Mechanism Of Sleep Related Problems Associated With Hypoxic-ischemic Encephalopathy(HIE)

Neonatal hypoxic-ischemic encephalopathy (HIE) can lead to severe chronic neurological deficits, which include psychomotor developmental retardation, epilepsy, and sleep-wake cycle disorders. Among these defects, very little is known about the molecular mechanisms underlying the circadian rhythm disturbance after HIE in newborns. Therefore, further investigation of the sleep problems in childrens with viraous degrees of HIE and their underlying mechanisms will definitely provide novel insights into the appropriate clinical treatments for children who suffer from HIE.Throughout their lifetime, mRNAs are escorted with a handful of RNA binding proteins and small non-coding RNAs. Among these trans-factors, microRNAs (miRNAs), a group of small non-coding RNAs, play a crucial role in regulating gene expression. Although miRNAs were reported to be associated with multiple diseases, how they affect the patholophysiology of HIE were largely unknown.In this study, we sought out to explore these two questions using data from both patients and animal models. Clinically, we investigated whether an increased risk of sleep problems is associated with perinatal HIE patients during development. In an animal model, we performed a genome-wide miRNA screening in pineal gland after HIE in neonatal rats and identified one miRNA (miR-182), whose expression was significantly reduced, that targeted the 3’-UTR of CLOCK gene and regulated its expression. These findings thereby shed light on the mechanisms of how miRNAs regulate circadian abnormality after neonatal HIE.Part I:Distinctive sleep problems in children with perinatal moderate or mild hypoxic-ischemiaAim:To investigate whether an increased risk of sleep problems is associated with various degrees of HIE.Methods:A total of 128 patients [moderate prenatal HIE (n= 44), mild HIE (n= 84)] and 88 normal, age/sex matched fulled termed children were included. All children were born between May 2010 and August 2013. Questionares were performed, analyzed for the correlation between different sleep problems and brain structure abnormalties with HIE with various degrees.Results:We discovered that sleep problems were highly associated with perinatal HIE. Further analysis revealed that patients with moderate HIE had a higher risk of sleep initiation andmaintenance issues, along with sleep-related breathing problems. In contrast, mild HIE patients had difficulties in maintaining regular sleep schedules.Conclusion:Our findings revealed novel pathophysiological characteristics especially in children with mild HIE, which are highly likely associated with the observed circadian rhythmic problems that affect sleep qualities. Our study therefore provides insightful views for the epidemiological studies of non-severe HI patients, who accounted for the majority HIE cases.Part Ⅱ:The role of miR-182 on CLOCK expression in pineal gland after hypoxia-schemia brain injury(HIBD) in neonatal ratsAim:Identification and analysis of the role of the pineal miR-182 after HIBDin rats.Methods:By performing a genome-wide miRNA screening in pineal gland after HIE in neonatal rats, we sought out to identify miRNAs (miR-182) whose expression were significantly reduced after HIE. Combining bioinformatics prediction and the luciferase reporter gene assay, we further confirmed the interaction between selected miRNA and its potential mRNA target.Results:Using high-throughput screening, we examined expression changes of miRNAs in pineal gland after neonatal HIBD. Among those significantly down-regulated miRNAs, we identified miR-182 that targeted the 3’-UTR of CLOCK gene. We further demonstrated that the elevation of CLOCK protein in cultured pinealocytes was abolished after oxygenglucose deprivation by adding miR-182 mimics, thereby providing direct evidence that miR-182 is involved in CLOCK gene regulation in pineal gland after neonatal HIBD.Conclusion:Our findings of the negative correlation between the miR-182 and CLOCK gene after HIBD clearly demonstrated that CLOCK is the target for miR-182, thereby shedding new light on the development of therapeutic treatment for circadian disturbances after HIBD.