Microglia Is Activated To Induce Autism-like Behavior In The Offspring Of Rats By LPS Exposure During Gestation

Objective: To investigate the activity of TLR4 signaling pathway,the changes of microglia subtypes and their relationship in the prefrontal cortex of autism-like behavior pups induced by lipopolysaccharide(LPS)during pregnancy.Methods: Pregnant rats(n=24)were randomly average divided into LPS treatment group and PBS control group.The pregnancy rats were injected intraperitoneally with 100?g/kg LPS or equal volume of PBS at the gestational day 9.5,respectively,and their pups were named as the LPS group or PBS group.Three-chamber sociability test,open field test,olfactory habituation/dishabituation test and Morris water test were used to assess offsprings' autism behavior and learning-memory function.The levels of IBA-1,iNOS and Arg-1 mRNA expressions in the prefrontal cortex were detected by real-time PCR.Western blotting was performed to examine the protein expression levels of Claudin-5,Occludin,IBA-1,iNOS,Arg-1,TLR4,Phospho-NF?B p65 and IKK? in the prefrontal cortex of offspring.The morphologic changes of microglia were observed in the prefrontal cortex of pups by immunofluorescence staining.The concentrations of LBP,TNF?(maternal serum)and TNF?,IL-10(offspring's cortex)were determined by ELISA kits.N9 microglia were cultured in vitro,the changes of TLR4?Phospho-NF?B p65?iNOS and Arg-1 expressions levels in the N9 cells induced by LPS following TAK242 treatment were measured by PCR and immunofluorescence observation.Results:(1)The LBP and TNF? levels in serum of gestational rats were significantly induced following LPS treatment(P(27)0.01,P(27)0.05),indicated that the immune response was activated in the pregnant rats.(2)Compared with PBS group,the offspring rats of LPS group spent longer time in the novel object chamber(P(27)0.001)and less time in the strange rat chamber(P(27)0.01).Meanwhile,the time of sniffing the peer urine in the LPS treated offspring was shorter than that of the PBS group(P(27)0.001),while there were no significant differences in the spending time of sniffing water and beer in both groups.Furthermore,in the open-field test,the number of line crossing was significantly reduced(P(27)0.01),and the time of self-grooming was statistically increased(P(27)0.001)in the LPS group compared with those of PBS group.In addition,the escape latency time in the LPS group were obviously longer than those in PBS group(P(27)0.001)during Morrior water test.(3)The levels of Occludin and Claudin-5 protein expressions associated with blood brain barrier were noticeably decreased in the prefrontal cortex of pups in the LPS group compared with those in the PBS group(P(27)0.001).(4)The concentration of inflammatory cytokine TNF? was induced in the prefrontal cortex of LPS pups(P(27)0.01),whereas the anti-inflammatory cytokine IL-10 was reduced(P(27)0.01).(5)The levels of IBA-1 and iNOS mRNA and protein expressions were significantly increased,while the Arg-1 mRNA and protein were meaningfully decreased in the prefrontal cortex of the LPS offspring.In addition,the microglia branch was decreased and the cell body displayed expansion and looseness in the LPS group.(6)The protein expressions of TLR4?Phospho-NF?B p65 and IKK? were remarkably increased in the prefrontal cortex of offspring treated by LPS compared with those treated by PBS(P(27)0.05,P(27)0.05,P(27)0.001).(7)TAK242 treatment suppressed the activation of TLR4/NF?B signaling pathway and decreased transformation of M1 subtype in the N9 microglia induced by LPS.Conclusion: It is concluded that TLR4/NF?B signaling pathway in the offspring's prefrontal cortical is activated by LPS exposure during mid-gestation,inducing microglia to transform M1 type and inflammatory cytokines secretion,which may be a risk factor for autism-like behavior in offspring rats.