MiR-126 and MiR-146a as Melatonin-Responsive Biomarkers for Neonatal Brain Ischemia

Neonatal encephalopathy (NE) resulting from hypoxia-ischemia (HI) poses significant challenges, often leading to long-term neurological deficits. Therapeutic hypothermia is currently the standard treatment but has limitations, prompting research into adjunct therapies like melatonin.Melatonin shows promise due to its neuroprotective properties. However, optimal dosing and timing post-injury are unclear. NE diagnosis relies on clinical signs and neuroimaging, but early detection remains challenging.We aim to identify early biomarkers of brain injury post-HI by examining miRNAs. miR-126 and miR-146a show potential, as alterations in their levels correlate with brain injury severity and respond to melatonin treatment in preclinical models.This research underscores the importance of finding reliable biomarkers to aid in NE management and improve outcomes for affected infants. Matherials and methods Rat cerebral Hypoxic-Ischemia and melatonin treatment This study utilized a preclinical model of Rat Cerebral Hypoxic-Ischemia (HI) to investigate the effects of melatonin treatment on microRNA (miRNA) expression. Pregnant Sprague-Dawley rats underwent surgical procedures in accordance with animal care regulations. On postnatal day 7, neonatal rats underwent unilateral carotid artery ligation followed by exposure to hypoxia. Melatonin was administered intraperitoneally post-HI induction. Serum and brain samples were collected at various time points post-HI and melatonin treatment. Bioinformatics analysis and quantitativa real-time PCR for Mature miRNA analysis A bioinformatics analysis using the miRNet tool identified miR-126 and miR-146a as potential biomarkers associated with neonatal cerebral HI. Quantitative Real-Time PCR was employed to assess miRNA expression levels in serum and cerebral cortex homogenates. Results showed significant alterations in miR-126 and miR-146a expression post-HI, with melatonin treatment attenuating these changes. Overall, the study suggests that miR-126 and miR-146a may serve as early biomarkers of brain injury following neonatal HI, and their modulation by melatonin highlights the potential therapeutic benefits of melatonin in this context. Data analysis Statistical analyses were performed by two-way ANOVA or one-way ANOVA using the Prism Computer program (GraphPad Software Inc.). Bartlett’s test was used to determine data homogeneity. The Bonferroni multiple comparison test or Newman–Keuls multiple comparison test was used to determine differences between groups. Results were considered to be significant when p ≤0.05. Results In our study, we utilized the miRNet bioinformatics tool to analyze a new NCHI pathway sensitive to melatonin, predicting miR-126 and miR-146a as the most important associated miRNA nodes. Subsequently, we performed qRT-PCR analysis on cerebral cortex and serum samples collected from animals sacrificed at 1, 6, and 24 hours post-HI and melatonin treatment. In the cerebral cortex, miR-126 expression decreased 1 hour post-HI, increased at 6 hours, and decreased again at 24 hours. Melatonin treatment significantly increased miR-126 expression at 1 and 6 hours post-injury compared to controls, but levels were equivalent to HI animals at 24 hours. Conversely, serum miR-126 levels increased post-HI and decreased after melatonin treatment. Similarly, miR-146a showed a down-regulation at 1 hour post-HI, up-regulation at 6 hours, and return to control levels at 24 hours in the cerebral cortex. Melatonin treatment increased miR-146a levels compared to controls at all time points and compared to HI animals at 1 and 24 hours. Serum miR-146a levels increased post-HI, while melatonin treatment maintained levels similar to controls at 1 and 6 hours and increased levels at 24 hours compared to controls. Overall, our results suggest that melatonin treatment modulates miR-126 and miR-146a expression in both cerebral cortex and serum, potentially contributing to its neuroprotective effects in neonatal cerebral hypoxic-ischemic injury. Discussion and conclusion Neonatal mortality, constituting 46% of under-five mortality globally, is significantly impacted by neonatal encephalopathy (NE), a condition often stemming from hypoxic-ischemic (HI) injury. Rapid and accurate diagnosis is crucial for reducing mortality and morbidity associated with NE. While biomarker research shows promise, bottlenecks hinder their integration into clinical practice. MiRNAs, particularly brain-specific ones, have emerged as potential biomarkers due to their expression in both the brain and peripheral blood. Using bioinformatics tools, we identified miR-126 and miR-146a as potential biomarkers for perinatal brain injury, suggesting their utility in diagnosis. Through a neonatal rat model of HI brain injury, we observed dysregulation of miR-126 and miR-146a expression in both brain tissue and serum. Melatonin treatment post-HI showed restoration effects on these miRNAs, indicating its neuroprotective potential. MiR-126, crucial for vascular integrity and angiogenesis, exhibited down-regulation in cerebral cortex post-HI, with contrasting upregulation in serum. Melatonin treatment restored cortical levels while maintaining serum levels similar to controls. MiR-146a, a negative regulator of inflammation, showed a similar trend, with early down-regulation and subsequent upregulation post-HI in the cortex, and higher levels in serum post-HI, reduced by melatonin. These findings suggest miR-126 and miR-146a as potential diagnostic biomarkers for neonatal HI brain injury, with their dysregulation detectable within hours post-HI insult, aligning with the therapeutic window for intervention. While further clinical validation is necessary, these results offer insights for improving bedside clinical management in neonatal NE cases.