| Objective:Developmental nicotine exposure is associated with many neuronal dysplasia in offspring.Microglia play an important role throughout the development of the brain.It is not clear how nicotine affects the development of microglia during neurogenesis and what effect this have on the development of neural stem cells?This study is intended to establish a mouse model of maternal nicotine exposure,and to explore the effects and mechanisms of maternal nicotine exposure on the polarization of hippocampal microglia,neural stem cell development and neural development in offspring at individual,tissue and molecular levels by using a variety of experimental methods,to provide scientific basis for the developmental toxicity of nicotine and helps pregnant women consciously avoid nicotine exposure.Methods:1.Modeling,neurodevelopmental index and behavioral testing:Seven-week-old female and male mice were paired with 3:1.Nicotine mice were provided with nicotine(200μg/ml)by drinking water starting from 2 weeks pre-mating until postnatal 20 day,while vehicle mice were received 1%saccharin.After birth,the early neurodevelopmental indicators(eye-opening time,olfactory reflex and auditory reflex)were measured.After PNE20,2-4 mice were selected from each litter at random for open field test.2.Gene expression levels detection:3 mice in two group were selected for whole genome microarray analysis,and the differentially expressed genes between the two groups were defined by P≤0.05 and Foldchange ≥1.5.The differentially expressed genes were analyzed by pathway analysis and GO analysis.Real-time quantitative PCR was performed on several representative genes(CXCL10,BDNF,JUNB,CCL12,IL-1β and IL-4).3.Histology detection:The brain of PD20 mice was fixed by heart perfusion,and the hippocampal coronal section was made by paraffin slicing machine with thickness of 5μm.Nissl staining was used to detect the changes of neurons and glial cells.The coronal section of hippocampus was performed by freezing microtome(thickness of 30μm),and Ibal immunofluorescence staining was performed.Ibal/BDNF double standard immunohistochemical staining was used to detect co-standard microglia and BDNF.The length and morphology of hippocampal neurite were detected by Tuj1 immunohistochemical staining.The level of NeuroD1 in mice hippocampus was detected by immunofluorescence staining;The expression levels of microglial markers Ibal,BDNF and synaptic related proteins PSD95 and GAD1 in the hippocampus of PD20 mice were quantitatively analyzed by Western blot.4.Cytology testing:After microglia cells were cultured、purified,blank control group,nicotine group,LPS+nicotine group and LPS group were set.The control group and nicotine group were treated with D-10 medium or 10μmol nicotine medium for 12 h and 24 h,respectively.The LPS+nicotine group was pretreated with 10μg/mL LPS for 30 min,followed by D-10 medium for 12 and 24 h.The LPS group was treated with LPS alone for 24h.After the supernatant was collected,the commercial cytokine array was used to determine the expression of cytokines.Results:1.Early neurodevelopmental index and behavioral testing results:The early neurodevelopmental indexes showed that eye opening time,olfaction and auditory maturation time were significantly prolonged.The results of open field test showed that the total movement distance in the nicotine group was longer than that in the control group(P<0.001),but the immobility time(P<0.01)and the residence time in the center area(P<0.05)were shorter than that of the control group.2.Gene expression levels detection results:there were 132 up-regulated genes and 163 down-regulated genes in nicotine group.BP analysis was carried out,and the results showed the top 10 biological processes of up-regulation and down-regulation,respectively.The top 5 pathways of up-regulation and down-regulation were shown by pathway analysis.GO analysis of differentially expressed genes showed that these genes mainly included inflammatory genes,neurotransmitters and synaptic related genes.The results of qPCR verification were consistent with those of gene microarray.3.Histology detection results:Nissl staining showed that glial cells in CA1 region increased significantly in nicotine group,while there was no significant difference in CA3 and DG region.Iba1 immunofluorescence staining showed that the number of M2 microglia in hippocampus increased,but the expression of NeuroD1 was decreased in nicotine group.Double immunohistochemical staining showed that the cells of Iba1+BDNF-and Iba1+BDNF+in the nicotine group were higher than those in the vehicle group.The axons of hippocampal neurons in the nicotine group were significantly prolonged by Tuj1 staining.Western blot showed that the expressions of Iba1,BDNF and postsynaptic membrane protein PSD95 in nicotine group were increased(P<0.05).4.Cytology testing results:nicotine significantly inhibited the downregulation of anti-inflammatory cytokines and up-regulation of pro-inflammatory cytokines induced by LPSConclusion:The results of this study showed that maternal nicotine exposure leads to offspring neurodevelopment delay and be anxiety-like behavior.In nicotine group,anti-inflammatory factors were up-regulated and pro-inflammatory factors were down-regulated.Maternal nicotine exposure induced the polarization of hippocampal microglia to M2,up-regulated BDNF but down-regulated NeuroD1.Experiment In vitro have also shown that nicotine induces microglia to secrete anti-inflammatory cytokines.These data suggest that maternal exposure to nicotine polarized hippocampal microglia toward M2 and secreted BDNF and anti-inflammatory cytokines,which are associated with neurodevelopmental disorder in offspring.Figure[17]table[1]reference[81]... |
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