The detection and quantification of highly reactive oxygen species using the novel HPF fluorescence probe in a rat model of focal cerebral ischemia.
A novel fluorescence probe, 2-[6-(4'-hydroxy) phenoxy-3H-xanthen-3-on-9-yl] benzoic acid (HPF) was used to investigate the generation of highly reactive oxygen species (hROS) under ischemia both in vitro and in vivo. In the in vitro study, HT 22 cells were used to demonstrate that was predominantly detected in the cytoplasm, which coincides with the location of the mitochondria and then its HPF fluorescence gradually increased from 6 to 24 h due to glutamate induced oxidative stress. In the in vivo study, the permanent and transient middle cerebral artery occlusion (MCAO) was induced in rats. Brain slices were incubated in an artificial medium containing HPF. The area of enhanced HPF fluorescence existed in both the ischemic core and the peri-infarct area at 4h after MCAO in both permanent and transient MCAO models. The area extended beyond the boundary of the ischemic damage into biochemically viable tissue. The enhanced fluorescent intensity following transient MCAO was higher than that observed in the permanent MCAO model. Hydroxyl radical scavenger, MCI-186 significantly suppressed the enhanced fluorescence intensity. This study demonstrated that HPF has a high sensitivity and specificity for the detection of hROS in focal cerebral ischemia as well as in a cellular model of oxidative stress.
Tomizawa
Shinichiro
S
Department of Neurosurgery, Gunma University Graduate School of Medicine, 3-39-22, Showa-machi, Maebashi, Gunma 371-8511, Japan. tomizawa-nsu@umin.ac.jp
Imai
Hideaki
H
Tsukada
Shota
S
Simizu
Tatsuya
T
Honda
Fumiaki
F
Nakamura
Mitsunobu
M
Nagano
Tetsuo
T
Urano
Yasuteru
Y
Matsuoka
Yuki
Y
Fukasaku
Noboru
N
Saito
Nobuhito
N
D000818:Animals; D001565:Benzoates / Q000737:chemistry; D001681:Biological Assay / Q000379:methods; D002545:Brain Ischemia / Q000378:metabolism* / Q000503:physiopathology; D002461:Cell Line, Transformed; D002544:Cerebral Infarction / Q000378:metabolism / Q000503:physiopathology; D004195:Disease Models, Animal; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D016166:Free Radical Scavengers / Q000494:pharmacology; D020244:Infarction, Middle Cerebral Artery / Q000378:metabolism / Q000503:physiopathology; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D009924:Organ Culture Techniques; D018384:Oxidative Stress / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000032:analysis* / Q000378:metabolism*
D001565:Benzoates; D005456:Fluorescent Dyes; D016166:Free Radical Scavengers; D017382:Reactive Oxygen Species