The electron microscopic cytochemical method, using CeCl3 as a capture agent to from electron - dense products of Ce - H2O2 complex, was adapted to localize ultrastructurally H2O2 produced by endotheelial cells of vessels afters hypoxia - reperfusion. Rats were perfused via their left ventricle by hypoxia buffer for 5, 10, and 20min respectively followed by 10min reperfusion wiith oxygenated buffer containing CeCl3. The ultrastructure alteration in endothelial cells of a common carotid artery was found to be more serious when the hypoxia duration was getting longer. After 20min hypoxia perfusion, the endothelial cells even dropped away from the endothelium. At the same time, the eelectron dense precipitate appeared on the luminal face of endotheial cells. The longer hypoxia lasted, the thicker precipitate formed. The area/length ratio (nm2/nm) of the precipitate was assessed by imaging analyzer. The precipitate were 150. 3±11. 4, 165. 6±9. 8 and 203. 5±6. 8nm2/nm(n = 5) respectively in 5, 10 and 20min group. The differences betweeen 20min and 5, 10min groups were very significant (P <0. 01), and the signficant difference between 5min and 10min group was also showed (P<0. 05). No precipitate appeared when CeCl3 did not be added in the reperfusion buffer. When catalase was added into perfusion buffers, the precipitate was decreased apparently or almost disappeared. Electron microscopic x- ray microanalysis of the endothelial electron dense precipitate revealed three characteristic peaks of Ce, indicating the presence of Ce. In connection with that catalase eliminated significantly the precipitate, it indicated that the precipitate was formed by H2O2 and Ce .This method may provide a new way to localize peroxide free radicals at the ultrastructural level.
Acta Biophysica Sinica
Hypoxia - reperfusion H_2O_2 CeCl_3 Vascular endothelial cell Electron cytochemistry