The Study Of Inflammation On The Development Of Immature Brain Injury

IntroductionCerebral palsy(CP) is a group of non-progressive motor impairment syndromes caused by lesions during early brain development. CP has become the leading cause of childhood disability in the world and has been a new public health and social issue. Intrauterine infection and hypoxia-ischemia brain injury have been suggested to be the mainly two causal factors of CP. And the inflammatory mehcanism produced by the two factors have been believed to play an important role in the development of CP. A large amount of cytokines in the process of inflammation was activated, interacted and mutual influenced, and jointly participate in pathophysiological process of regulating the body, which is the common pathway of different pathogenic factors that lead to brain injury. In addition, there is an overlap between inflammatory reaction and early immune response which can both participate in the body’s inflammatory process through the increased induction of cytokine release. Due to the immature neonatal immune function, the premature infants often have inmmune functions disorders in the central nervous system(CNS), including Th cells imbalances and Th related abnormal secretion of cytokines, which can cause the CNS inflammatory lesion and brain injury. Therefore, exploring the inflammatory response and the change of inflammatory cytokines is of great significance in the immature brain injury study.Inflammation that occurs in the fetal or neonatal period can persist to childhood, indicating that the occurrence of CP may be associated with the continuing role of inflammatory cytokines in the peripheral or CNS. However, the protein level of cytokines which produced by inflammation is regulated by gene polymorphisms. Thus it is necessary to study the relationship between gene polymorphisms and the susceptibility of CP.(Interleukin, IL)-6, IL-8, IL-10 and IL-17 A are polypeptide cytokines with a broad range of biological activities that plays a key role in systemic inflammatory process and has been believed to be strongly associated with neonatal brain injury and CP. But there still is no joint report for the relationship between gene single nucleotide polymorphisms(SNPs) of IL-6, IL-8, IL-10 and IL-17 A and CP.Staphylococcus epidermidis(SE) is a common nosocomial pathogen that causes bacteremia, late-onset sepsis, and intravascular catheter infections in preterm newborns. SE activates host inflammatory responses in part via Toll-like receptor 2(TLR2). Epidemiologic studies suggest a possible link between SE bacteremia and neonatal central nervous system(CNS) injury which might be due to the immature brain or suppressed immunity, but direct evidence is lacking.The immune system of neonates is immature and can still respone to inflammation to some extent. T-cells(CD4+ T and CD8+ T), B-cells and Th-type population have been suggested to play essential roles in the response to ischemic brain injury which indicate that innate immune response/inflammation play an important role in the development of immature brain injury.Overall, the study based the premature brain injury and its serious sequelae CP as the research objects, through the clinical and animals studies, from the gene regulation, infection and immune disorders caused by hypoxia ischemia, to explore the inflammatory response in the development of immature brain injury and provide theoretical basis for the prevention, diagnosis and treatment of CP.PartⅠ: The Association Between Sex-Related Interleukin-6 GenePolymorphisms and the Risk for Cerebral Palsy BackgroundThe relationship between genetic factors and the development of cerebral palsy(CP) has attracted much attention recently. Polymorphisms in the genes encoding proinflammatory cytokines have been shown to be associated with susceptibility to perinatal brain injury and development of CP. Proinflammatory cytokines such as interleukin-6(IL-6), IL-8, IL-10 and IL-17 A play a pivotal role in neonatal brain injury, and studies have been reported regarding the association of cytokines single nucleotide polymorphisms(SNPs) and CP. The purpose of this study was to analyze IL-6, IL-8, IL-10 and IL-17 A gene polymorphisms and protein expression and to explore the role of inflammation in the Chinese CP population.MethodsA total of 753 healthy controls and 713 CP patients were studied to detect the presence of five SNPs(rs1800796, rs2069837, rs2066992, rs2069840, and rs10242595) in the IL-6 locus; four SNPs(rs4073, rs2227307, rs2227306 and rs2227543) in the IL-8 locus; five SNPs(rs3024490, rs1800896, rs1800872, rs3021094 and rs1518110) in the IL-10 locus;five SNPs(rs3819025, rs3748067, rs2275913, rs8193036 and rs3819024) in the IL-17 A locus by mass ARRAY technique. Of these, 75 healthy controls and 85 CP patients were selected for measurement of plasma IL-6, IL-8, IL-10 and IL-17 by Luminex assay. The SHEsis program was used to analyze the genotyping data. For all comparisons, multiple testing on each individual SNP was corrected by the SNPSp D program. ResultsThere were no differences in allele or genotype frequencies between the overall CP patients and controls among the IL-6 and IL-10 polymorphisms(P>0.05), while significant differences in allele and genotype frequencies were found in the IL-17A(P = 0.009, P = 0.027 after SNPSp D correction; P = 0.005,P=0.015 after SNPSp D correction). There were no changes among the two groups in the IL-8 polymorphisms(P>0.05).However, subgroup analysis found significant sex-related differences in allele and genotype frequencies in the IL-6 gene. Differences were found between spastic CP and controls in males for rs2069837(P=0.007,P = 0.021 after SNPSp D correction); between CP with periventricular leukomalacia(PVL) and controls in males for rs1800796 and rs2066992(P = 0.014,P = 0.042 after SNPSp D correction; P = 0.013,P=0.039 after SNPSp D correction); for the IL-10 and IL-17 A gene, differences were found between tetraplegia CP and controls for IL-10 rs3024490 and for IL-17 A rs2275913 and rs3819024.Plasma IL-6, IL-10 and IL-17 levels were all higher in CP patients than in the controls. And the genotype from IL-6 and IL-10 has an effect on IL-6 and IL-10 synthesis, repectively. ConclusionsThe influence of IL-6 gene polymorphisms on IL-6 synthesis and the susceptibility to CP is related to sex. IL-6 is associated with the male CP with spastic subtype and PVL, IL-10 and IL-17 A are associated with tetraplegia. PartⅡ: Staphylococcus epidermidis bacteremia induces brain injuryin neonatal mice via TLR2-dependent and –independent pathways BackgroundStaphylococcus epidermidis(SE) causes late-onset sepsis in preterms. SE activates host responses in part via Toll-like receptor-2(TLR2). Epidemiologic studies link bacteremia and neonatal brain injury, but direct evidence is lacking. MethodsWild-type(WT) and TLR2-deficient(TLR2-/-) mice were injected intravenously with SE at postnatal day(PND)1 prior to measuring plasma and brain cytokines/ chemokines, bacterial clearance, brain caspase-3 activation, white/gray matter volume, and innate transcriptome. ResultsSE bacteremia spontaneously resolved over 24 hours without detectable bacteria in the cerebrospinal fluid(CSF). TLR2-/- mice demonstrated delayed SE clearance from blood, spleen and liver. SE increased white blood cells in CSF, IL-6, IL12p40, CCL2 and CXCL1 concentrations in plasma, CCL2 in brain, and caused rapid(within 6 hours) TLR2-dependent brain activation of caspase-3 as well as TLR2-independent white matter injury by PND14. ConclusionsSE bacteremia, in the absence of bacterial entry into the CSF, impairs neonatal brain development. SE bacteremia induced both TLR2-dependent and TLR2-independent brain injury, the latter occurring in the absence of TLR2, a condition in which bacterial burden increased. Our study indicates that the consequences of transient bacteremia in early life may be more severe than commonly appreciated and may inform novel approaches to reduce bacteremia- associated brain injury.Part Ⅲ: The immune response after hypoxia-ischemia in a mousemodel of preterm brain injury BackgroundPreterm brain injury consists primarily of periventricular leukomalacia accompanied by elements of gray-matter injury, and these injuries are associated with cerebral palsy and cognitive impairments. Inflammation is believed to be an important contributing factor to these injuries. The aim of this study was to examine the immune response in a postnatal day(PND) 5 mouse model of preterm brain injury induced by hypoxia-ischemia(HI) that is characterized by focal white and graymatter injury. MethodsC57Bl/6 mice at PND 5 were subjected to unilateral HI induced by left carotid artery ligation and subsequent exposure to 10% O2 for 50 minutes, 70 minutes, or 80 minutes. At seven7 days post-HI, the white/gray-matter injury was examined. The immune responses in the brain after HI were examined at different time points after HI using RT-PCR and immunohistochemical staining. ResultsHI for 70 minutes in PND 5 mice induced local white-matter injury with focal cortical injury and hippocampal atrophy, features that are similar to those seen in preterm brain injury in human infants. HI for 50 minutes resulted in a small percentage of animals being injured, and HI for 80 minutes produced extensive infarction in multiple brain areas. Various immune responses, including changes in transcription factors and cytokines that are associated with a T-helper(Th)1/Th17-type response, an increased number of CD4+ T-cells, and elevated levels of triggering receptor expressed on myeloid cells 2(TREM-2) and its adaptor protein DNAX activation protein of 12 k Da(DAP12) were observed using the HI 70 minute preterm brain injury model. ConclusionsWe have established a reproducible model of HI in PND 5 mice that produces consistent local white/gray-matter brain damage that is relevant to preterm brain injury in human infants. This model provides a useful tool for studying preterm brain injury. Both innate and adaptive immune responses are observed after HI, and these show a strong pro-inflammatory Th1/Th17-type bias. Such findings provide a critical foundation for future studies on the mechanism of preterm brain injury and suggest that blocking the Th1/Th17-type immune response might provide neuroprotection after preterm brain injury.