Mechanism Of Microglia Involved In The Early-Life-Stress Induced Aberrant Behaviors In Male Offspring

The event stimuli experienced of human or other mammals in the early stages of development,such as maternal neglect,abuse,food shortages,mental stress,diseases and infections,have already exhibited significant impacts on the cognition and psychology in later life.The immune memory and other traces generated after the individual experienced early stress events are preserved through complex molecular and cellular mechanisms,through epigenetic and cellular plasticity changes,and other ways to affect the response level of the central nervous system(CNS)to stress events in the later development period.Clinical studies have shown that the neuroinflammation is involved in the pathological process of psychological diseases.Exploring the mechanism of early life stress-induced changes of the internal environment in the CNS will provide important basis for drugs development and clinical application in stress-pathology model.In this study,we established an early life stress(ELS)model of C57BL/6J mice which performed maternal separation process during a postnatal period of 14 days,to investigate the changes of microglia phenotype and molecular markers in the CNS,then exploring the effects of regulating microglia phenotype on the development of pathological in offspring.The study was conducted mainly in the following aspects:1.Investigate whether ELS increases the sensitivity of offspring to a second stress.Although the postnatal maternal separation affected the performances of offspring in depression and anxiety-like behavior tests,there were no statistically significant differences between ELS and the control offspring.The comprehensive depression score also showed that there were no significant difference between ELS and control group.However,after two weeks of CRS during adolescent,ELS offspring showed significant depression and anxiety-like behaviors without the changes of motor ability.It suggests that ELS maybe induce significant behavioral changes in offspring via an indirect way,which increase their sensitivity to the stress event,thereby leading to depression and anxiety-like behaviors.2.Exploring the alteration of microglia phenotype in ELS mice,and examining the effects of regulating microglia phenotype on the behavior of ELS mice.The immunofluorescence assay showed that the microglia in the hippocampus of ELS offspring were more likely to be activated.After the second stress,there were more activated microglia in ELS offspring,the average volume of cell body was increased,the length and number of branches were decreased,and the expression of microglia marker CX3CR1 was decreased.Minocycline treatment significantly reduced the sensitivity of ELS offspring to the CRS,anxiety and depressive-like behavior tests showed there were no differences between ELS and control mice.Minocycline treatment significantly inhibited the response to the second stress of microglia in the ELS offspring,and the activated microglia were significantly lower than those in the untreatment group after the second stress.These results indicate that microglia are involved in the process of ELS induced mental abnormalities in the offspring,and regulation of microglial phenotype has showen the potential for improving the psychological diseases in ELS offspring.3.Exploring the effects of regulating microglia activation phenotype on homeostasis and neurogenesis in CNS of ELS offspring.Real-time PCR tests showed that the expression of proinflammatory markers in the hippocampus of ELS offspring were increased,and the expression of anti-inflammatory makers were decreased after the second stress.Western blot(WB)experiments also confirmed that the second stress resulted in increased protein level of proinflammatory cytokines(IL-6,IL-1β)and decreased anti-inflammatory cytokines(IL-4,TGF-β)in the hippocampus of ELS offspring.Minocycline administration significantly restored the homeostasis in the hippocampus,manifested decreased proinflammatory cytokines and the increased antiinflammatory cytokines.Proliferation and differentiation of neurons in the hippocampus were significantly reduced in ELS offspring after suffered the second stress,and the expression of newborn immature neurons marker DCX was also reduced.Inhibition of microglia restored the neuron proliferation and differentiation in the hippocampus of ELS offspring.BDNF was also increased after inhibiting the activation of microglia by minocycline,which would be beneficial to maintain normal neurogenesis in the hippocampus of ELS offspring.These results suggest that regulating microglia phenotype can rebalance the inflammatory cytokines of hippocampus in ELS offspring,and establish a friendly environment for neurogenesis.4.Exploring the role of nuclear transcription factor PPARγ in ELS-induced stress sensitivity processes.High sensitivity and low sensitivity ELS offspring were screened by behavioral tests.Then we detected the changes of microglia phenotype and PPARγ expression in the hippocampus and prefrontal cortex in different sensitivity ELS offspring groups.Enzyme-linked immunosorbent assay(ELISA)showed that the expression of PPARγ in the hippocampus and the cortex of high sensitivity ELS offspring were significantly higher than the control offspring.Furthermore,immunofluorescence staining showed that the proportion of activated microglia in the hippocampus of high sensitivity ELS offspring was higher than the control group.Correlation analysis between the sugar preference and forced swimming test results of offspring and PPARγ levels shows that PPARγ has a significant correlation with depressive-like behaviors in offspring mice.These results reveal that PPARγ,as a key regulator of microglial phenotype,is involved in the process of psychological abnormalities of ELS offspring.5.Exploring the effects of regulating PPARγ in the CNS on microglia phenotype and the behaviors of ELS offspring.PPARγ agonist pioglitazone administration significantly enhanced the expression of PPARγ in the hippocampus and cortex,and the stress sensitivity were also alleviated.Accordingly,pioglitazone administration prompted microglia shift to anti-inflammatory phenotype in the hippocampus and cortex of ELS offspring.In addition,we also investigated the role of PPARγ in the inhibition of microglial activation by minocycline,and found that minocycline significantly increased the m RNA and protein levels of PPARγ in the hippocampus and cortex of ELS offspring.Whereas inhibition of PPARγ expression through GW9662 significantly offset the regulation of the behaviors in ELS offspring by minocycline.These results demonstrate the important role of PPARγ in the regulation of microglia phenotype and the psychological development of ELS offspring.Together,microglia are involved in the process of ELS induced stress sensitivity.Regulation of the microglia phenotype promoted the rebalance of inflammatory cytokines in the neurogenesis niche of ELS offspring,which is beneficial to the neurogenesis in the hippocampus,exhibits therapeutic effects on ELS induced depression symptom.ELS leads to the change of PPARγ level,which in turn affects microglia phenotype and the homeostasis of neurogenesis niche.The PPARγ signaling involved in the regulation of the microglia phenotype and behaviors of ELS offspring.The results of the study reveal the important role of PPARγ signaling in regulating the activated phenotype of microglia in the early psychological development of individuals,and provide an important theoretical basis for neuropathological regulation and drug development targeting PPARγ and microglia.