The Study Of The Effect Of Hyperoxia On Immature Brain Mediated By Microglia Activation And The Sensitization Role Of Lipopolysaccharide

PART ONE EFFECT ON HYPEROXIA EXPOSURE ON THE FUNCTION OF N9MICROGLIA IN VITROObjectiveTo explore the hyperoxia exposure effects on viability and oxidative stress and proinflammatory function of N9microglia.MethodsAfter establishing injury model of N9microglia exposed to hyperoxia in vitro, the following are observed and detected during2h,6h,12h,16h,24h exposure to95%02:(1) morphologic change of N9microglia;(2) superoxide anion concentration variation via DHE-fluorescent probe;(3) ROS concentration variation via DCFH-DA-fluorescent probe;(4) the mRNA expression variation of TLR4, TNF-a, and IL-lβ;(5) TLR4protein expression variation via fluorescent immunohistochemistry;(6) the content of TNF-a, IL-1β in cellular supernatant via ELISA;(7) cell survival rate and apoptosis rate via Flow Cytometry (FCM).Results N9microglia transformed from quiescent state to activated state after hyperoxia exposure. The concentration of cellular ROS and superoxide anion are raised after2hour exposure compare to the control group (P<0.05), and increase with the extended exposure. The mRNA expression ofTLR4, TNF-α, and IL-lβ increase time dependently in a certain time range (p<0.05). However, the expression has sequence variance:TLR4mRNA increases at2h, TNF-a and IL-lβ mRNA increase at6h. The fluorescent immunohistochemistry results show that TLR4expression enhanced after12h exposure, mainly in plasma and cell membrane. The TNF-a and IL-lβ in cellular supernatant are increased after6h exposure with timely dependent (P<0.05). Cell apoptosis rate increases after12h exposure and reaches to the highest point after16h.ConclusionExposure to hyperoxia can activate vitro cultured N9microglia, enhance oxidative stress, facilitate inflammatory reaction, and lead to self-apoptosis. ROS-TLR4-proinflammatory mediators releasing pathway might be activated in microglia after hyeroxia exposure. PART TWO THE STUDY OF THE FUNCTION AND RELATIVE MOLECULAR MECHANISM OF MICROGLIA EXPOSED TO HYPEROXIA AFTER LPS TREATMENTObjectiveTo explore common effect on microglia exposed to hyperoxia after LPS treatment and the role of oxidative stress and TLR4Signaling pathway on microglia activationMethodsN9microglia(TLR4wild type) and EOC20microglia(TLR4knock-out type) were selected to establish cell injury model exposed to hyperoxia and LPS. First, N9microglia cell line cultured in vitro was randomly divided into six experimental groups(n=3):normoxia group, sLPS group(100ng/mL), hLPS group(1mg/L), hyperoxia group, hyperoxia+sLPS group(100ng/mL), hyperoxia+hLPS group(1mg/L). Each of the last two groups,30min after pretreatment with different level of LPS, was subjected to95%hyperoxia for various times (2h,6h,12h,24h and48h). The remanent groups was cultured in ambient O2in which sLPS group and hLPS group respectively was treated with100ng/mL and1mg/mL LPS in the cell supernatant. After treatment, at each time point, the cells of each group was harvested and the follow were observed and detected:(1) ROS concentration variation via DCFH-DA-fluorescent probe;(2) the mRNA expression variation of TLR4;(3) TLR4protein expression variation via western-blot;(4) the content of TNF-a in cellular supernatant via ELISA. Second, through NAC intervention and TLR4function knock-out(EOC20), to observe:(1)ROS content;(2) NF-κB activation;(3) TNF-a in cellular supernatant of microglia exposed to hyperoxia.ResultsROS activation and TNF-a mRNA expression of N9microglia exposed to hyperoxia after different concentration LPS treatment increase, while down regulation of TLR4mRNA. Changes have time-dependent and LPS concentration-dependent. The role of hLPS treatment is more significant than sLPS treatment. NF-κB activation and TNF-a content of N9microglia exposed to hyperoxia after the Antioxidant(NAC) intervention in cellular supernatant decrease and TLR4knock out(EOC20) have similar effect. The common effect of the two are more significant.ConclusionHyperoxia exposure and LPS treatment have synergistic effect on microglia activation. ROS or TLR4may be the key molecules that regulate hyperoxic effect on microglia. PART THREE LIPOPOLYSACCHARIDE SENSITIZE IMMATURE BRAIN INJURY OF NEONATAL MOUSE EXPOSED TO HYPEROXIAObjectiveTo explore the effect on immature brain injury of neonatal mouse exposed to hyperoxia after low concentration LPS treatment and the relative mechanism.MethodsForty eight PND3neonatal mouse are randomly divided into normal control group, LPS group(0.3mg/kg), hyperoxia group(hyperoxia exposure24h), hyperoxia+LPS group(hyperoxia exposure24after0.3mg/kg LPS treatment). At PND5, all neonatal mouse were killed and brain was removed to the following observation and detection:(1) morphological changes of microglia in the periventricular white matter via Tomato lectin staining;(2) MDA content in immature brain;(3) the mRNA expression variation of TNF-a;(4) caspase-3protein expression variation via western-blot;(5) ultrastructure morphological changes via electron microscope.ResultsMicroglia within the periventricular white matter partly transformed from quiescent state to activated state in LPS group, hyperoxia group and hyperoxia+LPS group comparison of the control group. Activated microglia number, MDA content, TNF-a mRNA expression and caspase-3protein expression in immature brain of hyperoxia group were higher than control group and LPS group. The effect on microglia activation exposed to hyperoxia after LPS treatment was the most significant in all goup. More obvious apoptosis in hyperoxia+LPS group than hyperoxia group and LPS group was confirmed via ultrastructure morphological oberservation.ConclusionHyperoxia causes immature brain injury mediated by microglia activation and LPS could sensitize the role.