Optical mapping of myocardial reactive oxygen species production throughout the reperfusion of global ischemia

Abstract

Reactive oxygen species (ROS) are short-lived, highly reactive chemical entities that play significant roles in all levels of biology. However, their measurement requires destructive preparation, thereby limiting the continuous measurement of ROS in a living tissue. We develop an optical mapping system to visualize ROS production in an isolated and perfused rat heart. By staining the heart with dihydroethidium (DHE), a 532-nm laser beam is directed to the epicardial surface, where we collect the red fluorescence (>600 nm) for semiquantitative analysis. With this system, ROS production as well as ventricular pressure and ECG in isolated perfused rat hearts are monitored throughout the reperfusion of global ischemia. Ischemia would decrease myocardial ROS production, while reperfusion would immediately result in sustained ROS overproduction. Optical mapping would provide information regarding the spatial distribution and temporal evolution of myocardial ROS production, which would enhance knowledge of the role of free radicals in cardiovascular biology.

Biophysical properties and functional consequences of reactive oxygen species (ROS)‐induced ROS release in intact myocardium

“…The experimental approach described in this manuscript assumes a regionally homogeneous distribution of O 2 − under baseline conditions of normal perfusion. Similar assumptions have been made by other investigators using similar techniques in the past (Salama et al 1987; Lu et al 2006). Moreover, our experimental design allowed for measurements of relative (not absolute) changes in ROS during the course of an experiment.…”

“…DHE loading of perfused rat hearts for 20 min resulted in a robust and sustained increase in background fluorescence intensity. Following dye washout, background fluorescence remained stable for at least 2 h. As such, H 2 O 2 ‐mediated changes in O 2 − levels across the heart could be quantitatively measured as a background‐subtracted and normalized value relative to the steady‐state, pre‐H 2 O 2 levels on a pixel‐by‐pixel basis, as has been performed by Lu et al (2006).…”

“…Dihydroethidium (DHE) fluorescence imaging was used to measure relative changes in O 2 − levels within the intact heart, as reported previously in ischaemia–reperfusion injury (Lu et al 2006). DHE, which is oxidized by O 2 − anions to ethidium, has been extensively exploited by multiple groups to monitor changes in myocardial O 2 − levels in response to oxidative injury (Kevin et al 2003; Camara et al 2004; Riess et al 2004; Lu et al 2006). Following cannulation, hearts were allowed to stabilize for 10 min at physiological temperature.…”

Optical imaging of mitochondrial function uncovers actively propagating waves of mitochondrial membrane potential collapse across intact heart

“…Similar assumptions have been made with regards to NADH and ROS levels by other investigators using similar techniques in the past [33, 34]. Because this is not a ratiometric imaging technique, we can only report relative (not absolute) changes in ΔΨ m during the course of an experiment.…”

Real-Time Fluorescence Measurements of ROS and [Ca2+] in Ischemic / Reperfused Rat Hearts: Detectable Increases Occur only after Mitochondrial Pore Opening and Are Attenuated by Ischemic Preconditioning

“…Others have monitored dihydroethidium (DHE) surface fluorescence of the perfused heart to detect ROS production during I/R [14,15]. However, major concerns have been expressed over the use of DHE as a ROS probe, both in terms of whether the fluorescent species monitored really detects superoxide [16] and its sensitivity to changes in mitochondrial membrane potential [17].…”