Research On The Apoptosis Mechanism Of PM_2.5 Exposure Induces Neurodevelopmental Damage In Mice Offspring

Objective:Pregnant mice PM_2.5（particle diameter≤2.5μm）models were established to observe the ultrastructural changes of neurons in the cortex and hippocampus of mice offspring.It detects the apoptosis of neurons,astrocytes,and microglia in the cerebral cortex and hippocampus,analyzes the expression changes of apoptosis-related factors in the cerebral cortex and hippocampus,and evaluates the cognitive,anxiety and depression-like emotions of the mice offspring.It further explores PM_2.5 exposure during pregnancy on the mechanism of neurodevelopmental damage in offspring and assesses the risk of fetal neurodevelopment exposure to air pollution during pregnancy.Methods:1 Animal model preparation Pregnant mice were randomly divided into blank group,the control group,PM_2.5 model groups（groups of low-,medium-,or high-dose）.Using rapid mice trachea drip improved method to instil PM_2.5suspension 30μL at different doses（low dose:0.2592μg/μL,medium dose:1.56695μg/μL,high dose:3.456μg/μL）respectively to the experimental groups,the control group with the same PBS volume,and don’t do anything deal with blank group.Drip once every 3 days during pregnancy,a total of 7 times.2 Detection of cell apoptosis 2.1 Distribution of apoptotic cells TEM slices of cerebral cortex and hippocampus were prepared on postnatal day 1 and the ultrastructure changes of neurons were observed by TEM.Frozen sections were prepared on postnatal day 14 and observed apoptotic rate by TUNEL staining in the frontal cortex and hippocampal CA1,CA3 and DG area.The location of NeuN/TUNEL,GFAP/TUNEL and Iba1/TUNEL double staining cells were found by double immunofluorescence staining and laser scanning confocal microscopy.2.2 The expression of apoptosis-related factors Cerebral cortex and hippocampus were partially isolated on postnatal day 14.The expression of apoptosis-related factors Caspase-8,Caspase-9,Bcl-2,Bax,p53 and c-Fos mRNA were detected by RT-qPCR.The expression of Cleaved Caspase-8,Cleaved Caspase-9,Bcl-2,Bax,p53 and c-Fos were detected by Western blot.2.3 Animal behavioral tests On postnatal day 30,the anxiety,depression-like mood and behavior of offspring in all groups were evaluated by the open field experiments and tail suspension experiments.Changes in cognitive function of learning and memory of offspring in all groups were evaluated by Mirror water maze experiments.Result:1 Mitochondrial crest fracture,local obfuscation,autophagosome and other ultrastructural changes were observed in the cerebral cortex neurons of the offspring in the medium-and high-dose group.The number of synaptic vesicles in the high-dose group was significantly lower than that in the control group,and the electron density of the presynaptic membrane and postsynaptic membrane decreased.There was a slight mitochondrial injury and a widening of the perinuclear space in the hippocampal neurons in the medium-and high-dose group.2 Compared with the control group,the positive TUNEL staining cells increased and the apoptosis ratios significantly increased in the medium-and high-dose group（P<0.05,P<0.01）in the frontal cortex and hippocampal CA1,CA3 and DG area on14 days after birth.Compared with the control group,NeuN fluorescence intensity decreased with the increase of PM_2.5 in the frontal cortex of the cerebral cortex（P<0.01）.In the DG area of the hippocampus,NeuN fluorescence intensity decreased with the increase of PM_2.5,and the high-dose group of PM_2.5 decreased significantly（P<0.01）.Compared with the control group,the positive number of NeuN/TUNEL significantly increased with different dose of PM_2.5 in the frontal cortex and hippocampal CA1,CA3 and DG area,especially in frontal cortex（P<0.01）.Compared with the control group,the fluorescence intensity of GFAP and Iba1increased with the increase of PM_2.5 in the frontal cortex of the cerebral cortex and the CA1,CA3 and DG regions.The positive numbers of GFAP/TUNEL and Iba1/TUNEL increased with the different dose of PM_2.5.3 Changes of apoptosis-related factors in cerebral cortex:compared with the control group,along with the increase of pregnancy PM_2.5 exposure dose,Caspase-8,Caspase-9,p53 and c-Fos mRNA and protein expression increased（P<0.05,P<0.01）,and the Bcl-2 and the Bcl-2/Bax mRNA and protein expression decreased（P<0.05,P<0.01）.In high dose group,mRNA levels and protein levels of Caspase-8,Caspase-9,p53 and c-Fos increased significantly（P<0.01）,and mRNA levels and protein levels of Bcl-2 and Bcl-2/Bax decreased with different dose of PM_2.5（P<0.05,P<0.01）.In medium dose group,mRNA levels of Caspase-9,p53 and c-Fos and protein levels of Caspase-8、Caspase-9 and c-Fos increased（P<0.05,P<0.01）.In low dose group,mRNA levels and protein levels of the apoptosis-related factors expression had no significant difference（P>0.05）.4 Changes of apoptosis-related factors in hippocampus:Compared with the control group,along with the increase in pregnancy PM_2.5 exposure dose in the offspring rat hippocampal Caspase-8,Caspase-9,p53 and c-Fos mRNA and protein expression increased（P<0.05,P<0.01）,and the Bcl-2 and the Bcl-2/Bax mRNA and protein expression（P<0.05,P<0.01）.In high dose group,mRNA levels and protein levels of Caspase-8,Caspase-9,p53 and c-Fos increased（P<0.05,P<0.01）,and mRNA levels and protein levels of Bcl-2 and Bcl-2/Bax decreased with different dose of PM_2.5（P<0.05,P<0.01）.In medium dose group,mRNA levels of Caspase-9,c-Fos increased significantly（P<0.01）and protein levels of Caspase-9,p53 and c-Fos increased（P<0.05,P<0.01）.In low dose group,mRNA levels and protein levels of Caspase-9,c-Fos increased（P<0.05,P<0.01）.5 Behavioral changes of mice offspring 5.1 The results of open field experiments showed that the total distance travelled by mice was shortened in medium-and high-dose groups,and decreased significantly in high dose group（P<0.01）,compared to control group.The central region activity distance decreased with different dose of PM_2.5,and decreased significantly in high dose group（P<0.05）,compared to control.5.2 Tail suspension experiments showed that static accumulation time lengthened significantly in medium-and high-dose group（P<0.05）,compared to control.But there was no obvious change in low dose group（P>0.05）.5.3 Morris water maze experiments showed that the escape latency of PM_2.5 model groups was gradually reduced,and there was no obvious change compared to control.The activity distance of the offspring of PM_2.5 high dose group was shortened,compared with the control group（P<0.05）.The time of mice entering the quadrant decreased significantly（P<0.05）,compared to control in the high-and medium-dose groups.Conclusion:1 PM_2.5 exposure during pregnancy induced ultrastructural changes of mitochondrial dysfunction and apoptosis in cerebral cortex and hippocampus of mice offspring.2 PM_2.5 exposure during pregnancy induced apoptosis of neurons,astrocytes and microglia increased in cerebral cortex and main areas of hippocampus of mice offspring,which may be related to activation of apoptosis-related genes.3 PM_2.5 exposure during pregnancy caused changes in cognition and social behavior,the anxiety,depression-like mood of mice offspring,which may be related to apoptosis of neurons and activation of astrocytes and microglia.