Intravital oxygen radical imaging in normal and ischemic rat cortex.
OBJECTIVE
We examined reactive oxygen species (ROS) generation on cerebral ischemia/reperfusion by intravital fluorescence imaging.


METHODS
In anesthetized adult rats, a fluorescent dye (5 microL), MitoSOX (5 micromol/L) for superoxide radical (.O2-), and hydroxyphenyl fluorescein (20 micromol/L) for hydroxyl radical (.OH), was injected into cortices by a pressurized bolus. Through a closed cranial window, fluorescent images were taken with a confocal microscope on 10-minute forebrain ischemia. Because hemoglobin absorbs excitation and emission lights, ischemia may affect the change in fluorescence intensity (FI) inside the brain. To examine the effects of ischemia on the FI change, fluoromicrospheres (0.2-microm diameter) were used to mimic a dye and FI was analyzed in the same manner as when using ROS indicators. Their FI increased to 129% during ischemia (n=3/mimicking each dye), and based on the results, FI of ROS indicators was corrected.


RESULTS
After correcting the FI of MitoSOX and hydroxyphenyl fluorescein, they showed no change during ischemia, whereas the raw data showed the increase. In the early period of reperfusion, FI significantly (n=5/each, P<.01) increased (to 183% in MitoSOX and to 189% in hydroxyphenyl fluorescein), and these increases were significant in the areas adjacent to the arteries. To test the feasibility of our imaging, edaravone (3.0 mg/kg) was used. The treatment completely scavenged .OH, but did not do so in .O2- generation.


CONCLUSIONS
ROS production increased in the early period of reperfusion but not during ischemia, which was location selective, being significant in the areas adjacent to the arteries. Our method was useful for investigating intracellular in situ ROS production.
Wang
Yong
Y
Photon Medical Research Center, Department of Neurosurgery, Hamamatsu University School of Medicine, Hamamatsu, Japan.
Yamamoto
Seiji
S
Miyakawa
Atsuo
A
Sakurai
Takashi
T
Ibaraki
Kyoko
K
Terakawa
Susumu
S
D000818:Animals; D002540:Cerebral Cortex / Q000378:metabolism* / Q000503:physiopathology; D004195:Disease Models, Animal; D005609:Free Radicals / Q000032:analysis / Q000378:metabolism; D017665:Hydroxyl Radical / Q000032:analysis / Q000378:metabolism; D020925:Hypoxia-Ischemia, Brain / Q000378:metabolism* / Q000503:physiopathology; D008297:Male; D008856:Microscopy, Fluorescence / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000032:analysis* / Q000378:metabolism*; D015427:Reperfusion Injury / Q000378:metabolism* / Q000503:physiopathology
D005609:Free Radicals; D017382:Reactive Oxygen Species; D017665:Hydroxyl Radical