The Impact Of Prenatal Alcohol Exposure To Innate Immune Mediated By Toll-Like Receptor4in Rat Brain

Ethanol is considered to be the most common substance affecting brain development.Prenatal ethanol exposure (PAE) is one of the main factors of birth defects, which lead toneonatal neurological retardation and/or mental retardation. Glial cells are all cells exceptneurons inside the nervous system, which could protect and/or nourish neurons and link toimmune function of nervous system. Due to the presence of the blood-brain barrier (BBB),material exchange of the central nervous system (CNS) and the blood is strictly limited,and the innate immunity plays an important role in the immune response in CNS. Theinnate immunity in CNS is closely related to the occurrence and development of variousneurological and psychiatric diseases. Toll-like receptor4(TLR4) is an importantmolecule that recognize pathogen and damaged molecule pattern, and the pathwaysmediated by TLR4has been intensively researched. This research consists of3parts. Allof them focus on the innate immune response mediated by TLR4in CNS. The aim of theresearch is to study on the impact of prenatal alcohol exposure to the development ofneonatal nervous system.Part1. Real time quantitative PCR and western blot were used to detect theexpressions of TLR4mRNA and MyD88and NF-κBp50proteins12h after injection oflipopolysaccharide (LPS) intraperitoneally (0.3mg/kg,0.01mg/ml, ip.) or equivalentvolume of NS and injection of LPS intracerebroventricularly (0.3mg/kg,1mg/ml, icv.) orequivalent volume of NS in neonatal rats (postnatal day1,3,7,14and21). The rat pups(postnatal day1,3and7) received an injection of0.3mg/kg LPS (0.01mg/ml, ip.) or an equivalent volume of NS as a control. At12h after LPS or NS, double immunolabelingfor Iba-1with RECA was performed to detect the morphology of the microglia and thedistribution of microvessel in the neonatal rat brain, respectively. At the same time theEvans blue (EB) albumin extravasation was investigated on microscope to detect tointegrity of BBB. Transmission electron microscope was used to investigate the ultramicrostructure of neonatal rat (P1and P3) brain12h after an injection of LPSperipherally.The results showed that the neonatal rat brain (even P1) strongly reacted to LPSi.c.v. The expressions of TLR4mRNA and MyD88and NF-κBp50proteins were allobviously up-regulated in the neonatal brain with an intracerebral injection of LPS.Double immunolabeling for Iba-1with RECA clearly showed that microglia was stronglyactivated12h after an intracerebral injection of LPS in neonatal rat brain. Although theactivated microglia distributed in all the area of the brain, there was no tendency ofcorrelation between the activation of the microglia and the distribution of the capillary.However, no microglia activation could be found in LPS i.p. and NS injection groups.Fluorescent microscopy showed that no red fluorescence of Evans blue (EB) was found inthe parenchyma of the neonatal rat brain at all ages except for non-BBB area.Transmission electron microscopy showed basically normal fine structure of the cerebralendothelia and other neural cells in both LPS and NS groups. There was no obviousalteration of endothelial tight junction12h after exposure to LPS i.p. in developing brains(P1and3).Part2. Prenatal ethanol exposure standard model in rats was established. Threegroups of pregnant rats were feded with ethanol liquid diet, pair liquid diet and control diet,respectively. Rats in the3groups were intraperitoneally injected with Brdu (200mg/kg)dissolved in0.9%NaCl (10mg/ml) once a day from P27to P29. Then doubleimmunolabeling for Brdu with Doublecortin was performed to detect the newly generatedneurons in these rats (P30) brains, which were chosen from PAE and control groups.Western blot was used to investigate the expressions of following proteins: TLR4, MyD88, TRAF6, TRIF, NF-κBp50, pI-κB, TNFα, IL-1β, IFN, in the neonatal (P30) rats brains,which were chosen from3groups,12before and after intracerebral injection of LPS (0.3mg/kg,1mg/ml). In addition, double immunolabelings for TRAF6and TRIF with NF-κBwere performed in neonatal rats (P30) brains, which were chosen from PAE and controlgroups,12before and after intracerebral injection of LPS (0.3mg/kg,1mg/ml).The results showed that: there were no significant differences founded inimmunolabeling for Brdu with Doublecortin in neonatal rats (P30) brains between PAEand control groups. Western blot showed that the expressions of9kinds of proteins wereall significantly up-regulated12after intracerebral injection of LPS (0.3mg/kg,1mg/ml)in neonatal rats (p30) brains of no-PAE groups. However, the expressions of TLR4, TRIFand IFN were down-regulated, and there were no significant changes in the expressions ofthe other6kinds of proteins12after intracerebral injection of LPS (0.3mg/kg,1mg/ml) inneonatal rats (P30) brains of PAE groups. The basic expressions of TRIF, TNFα, IL-1βand IFN were significantly higher in the rats (P30) brains of PAE group than the othergroups. The immunolabelings showed that TRAF6, TRIF and NF-κB were all activated inthe hippocampal dentate gyrus of the rats (P30) brains of no-PAE groups,12after i.c.v.injection of LPS (0.3mg/kg,1mg/ml). But there were no the same results founded in PAEgroups.Part3. Western blot was used to investigate the expressions of3kinds of proteis(TLR4, NF-κB, I-κB) in the rats (P30)brains, which were chosen from3groups,12h afteri.c.v. injection of MyD88homodimerization inhibitory peptide.The results showed that: the expressions of TLR4, NF-κBp50and pI-κB were allsignificantly down-regulated in the rats (P30) brains of no-PAE groups,12h afterintracerebral injection of MyD88homodimerization inhibitory peptide. But there were nothe same results founded in PAE groups.Conclusion:(1) The innate immune mediated by TLR4and the BBB in neonatal ratbrain (even at P1) has been well established.(2) There was no enough evidence to provethat PAE would change the number of newly generated neurons in the hippocampal dentate gyrus and the structure of neonatal rat (P30) brain.(3) The innate immunemediated by TLR4in PAE rat was inhibited. And the PAE may result in up-regulation ofsome inflammatory factors, such as TNFα, IL-1β and IFN.(4) The regulation mechanismof innate immune mediated by TLR4in PAE rat may be weakened or destroyed.Although the mechanism of impact of PAE to innate immune mediated by TLR4inrat brain are not clear, study in this area will be helpful for understanding the impact ofPAE to brain development. Maybe a new kind of effective pharmacological therapy willbe found to decrease the incidence of the birth defects of nervous system, even to curechronic neurological and psychiatric disorders.