The Toxic Effects Of Early Postnatal Exposure To Fine Particulate Matter On Brain Function And Underlying Mechanisms

PART? ALTERATION OF BRAIN FUCTION AND STRUCTURE FOLLOWING EARLY POSTNATAL EXPOSURE TO FINE PARTICULATE MATTERObjective: In this study,we aimed to clarify the toxic effects of early postnatal exposure to fine particulate matter on brain function and hippocampal structure.Methods: Neonatal rats were respectively divided into high dose and low dose and saline groups(n=20/group),neonatal rats in high dose group,low dose group and saline group were successively exposed to PM2.5 at concentration of 10 and 2mg/kg body weight and saline through intranasal instillation from postnatal day(PND)3 to 15,once a day,and control group was set in which neonatal rats were taken out only,Then following experiments were carried out in immature(PND28)and mature(PND60)rats separately:(1)Emotional behaviors were evaluated using the elevated plus maze and forced swimming test,cognitive ability was assessed by Morris water maze tests;(2)Hippocampal structure was observed by Hematoxylin-Eosin(HE)staining and Nissl staining.(3)Hippocampal neurons were collected from embryonic SD rats on day 18 of gestational age,the neurons in PM2.5 group on 3 day in vitro(DIV3)were treated with 0,10,50 and 250?g/ml PM2.5 sample,on DIV4 and DIV7,the cell viability were detected using Cell Counting Kit-8(CCK8)test,and on DIV7,the shape of neurons were observed by immunofluorescence staining.Results:(1)The elevated plus maze test results showed that in immature rats,compared with saline group,the high? and low?dose groups showed a significantly lower percentage of number of entries into the open arms(p<0.05),and in mature rats,the high dose group showed significantly lower the percent of number of entries into open arms and time spent in open areas compared to saline group(p<0.05 respectively).(2)The forced swimming test results indicated that in mature rats,the latency to immobility sharply decreased in high dose group compared with saline group(p<0.05).(3)Morris water maze test results showed that for immature rats,the escape latency was increased apparently in high dose group compared to saline group on training days 4 and 5(p<0.01,p<0.05).during the probe test,in immature rats,the number of entries into the platform zone was sharply lower in the high dose group than in the saline group(p<0.05).(4)HE and Nissl staining results indicated that in immature rats,the cell number was decreased,structures became disorder,and Nissl body number were reduced in hippocampal CA1 and CA3 area after exposing to high-and low dose PM2.5,in mature rats,structure was disorder,and Nissl body number were decreased in hippocampal CA1 and CA3 area in high dose group compared to saline group.(5)the percent of NF200-positive cells in all cells exceeded 90%,which proved primary hippocampal neurons were cultured successfully.(2)CCK8 test results indicated that the neuron viabilities were significantly decreased after exposing to PM2.5 at 50?g/ml and 250?g/ml on both DIV4(p <0.01,p <0.001)and DIV7(p <0.05,p <0.001).(3)Immunofluorescence staining displayed that neurons were clustered slightly after 50?g/ml PM2.5 treatment,and neurons number was decreased,the neuron shape was abnormal,and neurons were clustered seriously after 250?g/ml PM2.5 treatment,so 50?g/ml was chosen as intervention concentration.Conclusion: The present study demonstrated that PM2.5 exposure during brain development stage could impair emotional behaviors,cognitive ability and hippocampal structure,and could damage neuronal viability and shape,The neurotoxic effects of PM2.5 seemed to be dose-dependent and time-dependent,and the influences of high dose PM2.5 exposure on neurodevelopment could last to adulthood,showing some improvements compared with childhood.PART ? ALTERATION OF SYNAPTIC STRUCTURE AND FUNCTION FOLLOWING EXPOSURE TO FINE PARTICULATE MATTERObjective: We aimed to determine the toxic effects of exposure to PM2.5 on synaptic structure and function.Methods: Neonatal rats were respectively divided into four groups: control,saline,high dose and low dose(n=28/group),the animal model were established based on part?.Then long term potentiation(LTP)was determined by electrophysiological experiment,synaptic structure was observed by transmission electron microscopy(TEM),the expressions of postsynaptic density-95(PSD95),growth associated protein-43(GAP43),and synaptophysin(SYP)were detected by western blot in immature(PND28)and mature rats(PND60)separately.At the same time,hippocampal neurons were divided into control group and PM2.5 group,the neurons in PM2.5 group on 3 day in vitro(DIV3)were treated with 50?g/ml PM2.5 sample,on DIV7,the synaptic ultrastructure of neurons in two groups were observed by TEM,on DIV4 and DIV7,the expressions of PSD95,GAP43,and SYP proteins of neurons were detected by immunofluorescence staining and Western blot.Results:(1)Electrophysiological experiment results showed that in immature rats,LTP stimulated in hippocampal CA3 area and recorded in CA1 area were much lower in high dose group and low group than in saline group(p<0.001,p<0.01),in mature rats,LTP was decreased in high dose group compared with saline group(p<0.001).(2)TEM results demonstrated the number of synapses,the thickness of PSD and length of active zone were decreased in the high dose group compared to the saline group in both immature and mature rats(p<0.05 respectively).(3)Western blot results showed that in immature rats,the expression levels of PSD95,GAP43,and SYP were apparently lower in high dose group than in saline group(p<0.001,p<0.01),and the expression of PSD95 was reduced in low dose group compared to saline group(p<0.05),in mature rats,the expressions of PSD95 and SYP were decreased compared with saline group(p<0.05 respectively).(4)TEM results showed that compared with control group,the length of active zone of synapse was reduced and the thickness of PSD was also decreased after 50?g/ml PM2.5 treatment on DIV3.(5)immunofluorescence staining and Western blot indicated that the expression levels of PSD95,GAP43,and SYP protein of neurons were lower in PM2.5 group than in control group on both DIV4 and DIV7(p<0.05 respectively).Conclusion: The vivo and vitro study showed that PM2.5 exposure could impair synaptic structure and function.The synaptic-toxic effects of PM2.5 seemed to be dose-dependent and time-dependent,and the influences of high dose PM2.5 exposure on synaptic plasticity could last to adulthood,showing some improvements compared with childhood.PART ? THE ROLE OF PKA/CREB/BDNF SIGNALING PATHWAY IN FINE PARTICULATE MATTER-INDUCED NEURONAL INJURIES.Objective: We aimed to investigate whether PKA/CREB/BDNF signaling pathway was involved in PM2.5-induced neuronal injuries and upor down-regulating PKA/CREB/BDNF signaling pathway could affect the alteration of PM2.5-treated primary hippocampal neuronMethods: Neonatal rats were respectively divided into four groups: control,saline,high dose and low dose(n=12/group),the animal model were established according to part?.Then the phosphorylation of c AMP response element binding protein(CREB)and protein kinase A(PKA)and BDNF expression were detected by western blot in immature(PND28)and mature rats(PND60)separately.At the same time,hippocampal neurons were divided into control group and PM2.5 group,the neurons in PM2.5 group were treated with 50?g/ml PM2.5 sample on DIV3,on DIV4 and DIV7,the phosphorylation levels of PKA and CREB and the expression level of BDNF were determined using Western blot.After identifying PKA/CREB/BDNF signaling pathway was involved in the neuron injuries caused by PM2.5,the concentrate of synaptamide(pathway activator)was screened by CCK8 test and Western blot,then the neurons on DIV3 were divided into four groups: control group,PM2.5 group,synaptamide intervention group,and H-89(pathway inhibitor)intervention group,control group: neurons without any treatment,PM2.5 group: PM sample was added to the neuron culture medium at 50?g/ml,synaptamide and H-89 intervention group: 50?g/ml PM2.5 sample was added to the neuron culture medium,while 10?M synaptamide or 30?M H-89 were added to the medium separately,on DIV4 and DIV7,the neuron viabilities in each groups were assessed by CCK8 test and the expression levels of PSD95,GAP43,and SYP protein of neurons in each group were tested by immunofluorescence staining and Western blot,on DIV7,the synaptic ultrastructure of neurons in each groups were observed by TEM.Results:(1)Western blot results showed in immature rats,the phosphorylation levels of PKA and CREB and expression of BDNF were significantly decreased in high dose group compared to saline group(p<0.01 or p<0.001),and the phosphorylation levels of CREB and BDNF expression were lower in low dose group than in saline group(p<0.05 respectively),in mature rats,the phosphorylation levels of PKA and CREB and expression of BDNF were sharply reduced in high dose group compared to saline group(p<0.05 respectively).(2)Western blot showed that the phosphorylation levels of PKA and CREB and the expression level of BDNF in PM2.5 group were reduced sharply compared with control group on both DIV4(p<0.05 respectively)and DIV7(p<0.001 respectively).(3)Western blot indicated that on DIV4,the phosphorylation levels of PKA of neurons were raised after 10?M synaptamide treatment compared with 0.1?M synaptamide group and control group(p<0.05,p<0.001),the phosphorylation levels of CREB of neurons were higher in 10?M and 1?M synaptamide group than in control group(p<0.001,p<0.01),and the BDNF expression were increased in 10?M synaptamide group compare to control group(p<0.01),so 10?M was chosen as synaptamide intervention concentration.(4)Western blot showed that on DIV4,the phosphorylation levels of PKA and CREB and the expression level of BDNF were higher in synaptamide intervention group than in PM2.5 group(p<0.05 respectively),the phosphorylation levels of PKA and CREB and the BDNF expression in H-89 intervention group were decreased compared with PM2.5 group(p<0.05 respectively).(5)CCK8 test demonstrated that the neuron viability was higher in synaptamide intervention group than in PM2.5 group on DIV4(p<0.01),and the neuron viability in synaptamide intervention group was increased compared with PM2.5 group,but decreased compared to control group on DIV7(p<0.01,p<0.05).(6)TEM results showed the thickness of PSD in synaptamide intervention group was raised compared with PM2.5 group,but the length of AZ was lower in synaptamide intervention group than in control group.(7)Immunofluorescence staining and Western blot results demonstrated that on DIV4,the expression levels of PSD95,GAP43,and SYP in synaptamide intervention group were apparently increased compared to PM2.5 group(p<0.05 respectively),and PSD95 and SYP protein expressions in H-89 intervention group were decreased compared with PM2.5 group(p<0.05 respectively),on DIV7,the expression levels of PSD95 and SYP were significantly higher in synaptamide intervention group than in PM2.5 group(p<0.05 respectively),PSD95 protein expressions was lower in H-89 intervention group than in PM2.5 group(p<0.05).Conclusion: The present study demonstrated that PM2.5 exposure could inhibit PKA/CREB/BDNF signaling pathway in vivo and in vitro.Up-regulating PKA/CREB/BDNF signaling pathway could alleviate the decrease of neuron viability and damage of synapse induced by PM2.5 exposure,and down-regulating PKA/CREB/BDNF signaling pathway exerts little influence on the alteration of neuron viability caused by PM2.5 exposure,but could aggravate the injury of synapse caused by PM2.5 exposure.