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 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.
Sung
Dong Kyung
DK
Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea.
Chang
Yun Sil
YS
Kang
Sam
S
Song
Hwa Young
HY
Park
Won Soon
WS
Lee
Bong Hee
BH
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
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