Clinical management of neonatal hypoxic-ischemic encephalopathy (HIE) suffers from the lack of reliable surrogate marker tests. Proteomic analysis may identify such biomarkers in blood, but there has been no proof-of-principle evidence to support this approach. Here we performed in-gel trypsin digestion of plasma proteins from 4 groups of 10-day-old mice (untouched and 24 h after low-dose lipopolysaccharide [LPS] exposure, hypoxia-ischemia [HI], or LPS/HI injury; n=3 for each group) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and bioinformatics analysis to search for HI- and LPS/HI-associated brain injury biomarkers. This analysis suggested the induction of plasma osteopontin (OPN) by HI and LPS/HI, but not by the sham and injury-free LPS-exposure. Immunoblot confirmed post-HI induction of OPN protein in the brain and blood, whereas the Opn mRNAs was induced in the brain, but not in blood. This disparity suggests brain-derived plasma OPN after HI injury. Similarly, immunostaining showed the expression of OPN by Iba1+ microglia/macrophages in HI-injured brains. Further, intracerebroventricular injection of LPS activated microglia and up-regulated the plasma OPN protein. Importantly, the induction of plasma OPN after HI is greater than those of matrix metalloproteinase 9 (MMP-9) and glial fibrillary acid protein (GFAP). The plasma OPN levels at 48 h post-HI also parallel the severity of brain damage at 7 d recovery. Together, these results suggest that the OPN may be a prognostic blood biomarker in HIE by monitoring the brain microglial activation.
Significance Statement: There are no reliable blood biomarkers for neonatal brain injury in HIE to date, and the majority of previous studies tested candidate biomarkers individually. Here we report, for the first time, proof-of-principle evidence that mass spectrometry-based quantitative proteomic methods may efficiently identify blood biomarkers in HIE. Using this method, we show that osteopontin (OPN), an integrin-binding protein secreted by activated microglia, rises in blood and correlates with the severity of brain damage in experimental HIE. These data suggest that OPN may be a blood biomarker in HIE.
Authors report no conflict of interest.
This work was supported by the NIH grants (NS084744, HD080429, NS095064 to C-Y.K.), an American Heart Association fellowship (to Y-Y.S.), and supported in part by the Proteomics Core of the Emory Neuroscience NINDS Core Facilities grant (P30NS055077).