The Detection Of Reactive Oxygen Species In Single Ventricular Myocyte By Chemiluminescence Method

Objectives: The aims of this study were 1. Establishment of method to detect the concentrations of reactive oxygen species (ROS) in single adult rat cardiomyocyte with Ion Imaging System; 2. To test the availability of the method, we measured the changes of intracellular ROS concentrations using double wavelengths ratio by imitating the models for single adult rat cardiomyocyte ischemia reperfusion and oxidative stress, in the presence or absence of glutathione (GSH), a kind of antioxidant .Methods: 1. Adult rat cardiomyocytes were obtained by enzymatic dissociation procedure. Cardiomyocytes were suspended in normal Tyrode's solution in which the Ca2+ concentration was 1.8mM. The cells were incubated with 10μM 2',7'-Dichlorofluorescin diacetate (DCFH-DA) for loading of 2',7'-Dichlorofluorescin (DCFH) for 15 min at 35℃. Then the cells were washed with the normal Tyrode's solution three times to remove remained extracellular dye. 2. The oxidative stimulus was initiated by the addition of 10mM H2O2 to each dish at room temperature. Fluorescence images of the single adult rat cardiomyocyte were recorded at excitation wavelengths from baseline 340nm to 580nm with the interval of 20nm by the ion imaging system. The changes of fluorescence signal were analyzed by using dedicated image software. By these protocols to find the two excitation wavelengths which made the fluorescence signal intensities were opposite, one is the biggest and the other is the smallest. 3. Imitated the model for single adult rat cardiomyocyte ischemia reperfusion: cardiomyocyte was perfused with the mimic ischemia Tyrode's solution (in which the Lactic Acid was 20mM, pH was 6.6 ) for 20 minutes, then the cardiomyocyte was reperfused with the normal Tyrode's solution. Established cardiomyocyte the model for oxidative stress by perfusing with 10 mM H2O2..Intracellular ROS concentrations (expressed as double wavelengths ratio) were measured using ion imaging system in ischemia reperfusion and oxidative stress conditions , in the presence / absence of GSH.Results: 1. The two excitation wavelengths to detect the concentrations of reactive oxygen species (ROS) in single adult rat cardiomyocyte in which the two fluorescence intensities were changed in opposite were 480nm and 420nm. The values of the fluorescence intensity at 480nm and 420nm were the biggest and smallest, respectively. So we used F480/F420 to express the intracellular ROS concentrations. 2. We imitated adult rat cardiomyocytes ischemia reperfusion and applied GSH to detect the changes of intracellular ROS concentrations: during perfusion with the normal Tyrode's solution (control group), the intracellular ROS fluorescence intensity (F480/F420) was stable. After mimic ischemia, intracellular ROS fluorescence intensity (F480/F420) increased progressively to 115.27±4.52% of pre-ischemic at the end of 20 minutes...