Comparative evaluation of hypoxic-ischemic brain injury by flow cytometric analysis of mitochondrial membrane potential with JC-1 in neonatal rats

Abstract

We assessed the validity of monitoring changes in mitochondrial membrane potential (Delta Psi) with a fluorescent probe JC-1 (5 5 6 6 -tetrachloro-1 1 3 3 -tetraethyl benzimidazolo-carbocyanine iodide) for the quantitative evaluation of neonatal hypoxic-ischemic brain injury Seven-day-old rat pups were subjected to 2 h of 8% oxygen following unilateral carotid artery ligation Brain tissue was obtained for JC-1 staining at 24h after hypoxia ischemia (HI) and the results were compared with those of other simultaneous measurements such as flow cytometry with fluoresceinated annexin V/propidium iodide (PI) terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) staining triphenyl tetrazolium chloride (TTC) infarct area and western blot for cytosolic cytochrome c Flow cytograms of JC-1 showed two distinct sub-populations with different Delta Psi red with high Delta Psi and green with low Delta Psi at 24h after HI This shift of JC-1 fluorescence from red to green indicated a collapse of Delta Psi The increased percentage of low Delta Psi with JC-1 showed a significant positive correlation with a simultaneous increase in annexin V+/PI+ necrotic cells TUNEL-positive cells TTC infarct area and western blot of cytosolic cytochrome c and negative correlation with annexin V-/PI- live cells In summary low Delta Psi measured with JC-1 was significantly correlated with results from other methods used to assess the extent of brain damage after HI Therefore fluorocytometric analysis of Delta Psi with JC-1 might be a sensitive and reliable technique in the quantitative evaluation of neonatal brain injury (C) 2010 Elsevier B V All rights reserved