title: 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 (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 2h 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 ΔΨ, red with high ΔΨ and green with low ΔΨ, at 24h 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.
authors.affiliation: Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea.
authors.forename: Dong Kyung
authors.initials: DK
authors.lastname: Sung
authors.forename: Yun Sil
authors.initials: YS
authors.lastname: Chang
authors.forename: Sam
authors.initials: S
authors.lastname: Kang
authors.forename: Hwa Young
authors.initials: HY
authors.lastname: Song
authors.forename: Won Soon
authors.initials: WS
authors.lastname: Park
authors.forename: Bong Hee
authors.initials: BH
authors.lastname: Lee
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D017304:Annexin A5 / Q000378:metabolism; D001562:Benzimidazoles; D001921:Brain / Q000473:pathology; D020520:Brain Infarction / Q000209:etiology / Q000473:pathology*; D002232:Carbocyanines; D016923:Cell Death / Q000502:physiology; D045304:Cytochromes c / Q000378:metabolism; D004195:Disease Models, Animal; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D020925:Hypoxia-Ischemia, Brain / Q000150:complications / Q000175:diagnosis*; D020287:In Situ Nick-End Labeling / Q000379:methods; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects / Q000502:physiology*; D009474:Neurons / Q000378:metabolism / Q000473:pathology; D011419:Propidium; D051381:Rats; D017207:Rats, Sprague-Dawley; D013778:Tetrazolium Salts; D013997:Time Factors
chemical_list: D017304:Annexin A5; D001562:Benzimidazoles; D002232:Carbocyanines; D005456:Fluorescent Dyes; D013778:Tetrazolium Salts; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; D011419:Propidium; C009591:triphenyltetrazolium; D045304:Cytochromes c