Reactive oxygen species generated by mitochondrial injury in human brain microvessel endothelial cells

Yoshiaki Itoh, Rie Takaoka, Masayuki Ohira, Takato Abe, Norio Tanahashi, Norihiro Suzuki

Research output: Contribution to journalArticlepeer-review

19 Citations (Scopus)


Generation of reactive oxygen species (ROS) and their detrimental effects on the brain after transient ischemia are widely recognized. We studied ROS production from mitochondria in human brain microvessel endothelial cells (HBEC) under chemical hypoxia. HBEC in confluent conditions were incubated for 30 min with 10 μM 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein (DCF) diacetate, which was hydrolyzed and trapped inside the cells. ROS were measured with a fluorescent microscope, a CCD camera and an image analyzing system. Injury to mitochondrial respiratory chain was induced either with rotenone (an inhibitor of mitochondrial complex I) or with m-chlorocarbonyl cyanide phenylhydrazone (CCCP) (an uncoupler of ATP synthetase). Shortly after application of 10 μM rotenone, fluorescent intensity started to increase and the gradual increase continued for 10 min. Similarly, CCCP (10, 50 and 100 μM) dose-dependently increased the fluorescent intensity (p<0.01). Edaravone, a free radical scavenger widely used for treatment of cerebral infarction in Japan, at 100 μM successfully suppressed this ROS production (p<0.05). These data show that chemical hypoxia with normal concentration of oxygen in the medium induced free radicals generation in HBEC. Importance of endothelial mitochondria as a source of free radicals after reperfusion is suggested.

Original languageEnglish
Pages (from-to)163-168
Number of pages6
JournalClinical Hemorheology and Microcirculation
Issue number1-2
Publication statusPublished - 2006


  • Dichlorodihydrofluorescein
  • Endothelial cell
  • Human brain
  • Microvessel
  • Reactive oxygen species (ROS)

ASJC Scopus subject areas

  • Physiology
  • Hematology
  • Cardiology and Cardiovascular Medicine
  • Physiology (medical)


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