Early Assessment And Prevention Research Of Adverse Neurological Outcome In Preterm Infants

Introduction About 15 million preterm neonates are currently born every year globally.Modern advances in prenatal and neonatal intensive care have led to an increase in the survival rate of these premature infants, especially very and extremely low birth weight infants. However, neonates born preterm have increased risk of both brain damage and long-term neurodevelopmental disorders such as cerebral palsy,intellectual disabilities, severely visual and auditory disorders, which seriously affect the quality of life in premature infants. Early diagnosis and early treatment of brain damage in preterm infants has the potential to improve their long-term quality of life.Prevention and treatment of adverse prognosis in premature infants is the focus of the current study of neonatal medicine.Amplitude-integrated electroencephalography(a EEG) has the advantages of being simple to perform, of allowing continuous bedside monitoring, and of providing easily interpreted results, all of which make this an important method in the neonatal intensive care unit for monitoring brain function. It has also been shown that a EEG classifications correlate strongly with the clinical degree of hypoxic-ischemic encephalopathy and neurological outcomes in full-term infants. The role of a EEG for predicting long-term adverse neurological outcome in preterm infants needs is not clear and previous studies have only included limited numbers of patients.Erythropoietin(EPO) is a kind of endogenous glycoprotein produced by the kidney under hypoxia. EPO was first recognized for its hematopoietic properties.Experimental studies have reported that rh EPO possesses neuroprotective properties in different neonatal brain injury animal models. Clinical studies have shown that rh EPO treatment of mild to moderate hypoxic-ischemic encephalopathy in term infants improved long-term neurological outcomes. Although recent studies have confirmed that rh EPO has a neuroprotective effect on preterm infants, the timing,dose, and duration of rh EPO treatment are not clear, the long-term follow-up data are limited, and the safety is not clear. It is confirmed that EPO can through the blood brain barrier both in animal and human. However, the effective neuroprotective concentration range in different reports was of great difference. Studies on the rh EPO pharmacokinetics in blood and cerebrospinal fluid of premature infants was rare.Metabolomics is the science about quantitatively scrutinizing metabolites and metabolic pathways in biosystems in high-throughtput manners, which became very popular in last twenty years. Along with genomics, transcriptomics and proteomics,metabolomics is a part of systematic biology. The main application of metabolomics in the field of medicine are in two aspects: First, to explore the mechanism of disease or drug action. Second, to search for molecular markers that can be used for diagnosis,prognosis and drug efficacy monitoring. Currently, the application of metabolomics in cancer, cardiovascular disease, diabetes, and schizophrenia disease has found important biological markers, which deepened the understanding of disease mechanisms, and successfully used for diagnosis and typing. However, there is no report on the application of metabolomics in the study of cerebral palsy.Proteomics is an organic part of systematic biology. The purpose of proteomics is to overall analysize the components, expression and modification of intracellular proteins, to explore the interaction and association between proteins, and to reveal the function of proteins and regularities of cellucar activities. Proteomics is an effective way to screen the biomarkers of diseases. At present, this method has not been applied in the study of the biological markers of cerebral palsy.Overall, the study using early a EEG, early rh EPO treatment and based on metabolomics and proteomics, through clinical study and fundamental experiments,to investigate the early evaluation and prevention measures of adverse neurological outcome in preterm infants.PartⅠ: Early amplitude-integrated electroencephalography predicts brain injury and neurological outcome in very preterm infantsObjective We evaluated how well early a EEG could predict brain damage and long-term neurodevelopmental outcomes in very preterm infants compared with brain imaging assessments.Methods346 neonates with GA < 32 weeks admitted to the intensive care unit within 2h after birth were included in the study conducted between January 2012 and January2013. Neonates with congenital brain malformations, chromosomal diseases, genetic diseases, or metabolic diseases were excluded. a EEG traces were recorded within 72 h,head ultrasound(HUS) was performed within 3 days after birth and then weekly until4 weeks after birth, head magnetic resonance imaging(MRI) was performed at 40 weeks corrected age. All of the preterm infants were tracked until 18 months corrected age. Mental or psychological development was evaluated with the Bayley Scales of Infant Development, Second Edition. Poor outcome was defined as survival with one or more of cerebral palsy, hypophrenia, or audio-visual disorder or death due to brain damage.Results We found that severe a EEG abnormalities(p = 0.000) and a EEG total score < 5(p = 0.006) within 72 h after birth were positively correlated with white-matter damage, but a EEG abnormalities were not associated with intracranial hemorrhage(p= 0.186). Severe abnormalities in a EEG recordings, head ultrasound, and cranial magnetic resonance imaging were all positively correlated with poor outcome at 18 months corrected age. The predictive power of poor outcomes of the a EEG and MRI combination was the same as the a EEG, MRI, and head ultrasound combination with a sensitivity of 52.4%, specificity of 96.2%, positive predictive value of 78.6%, and negative predictive value of 88.4%.Conclusion These results indicate that severely abnormal a EEG recordings within 72 h after birth can predict white-matter damage and long-term poor outcomes in very preterm infants. Thus a EEG can be used as an early marker to monitor very preterm infants.PartⅡ: Recombinant human erythropoietin improves neurological outcomes in very preterm infantsObjective The objective of this trial was to evaluate the efficacy of repeated low-dose rh EPO in the improvement of neurological outcomes and its safety in very preterm infants, and to observe the pharmacokinetic of rh EPO in serum and cerebrospinal fluid(CSF).Methods A total of 800 infants of ≤32 weeks gestational age who had been in an intensive care unit within 72 h after birth were included in a placebo-controlled trial conducted between January 2009 and June 2013. Infants with genetic metabolic diseases,congenital abnormalities, polycythemia, severe infection, unstable vital signs, or grade III/IV intracranial hemorrhage before randomization were excluded. Preterm infants were randomly assigned to receive rh EPO(500 IU/kg) or placebo(equivalent volume of saline) intravenously within 72 h after birth and then once every other day for 2 weeks. The primary outcome was death or moderate to severe neurological disability assessed at 18 months of corrected age. Sixty infants in the rh EPO treatment group with gestational age of 29-30 weeks were randomly selected and divided into 6 groups with 10 cases in each group. Venous blood and CSF were collected before and at 3 h, 6 h, 12 h, 24 h, and 48 h after the first dose of rh EPO.EPO concentrations in serum and CSF were determined by enzyme linked immunosorbent assay.Results Death and moderate/severe neurological disability occurred in 91 of 338 very preterm infants(26.9%) in the placebo group and in 43 of 330 very preterm infants(13.0%) in the rh EPO treatment group(OR = 0.40 [95% CI = 0.27–0.59], p < 0.001)at 18 months of corrected age. Subgroup analyses showed that rh EPO improved long-term neurological outcomes for infants with different gestational age(<28w,28–296/7w, 30–32w), different birth weight(<1000g, 1000-1499 g, ≥1500g), boys and girls(p < 0.001). The rate of neonatal complications, such as intracranial hemorrhage grade III–IV(OR = 0.38 [95% CI = 0.23–0.62], p = 0.000), periventricular leukomalacia(OR = 0.52, [95% CI = 0.35–0.79], p = 0.002), necrotizing enterocolitis(OR = 0.44 [95% CI = 0.27–0.73], p = 0.001), and sepsis(OR = 0.65, [95% CI =0.46–0.92], p = 0.015) were all reduced in the rh EPO group compared to the placebo group at term equivalent age, and no excess adverse events were observed. The EPO level in serum(480.32 ± 7.97 m IU/ml) and CSF(42.77 ± 8.42 m IU/ml) both reached peak at 3h after the first dose of rh EPO, There was a positive correlation(coefficient0.881, p < 0.05) between serum and CSF EPO levels.Conclusion Repeated low-dose rh EPO treatment reduced the risk of long-term neurological disability in very preterm infants, and with no obvious adverse effects. EPO can pass through the blood-brain barrier in very preterm infants.Part Ⅲ: Applications of metabolomics in cerebral palsy research of preterm childrenObjective In this study, we used the method of metabolomics to identify the character of metabolites in premature children with cerebral palsy, to explore the pathogenesis and the diagnostic biomarker of cerebral palsy.Methods Cerebral palsy children between 12-36 months who were born less than 37 weeks of gestation age and with periventricular leukomalacia were included in the disease group. Premature children of the same ages without nervous system disease were included in the control group. GC-TOF-MS based untargeted metabolomics was utilized to observe metabolites in blood of the two groups. Metabolites changes in preterm children with cerebral palsy were identified by univariate analysis,multivariate analysis, and biological information enrichment analysis.Results We have collected 11 cerebral palsy children and 11 normal children, detected177 metabolites, and 42 metabolites were finally used for bioinformatical analysis.Compared with normal preterm children, blood inosine and energy metabolism pathway appeared abnormal in preterm children with cerebral palsy. Inosine decreased significantly, aconitic acid increased significantly, the function of the tricarboxylic acid cycle impaired, the pentose phosphate pathway and the galactose metabolism pathway activated. A total of 1 + 3 principal components with the Q2 value of 0.66 were obtained by OPLS-DA analysis. Thus, premature cerebral palsy children were clearly separated with premature normal children by the first principal component, and the quantitative diagnostic model was successfully established.Conclusion Metabolomics facilitates our understanding of cerebral palsy’s metabolic disturbance, helps to comprehend the pathogenesis. We provide a quantitative diagnostic model for early assessment of cerebral palsy in premature infants.Part IV: Applications of proteomics in cerebral palsy research of preterm childrenObjective In this study, we used the method of proteomics to identify the differentially expressed proteins in preterm children with cerebral palsy, and to explore the pathogenesis and early diagnostic biomarkers of cerebral palsy.Methods Cerebral palsy children between 12-36 months who were born less than 37 weeks of gestation age and with periventricular leukomalacia were included in the disease group. Premature children of the same age without nervous system disease were included in the control group. i TRAQ based proteomics was utilized to identify the differentially expressed proteins in plasma of the two groups. GO and enrichment analysis were used to further explore the function of differential proteins and related pathways.Results We have collected 11 cerebral palsy children and 11 normal children, detected819 differential proteins, and 66 significantly differential proteins. GO analysis showed that the differential proteins mainly involved in antioxidation, catalytic activity, transporter activity, receptor activity, biological adhesion, cellular process,growth, developmental process, immunity, metabolic process and other biological processes. Pathway enrichment analysis showed integrin signaling pathway,cytoskeletal regulation pathway, inflammation mediated by chemokine and cytokine signaling pathway, which may be involved in the pathogenesis of cerebral palsy of premature infants.Conclusion Differential proteins related signal pathways deepen our understanding of the pathogenesis of cerebral palsy. Differential proteins may be helpful to early diagnosis of cerebral palsy, and may become potential therapeutic targets.