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Intravital Oxygen Radical Imaging in Normal and Ischemic Rat Cortex

Wang, Yong MD, PhD; Yamamoto, Seiji MD, PhD; Miyakawa, Atsuo PhD; Sakurai, Takashi PhD; Ibaraki, Kyoko PhD; Terakawa, Susumu MD, PhD

Neurosurgery:
doi: 10.1227/01.NEU.0000370055.99998.6B
Experimental Studies: Editor's Choice
Abstract

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 μL), MitoSOX (5 μmol/L) for superoxide radical (·O2), and hydroxyphenyl fluorescein (20 μmol/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-μm 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.

CONCLUSION: 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.

In Brief

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 &amp;#x03BC;L), MitoSOX (5 &amp;#x03BC;mol/L) for superoxide radical (&amp;#x00B7;O2-), and hydroxyphenyl fluorescein (20 &amp;#x03BC;mol/L) for hydroxyl radical (&amp;#x00B7;OH), was injected into cortices by a pressurized bolus. Through a closed cranial window, fluorescent images were taken with a confocal microscope after 10-minute forebrain ischemia. Fluoromicrospheres (0.2-&amp;#x03BC;m 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, there was no change during ischemia. In the early period of reperfusion, FI significantly (n = 5/each, P &amp;#x003C; .01) increased (to 183% in MitoSOX and to 189% in hydroxyphenyl fluorescein), and these increases were significant in the areas adjacent to the arteries. Furthermore, edaravone (3.0 mg/kg) treatment completely scavenged &amp;#x00B7;OH, but did not do so in &amp;#x00B7;O2- generation. CONCLUSION: 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.

Author Information

Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu, Japan (Wang) (Miyakawa) (Sakurai) (Ibaraki) (Terakawa)

Department of Neurosurgery, The First Affiliated Hospital of China Medical University, Shenyang, China (Wang)

Photon Medical Research Center, Department of Neurosurgery, Hamamatsu University School of Medicine, Hamamatsu, Japan (Yamamoto)

Reprint requests: Seiji Yamamoto, MD, PhD, Photon Medical Research Center, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu 431–3192, Japan. E-mail: seijiy@hama-med.ac.jp

Received, March 11, 2009.

Accepted, January 11, 2010.

Copyright © by the Congress of Neurological Surgeons