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

We assessed the validity of monitoring changes in mitochondrial membrane potential (ΔΨ) 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 24 h 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 ΔΨ, red with high ΔΨ and green with low ΔΨ, at 24 h after HI. This shift of JC-1 fluorescence from red to green indicated a collapse of ΔΨ. The increased percentage of low ΔΨ 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 ΔΨ 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 ΔΨ with JC-1 might be a sensitive and reliable technique in the quantitative evaluation of neonatal brain injury.

Research highlights▶ Two distinctive populations of cells with both high and low ΔΨ were identified 24 h after HI. TUNEL-positive cells and TTC infarct area showed significant correlation with the percentage of low ΔΨ JC-1. ▶ Western blot band intensity of cytosolic cytochrome c was significantly correlated with the percentage of low ΔΨ JC-1.