APP/PS transgenic(3.5 months old)respectively were randomly divided into two groups.The control group and PM2.5 group were given sterile saline or PM2.5 suspension intra-tracheally(100μg/mice,every other day for one month).The next day after the molding,learning and memory behavior(Water Maze and Y-Maze)and stress anxiety behavior(Elevated Plus Maze and sugar water preference experiment)were tested.After that,blood was taken from the angular vein.Fresh spleen tissue,brain tissue was taken after anaesthesia.Blood routine examination of peripheral blood was examined by automated blood cell analyzer.Flow cytometry was used to detect the percentage of monocyte of splenocyte.One side of the brain tissue was fixed by 4% paraformaldehyde for immunohistochemical staining;the other side of the brain tissue was stored at-80°C for q PCR.2.Nissl staining and immunohistochemical staining: Observe the morphological characteristics of neurons by Nissl staining.Immunohistochemical staining was conducted with floating method.Microtubule associated protein 2(MAP2)was used to observe the morphological characteristics of neurons and nerve fibers.The amyloid protein 1-42(Beta-amyloid 1-42,Aβ1-42)was used to observe the characteristics and quantity of Aβ plaques.The number and morphology of microglia were observed by ionized calcium binding adapter molecule-1(IBA-1).3.The expression of M1/M2 microglia markers and releases cytokines in hippocampus and cortex of mice were detected by q PCR.Result:1.Behavioral resultsNo significant difference was observed in body weight after PM2.5 exposure in all groups of mice.The elevated plus maze(EPM)test and sucrose preference test(SPT)were used to measure the emotion of mice.Results of the EPM showed that PM2.5 caused a reduction of the percentage of time spent in the open arms: after PM2.5exposure,the APP/PS transgenic animals decreased in the open arm time by 43%(P<0.05);mice of young and old groups showed a decreasing trend(8% and 13%).In addition,after PM2.5exposure,the open arm time of mice of young and old groups were reduced by41% and 50% compared with that of the young control group(P<0.05).The results of SPT showed that the sugar drinking rate of each control group was between 75% and 85%.The sucrose preference decreased was about 24% and only significantly in the old group after PM2.5 exposure(P<0.001),and no significant change was observed in the other groups.The results of the Morris water maze test showed that PM2.5 exposure did not changed the swimming speed of mice in the learning phase.Compared with the control group,the escape latency,time to reach to the hidden platform,was longer after PM2.5exposure,especially in old and APP/PS transgenic group.Take 5th day for example,compared with the control group,the escape latency after PM2.5 exposure was prolonged by 40%(young group,P<0.05),16%(old group,P<0.05)and 35%(APP/PS,P<0.05).The time percent spent in the platform quadrant decreased after PM2.5exposure.The results of the swimming distance percent in the platform quadrant and the number of times crossings the target quadrant were also decreased significantly after PM2.5 exposure.On the condition that there was no significant difference in the total number of arm entries between the PM2.5 treatment groups and the corresponding control groups,the results of Y maze showed that the frequency of spontaneous alternation in young,old and APP/PS experimental groups were decreased by 25%(P<0.01),33%(P<0.05),and28%(P<0.05).2.Detection results of Complete Blood Count and activated monocyte in spleen The results of peripheral hemogram showed an increased leukocytes after long term PM2.5 exposure especially in APP/PS transgenic AD model(1.6 times,P<0.01).PM2.5aspiration also increased the platelets level in peripheral blood of the young and APP/PS transgenic groups(P<0.05).There was no significant difference in the number of RBC in each group.The results of flow cytometry showed that CD11b+ monocytes in the spleen of each control group were about 70%.PM2.5 exposure increased the percentage of CD11b+splenocytes in the young,old and the APP/PS transgenic AD groups significantly,and the increasing rate were about 23.5%-27%(P<0.01).3.Nissl staining and immunohistochemistry staining resultsCompared with the corresponding control group,the neurons in the hippocampal CA1 area of young,aged and APP/PS transgenic mice treated with PM2.5 were significantly damaged,and the density of nissl positive neurons was decreased by 35%,78% and52%(P<0.01),the MAP2 positive neurons was respectively decreased by 22%(P<0.01),59% and 38%(P<0.001),and the number of Aβ1-42 deposition was increased significantly after PM2.5 exposure in aged and APP/PS transgenic groups by 277% and262%(P<0.001).The above changes were not obvious in the cortex of mice.In addition,compared with young PM2.5 treatment group,the density of nissl positive cells and the number of MAP2 positive cells in the hippocampal CA1 area of the old and APP/PS transgenic groups were significantly reduced,by 76%(P<0.001)and 40%(P<0.01)in nissl staining,by 50%(P<0.001)and 24%(P<0.01)in MAP2 staining.While the Aβ1-42 deposition increased significantly in the same groups after PM2.5exposure,increasing by 244%(P<0.001)and 296%(P<0.001).4.Microglia activation,phenotype and expressionThe staining results of IBA-1 showed that the number and morphology of microglia in the hippocampal area of the mice treated with PM2.5 significantly changed,and the number of cells,size of cell body and branch complexity all increased,showing an obvious activation state.The integrated optical density(IOD)of microglia in young,old and APP/PS transgenic groups were increased by 57%(P<0.01),31%(P<0.05),and43%(P<0.05)after PM2.5 exposure.The IOD values of cortical microglia after PM2.5 exposure were not significantly different from those in the corresponding control group.In addition,Compared with young PM2.5 treatment group,PM2.5 exposure increased the IOD values of old and APP/PS transgenic animals significantly by 230%(P<0.001) and 38%(P<0.05)in hippocampus,100%(P<0.05)and 80%(P<0.01)in cortex.qPCR results showed that PM2.5 increased the expression of M1 marker CD86 and M1 cytokines IL-1β and NOS2 in hippocampus of each group,decreased the expression of M2 marker CD206 and M2 cytokines Arg1,TGF-β2.The effect of PM2.5 on the activation and expression of M1 and M2 microglia in the prefrontal cortex of young and aged animals was similar to hippocampus,but PM2.5 can increase the expression of both M1 and M2 microglial markers and their cytokines in the cortex of APP/PS transgenic mouse.Conclusions:Long-term PM2.5 exposure could impair learning and memory,damage hippocampal neurons and induce AD-like pathological changes,the abnormal activation and release of microglia in the hippocampus and cortex in the mice of all the groups.The aged and APP/PS transgenic mice showed more obvious behavior and brain pathological changes.This study provides experimental evidence for long-term air pollution as a predisposing factor for learning and memory impairment and aged and AD genetically predisposed population are the main objects of protection.The mechanism of activation and typing of microglia induced by PM2.5 provides further research clues and clinical therapeutic targets for prevention and treatment of learning and memory disorders and their brain changes caused by PM2.5.