Cerium-based Histochemical Demonstration of Oxidative Stress in Taurocholate-induced Acute Pancreatitis in Rats: A Confocal Laser Scanning Microscopic Study

Abstract

Direct in vivo histological detection of oxygen-derived free radicals (OFRs) in inflammatory conditions is not fully resolved. We report an application of cerium histochemistry (in which capture of OFRs by Ce atoms results in laser-reflectant cerium-perhydroxide precipitates) combined with reflectance confocal laser scanning microscopy (CLSM) to demonstrate the evolution of oxidative stress in taurocholate-induced acute pancreatitis (AP) in rats. Animals were perfused with CeCl3in vivo and cryostat sections of pancreata were studied by CLSM. Vascular endothelium was immunolabeled for PECAM-1. OFR production by isolated polymorphonuclear leukocytes (PMNs) incubated in vitro with CeCl3was quantified by image analysis. In the pancreas, strong OFR-derived cerium reflectance signals were seen in acinar cells at 1-2 hr, capillaries and small venules were frequently engorged by cerium precipitates, and adherent PMNs presented weak intracellular reflectance signals. At 8-24 hr, acinar cell OFR production decreased, whereas adherent/transmigrated PMNs displayed abundant intra- and pericellular reflectance. PECAM-1 expression was unchanged. PMNs from ascites or blood showed significant ( p<0.01) time-dependent OFR production, plateauing from 2 hr. The modified cerium capture/CLSM method allows the co-demonstration of in vivo oxidative stress and cellular structures labeled with fluorescent markers. In vivo oxidative stress was shown histologically for the first time in experimental AP.