Serial blood cytokine and chemokine mRNA and microRNA over 48 h are insult specific in a piglet model of inflammation-sensitized hypoxia-ischaemia

Background Exposure to inflammation exacerbates injury in neonatal encephalopathy (NE). We hypothesized that brain biomarker mRNA, cytokine mRNA and microRNA differentiate inflammation (E. coliLPS), hypoxia (Hypoxia), and inflammation-sensitized hypoxia (LPS+Hypoxia) in an NE piglet model. Methods Sixteen piglets were randomized: (i) LPS 2 mu g/kg bolus; 1 mu g/kg infusion (LPS;n = 5), (ii) Saline with hypoxia (Hypoxia;n = 6), (iii) LPS commencing 4 h pre-hypoxia (LPS+Hypoxia;n = 5). Total RNA was acquired at baseline, 4 h after LPS and 1, 3, 6, 12, 24, 48 h post-insult (animals euthanized at 48 h). Quantitative PCR was performed for cytokines (IL1A,IL6,CXCL8,IL10,TNFA) and brain biomarkers (ENO2,UCHL1,S100B,GFAP,CRP,BDNF,MAPT). MicroRNA was detected using GeneChip (Affymetrix) microarrays. Fold changes from baseline were compared between groups and correlated with cell death (TUNEL) at 48 h. Results Within 6 h post-insult, we observed increasedIL1A,CXCL8,CCL2andENO2mRNA in LPS+Hypoxia and LPS compared to Hypoxia.IL10mRNA differentiated all groups. Four microRNAs differentiated LPS+Hypoxia and Hypoxia: hsa-miR-23a, 27a, 31-5p, 193-5p. Cell death correlated withTNFA(R = 0.69;p < 0.01) at 1-3 h andENO2(R = -0.69;p = 0.01) at 48 h. Conclusions mRNA and miRNA differentiated hypoxia from inflammation-sensitized hypoxia within 6 h in a piglet model. This information may inform human studies to enable triage for tailored neuroprotection in NE. ImpactEarly stratification of infants with neonatal encephalopathy is key to providing tailored neuroprotection. , , , and mRNA are promising biomarkers of inflammation-sensitized hypoxia. mRNA levels differentiated all three pathological states; fold changes from baseline was the highest in LPS+Hypoxia animals, followed by LPS and Hypoxia at 6 h. miR-23, -27, -31-5p and -193-5p were significantly upregulated within 6 h of a hypoxia insult. Functional analysis highlighted the diverse roles of miRNA in cellular processes