Background
Reactive oxygen species (ROS) have been implicated in the pathogenesis of various neurological disorders, such as ischemia, trauma, and degenerative disease [
1‐
3]. Ischemic stroke can induce pathophysiological events such as amino acid excitotoxicity, ionic imbalance, and oxidative stress [
4,
5]. Cumulative evidence suggests that ROS are important mediators of cell injury in the ischemic brain [
6,
7]. Oxidative stress can primarily lead to the formation of superoxide radical (O
2 · −) and nitric oxide (·NO), and then, highly reactive ROS including hydroxyl radical (OH · −) and peroxynitrite (ONOO−) are generated by ischemia and reperfusion [
8,
9]. ROS which induce lipid peroxidation, proteins, and DNA damage in ischemic brain tissue trigger molecular pathways leading to necrosis, apoptosis, and neuroinflammation with subsequent neuronal death and memory and/or motor dysfunction. Therefore, it is important to know the ROS generation in the brain following ischemia/reperfusion in the pathophysiologic process leading to ischemic tissue damage.
To obtain direct evidence of ROS generation in disease pathogenesis, highly sensitive and specific optical probes (fluorescent, luminescent, or chemiluminescent probes) for detecting ROS are being developed [
10‐
13]. However, it is very difficult to directly detect ROS in the brain because they are extremely reactive and their life span is very short. In the brain during or after ischemia, ROS generation has been detected by using electron spin resonance and a microdialysis method [
14,
15]. Murakami et al. [
16] have reported that hydroethidine can detect the O
2 · − produced by occlusion of the middle cerebral artery using mutant mice with a heterozygous knock-out gene encoding mitochondrial manganese superoxide dismutase (Mn-SOD). The areas of ROS in both the ischemic core and the peri-infarct area in the permanent and transient middle cerebral artery occlusion (tMCAO) model were detected by using a novel fluorescence probe [
17]. Striatal ROS generation in ischemic brain was measured by using salicylate, a hydroxyl radical trapping agent, and a microdialysis method [
18].
We have recently reported the usefulness of the radical trapping radiotracer, [
3H]-labeled
N-methyl-2,3-diamino-6-phenyl-dihydrophenanthridine ([
3H]hydromethidine) for detecting ROS generation in the brain [
19]. We have already shown that [
3H]hydromethidine is converted to oxidized products by a superoxide radical (O
2 · −) and a hydroxyl radical (OH · −). Brain ROS generation induced by cerebral microinjection of sodium nitroprusside could be autoradiographically detected by intravenous administration of [
3H]hydromethidine. We concluded that [
3H]hydromethidine rapidly and freely penetrated into the brain where it was rapidly converted to oxidized forms, which were trapped there in response to ROS.
In the present study, we investigated the spatiotemporal changes of ROS generation after ischemia/reperfusion in the tMCAO model mouse using the radical trapping radiotracer [3H]hydromethidine. In addition, the effects of 1,3-dimethyl-2-thiourea (DMTU), a hydroxyl radical scavenger, on ROS generation and ischemic damage following tMCAO in mice were also assessed to determine whether its effect might be responsible for hydroxyl radical scavenging action.
Discussion
In the present study, we tried to detect the spatiotemporal change of ROS generation in mouse brain following ischemia/reperfusion. [
3H]Hydromethidine, a radical trapping radiotracer, was used for ROS detection. In addition, in order to determine whether accumulation of radioactivity after injection of [
3H]hydromethidine is attenuated by a free radical scavenger in the ischemic region, we investigated the effects of DMTU, a hydroxyl radical scavenger, on ROS generation and brain injury after tMCAO in mice. As shown in Figs.
2 and
3, we found that tMCAO produced a marked increase in ROS production, which peaked at 5 h after reperfusion. Accumulation of radioactivity, indicating ROS generation, was observed in the ipsilateral hemisphere of the tMCAO model mouse. In the striatum, the accumulation was first observed 1 h after reperfusion. The increase of radioactivity was attenuated at 2 h after tMCAO and then reached maximum at 5 h. The accumulation of radioactivity was maintained until 7 h after tMCAO although the level was lower than that at 5 h. The time course of cortical accumulation was also similar to that of the striatum. Hippocampal and thalamic accumulation was also observed at 5 and 7 h after reperfusion in some mice.
Many techniques have been used to detect the generation of ROS in the brain. It is generally detected by indirectly measuring secondary products such as oxidized protein, peroxidized lipids, and oxidized DNA. Direct detection of ROS has been done by using chemiluminescence, fluorescence, spin trapping, and microdialysis [
15,
18,
22]. In vivo microdialysis study showed that the formation of 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA), products of salicylate trapping of hydroxyl radicals, increased in the striatum during acute ischemia and reperfusion [
18]. Increase of 2,3-DHBA and 2,5-DHBA was observed during ischemia, with high levels being maintained until 2 h after reperfusion.
Temporal and spatial profiles of O
2 · − after permanent focal ischemia have been examined in mice using in situ staining with dihydroethidium (DHE) [
23]. They showed that O
2 · − generation increased 1 h after MCAO in the ischemic core of the brain and in the boundary area of the infarct zone between 3 and 6 h after permanent focal brain ischemia. Fluorescence imaging using fluorescence probe, 2-[6-(40-hydroxy)phenoxy-3H-xanthen-3-on-9-yl] benzoic acid (HPF) revealed areas of enhanced HPF fluorescence in both the ischemic core and peri-infarct area at 4 h after MCAO in both the permanent and transient MCAO models; the regions generating ROS were more widespread than the areas of ischemic damage [
17]. These previous and our findings suggest that ROS generation might be observed at several hours after ischemia/reperfusion. The accumulation of [
3H]hydromethidine is thought to be due to ROS production because [
3H]hydromethidine was rapidly converted to oxidized forms, which were trapped there in response to the production of ROS. The amount of trapped oxidized form were mainly dependent upon three factors, the delivery process from plasma (regional cerebral blood flow), the oxidation rate in the brain, and the washout rate of unoxidized [
3H]hydromethidine from the brain. Therefore, in ischemic brain, the accumulation of [
3H]hydromethidine might be influenced by cerebral blood flow and BBB permeability. It has been reported to decrease cerebral blood flow in the ischemic side for more than several hours after ischemia/reperfusion [
24,
25]. These reports suggest that the accumulation of [
3H]hydromethidine in the ischemic side was independent of regional cerebral blood flow because regional cerebral blood flow seems to decrease in the ischemic area. On the other hand, BBB disruption has been observed at several hours after ischemia/reperfusion [
26,
27]. In the present study, we found that BBB disruption was not observed at 1 or 5 h after ischemia/reperfusion in the tMCAO (90 min) model. These data indicate that the accumulation of [
3H]hydromethidine at 1 or 5 h after tMCAO might not be affected by BBB disruption. In contrast, the accumulation of [
3H]hydromethidine at 7 h might be influenced by BBB disruption. However, DMTU reduced the accumulation of [
3H]hydromethidine although it could not improve striatal BBB disruption at 7 h after ischemia/reperfusion. Thus, our result suggests that the accumulation of [
3H]hydromethidine in the ischemic area could be due to ROS generation rather than input function such as regional blood flow or BBB disruption. Our interesting finding was that ROS generation was attenuated by the control level at 2 h after reperfusion in the striatum and cortex, ischemic core area. We clearly showed that the accumulation was first observed at 1 h after reperfusion. The increase of radioactivity was attenuated at 2 h after reperfusion, and then, it peaked at 5 h. Antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GSHP), have been reported to play an important role for ROS generation of tissue affected by ischemia/reperfusion. An increase in GSHP was observed in the parietal cortex at 1 h after ischemia/reperfusion in the tMCAO rat model [
28]. These previous findings suggest that ROS generation might be regulated by endogenous antioxidant activity in the brain after ischemia/reperfusion.
ROS such as O
2 · −, OH · −, and ONOO− are highly reactive molecules that have been implicated in the pathogenesis of secondary neuronal damage after ischemia/reperfusion [
29]. The superoxide radical (O
2 · −) is usually the primary ROS produced and is subsequently converted into hydrogen peroxide (H
2O
2) through spontaneous or SOD-catalyzed dismutation. The reaction of O
2 · − and NO generates the powerful oxidant ONOO−. Reaction of H
2O
2 and ONOO− can generate the highly reactive hydroxyl radical (OH · −) [
30,
31]. The accumulation of radioactivity observed in the ipsilateral hemisphere suggests that [
3H]hydromethidine might be oxidized by ROS and then the oxidized form was trapped in the tissue. We recently reported that [
3H]hydromethidine reacted with O
2 · − and OH · − but not H
2O
2 from the results of an in vitro study. Considering these in vitro results, the accumulation of radioactivity in the brain of our ischemic model strongly suggests that the oxidized form of [
3H]hydromethidine produced by OH · − or O
2 · − was trapped in the brain.
We also investigated the effects of DMTU, a hydroxyl radical scavenger, on ROS generation and brain injury after tMCAO in mice. DMTU is often used as a free radical scavenger and can reduce brain damage due to ischemia [
32]. It has also been reported that DMTU reduced brain infarction and edema after permanent MCAO in rats [
33]. We also showed that DMTU could attenuate brain infarction at 24 h after ischemia/reperfusion in the mouse focal ischemia model. In addition, DMTU attenuated the accumulation of radioactivity in the ipsilateral side of the brain after reperfusion. These results suggest that the accumulation of radioactivity induced by tMCAO is to be mainly due to oxidative conversion of [
3H]hydromethidine by ROS such as OH · −. On the other hand, DMTU increased radioactivity concentration of [
3H]hydromethidine in the contralateral side at 7 h after ischemia/reperfusion. The increase of radioactivity concentration might be due to increased cerebral blood flow induced by DMTU treatment. Further studies will be needed to assess the effect of DMTU on cerebral blood flow.
Reactive nitrogen species such as NO and ONOO− play an important role in the process of cerebral ischemia/reperfusion injury [
34]. Superoxide can react with NO to produce ONOO− that causes mitochondrial dysfunction, and this leads to brain damage. We recently found that [
3H]hydromethidine was oxidized by ONOO− in in vitro study (in-house data). Therefore, it is possible that [
3H]hydromethidine was oxidized by ONOO− in the brain after ischemia/reperfusion. In addition, lipid peroxidation of the cell membrane induced by ROS is considered to oxidize [
3H]hydromethidine in ischemia/reperfusion in the brain. Thus, it might be difficult to identify the involvement of specific species in brain injury after ischemia/reperfusion because [
3H]hydromethidine is oxidized by ROS such as O
2 · −, OH · −, or ONOO−. The present results suggest [
3H]hydromethidine to be a useful probe for assessing the role of ROS in the ischemic brain state. Time-dependent change of ROS could be detected in the same animals using positron-labeled hydroethidine-related compounds because hydromethidine can be labeled using [
11C]methylation instead of [
3H]-labeling. Further studies including small-animal PET studies using [
11C]hydromethidine should be very useful for studying the pathophysiological roles of ROS in diseases such as ischemic stroke.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
KA, MT, NT, NI, and SM participated in the design of the study and carried out the animal studies. MI performed the tMCAO. TR participated in the MRI scans. KF and KM participated in the radiochemical synthesis. KA drafted the manuscript. OI participated in the study design and drafted the manuscript. All authors read and approved the final manuscript.