Intrauterine Subclinical Inflammation Sensitizes Hypoxic Ischemia-induced Injury In The Immature Rat Brain And The Mechanisms

Background and ObjectiveWith the improvement of medical technology, the survival rate of pretermchildren significantly increased, while the incidence of brain injury in pretermchildren was also found increased year by year. Brain injury in premature infantmainly includes ventricle or intraventricular hemorrhage (PIVH) and periventricularleukomalacia (PVL), and PVL is the major pathological type in preterm infants braininjury. Epidemiology data shows that hypoxia ischemia (HI) and infection are the twoimportant factors for PVL or cerebral palsy. Although single HI and infection is notenough to lead to brain damage, combination of both during the perinatal period maycause serious damage to immature brain. Maternal chorioamnionitis, prematurerupture of membranes and asphyxia of premature infants are common in clinical work,and often coexist.There were much researches about the synergy of HI combined withinfection abroad, but no clear reports about whether intrauterine subclinical infectionsensitizes HI-induced injury to the immature rat brain at home or abroad. Therefore,in this study, we first establish animal model of intrauterine subclinical inflammationsensitizes hypoxic ischemia in immature rat brain, and certificate the existence ofbrain injury by the marker of MBP, MAP-2and TNF-a, and further to verify theprotective effect of EPO on white matter injury, and to detect the dynamic changes ofhistones acetylation enzyme (HDACs) and histone acetylation enzyme1mRNA(HDAC1mRNA) expressions, to investigate the possible mechanism of immaturebrain injury from the aspects of epigenetics. Materials and methodsAnimals Adult clean grade SD rat,30of female and10of male, weighing350g-450g, purchased from experimental animal center of henan province [batchnumber: SCXK (prepare)2012-2012].Caged female and male rats as3:1proportion,detected vaginal smear at8o ’clock every morning since the second day, note theemerge of vaginal plug or with sperm full vision by optical microscope as the first dayof pregnance, kept separately until the15th day of pregnance.Neonatal rat model of brain injury15days of pregnant SD rats were randomlyassigned into2groups:Injected LPS(0.3mg/kg) or normal saline(N.S)intraperitoneally,continue to raise until delivery. At postnatal day5, pups wererandomly assigned into control group(NS injection without HI), LPS (LPS injectionwithout HI), HI (NS injection with HI) and LPS+HI (LPS injection with HI).Intervention group was divided into LPS+HI+N.S and LPS+HI+EPO group.Unilateral HI was induced by left carotid artery ligation followed byhypoxia(according to the Rice-Vannucci model), mice were anesthetized with ether,and the duration of anesthesia was<5min. After surgery, the pups were allowed torecover for1h. They were then placed in an airtight container perfused with ahumidified gas mixture (8%oxygen in nitrogen) for40min, keep the containertemperature at36°C, pups were returned to their dam after hypoxic exposure. Theintervention groups was intraperitoneally injected EPO (5000u/kg) or isodose N.S4hbefore and immediately after HI, and continue to inject once a day until the forth dayafter HI.Sample preparation and detection Rats were killed at6h,24h and7d after HI. Theexpression of TNF-a and HDACs in the brain homogenate were measured by ELISA,The level of MBP and MAP-2were detected by immunohistochemistry staining.The expression of MAP-2mRNA and HDAC1mRNA was detected by Real TimePCR.Results1. General condition between two pregnant rats groupThere were no differences in activity, diet and stillbirth rate between twopregnant rats, neither groups appear premature delivery. It showed no differences inthe birth weight between N.S group (6.560.33g) and LPS group (6.350.37g).2. The dynamic changes of TNF-a, MBP expressions after HIAt6h and24h after HI, TNF-a levels in neonatal rat brain in LPS+HI group washigher than that in the other three groups, and the differences were statistically significant (P <0.05), while compared among the other three groups, the differenceswere not statistically significant. At7d after HI, compared with the other three groups,MBP expression levels of LPS+HI group was the lowest, and the differences werestatistically significant (P <0.05), but no statistically significant differences among theother three groups.3. The dynamic changes of MAP-2、MAP-2mRNA expressions after HIThe MAP-2immunohistochemical staining showed scattered necrosis at24hafter HI in LPS+HI group, but not in the remaining three groups. MAP-2mRNAexpression reduced at6h after injury in LPS+HI group, and gradually increased, thedifferences were statistically significant compared with other groups (P <0.05).4. Protective effect of EPO on the immature brain injuryAt7d after HI, the IOD of MBP levels in LPS+HI+EPO group (131.592.24)was significantly higher than that in LPS+HI+N.S group (103.363.62), and thedifference was statistically significant (P <0.01).5. Epigenetic changes after the immature brain injuryAt different time points after HI, HDACs and HDAC1mRNA expression levelsin LPS+HI group were the highest compared with other groups,and differences werestatistically significant (P <0.05), but existed no statistically significant differencesbetween the other three groups.Conclusions1. Intrauterine subclinical inflammation sensitizes HI-Induced injury in the immaturerat brain.2. EPO plays a protective role to white matter after brain damage.3. The mechanism of immature brain damage associated with epigenetics.