Proteomics of Brain Injury in a Neonatal Model of Deep Hypothermic Circulatory Arrest.
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Objective: Deep Hypothermic Circulatory Arrest (DHCA) is an important strategy in repairs of complex congenital heart disease. Concern over cerebral injury limits safe duration of arrest. Proteomics is a novel and powerful technique to study global protein changes in a given protein system. Employing a model of DHCA, we sought to characterize the protein changes associated with DHCA brain injury.
Methods: Ten neonatal piglets (1 week age, 3kg) were randomized to cardiopulmonary bypass (CPB) with DHCA or sham operation. DHCA animals underwent induction of CPB (100cc/kg/min) with systemic cooling to 18 C then DHCA for 60 minutes. Animals were then rewarmed to normothermia, weaned from CPB and harvested after 30 minutes off bypass. Control animals underwent sternotomy and were harvested after an equal period of time without instrumentation. Plasma samples were taken prior to CPB and prior to harvest. Protein solubilization of cerebral cortex homogenates was followed by 2D Difference Gel Electrophoresis (DIGE) using fluorescent Cyanine dyes. Software analysis of the gels identified protein spots differentially expressed between the 2 groups. These spots were excised and mass spectrometry (MS) utilized to identify the proteins. A second group of 4 piglets were similarly randomized and tissues prepared for light and electron microscopic examination by perfusion fixation at the conclusion of the experiment.
Results: Control brain showed normal neuronal architecture. DHCA caused extensive neuropil vacuolation, neuronal swelling; both neuronal and glial cells exhibited severe organelle swelling. Proteomic analysis of cerebral cortical tissue found 10 protein spots to be differentially expressed (p<0.05); 8 were identified by MS to represent 6 proteins. Five proteins showed increased expression in DHCA brain tissue: Apolipoprotein A-1, protamine P1, neurofilament triplet M protein and 2 hypothetical proteins. Enolase showed decreased expression. Protein disulfide isomerase showed increased expression of borderline significance. Apolipoprotein A-1 was also found in the proteomic analysis of the plasma (decreased expression in DHCA).
Conclusions: This study has characterized the protein changes seen in cortical brain injury in a neonatal model of DHCA. These changes may provide important mechanistic clues as to the molecular pathogenesis of cerebral injury and direct further research aimed at attenuating injury seen with DHCA.
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