No abstract

The aim of this study was to analyze, using electron microscopy, the morphological alterations that progressively appear in the pancreas of rats with acute pancreatitis induced by bile-pancreatic obstruction over 48 h. In addition, in order to ascertain the capability of pancreas regeneration at different stages of pancreatitis, the distribution of pancreatic cells throughout the different phases of the cell cycle was also analyzed by flow cytometry using propidium iodide staining. Interstitial edema, macrophage infiltration, vacuolization, and dilatation of endoplasmic reticulum were observed from 1.5 h after obstruction onward. Interestingly, cell cycle studies showed an increased proportion of S-phase cells at early stages of pancreatitis (1.5 h after obstruction), which leads to a significant increase in cells in G2/M phase 12 h after pancreatic obstruction. Histological studies revealed severe alterations in pancreas of rats with obstruction maintained over 48 h which affects the nuclear structure. Intracellular disorganization, apoptosis, and focal necrosis were observed at this stage. Furthermore, flow-cytometric analysis of cell DNA contents showed a significant decrease in the proportion of S and G2/M cells and a significant increase in G0/G1 cells, suggesting an arrest of almost all cells in quiescent states. These results suggest that rat pancreas cells are able to recover during the first 12 h after pancreatic obstruction. However, the gland would lose its ability to regenerate if the obstruction was maintained for longer periods.

Trypsinogen and amylase content has been analysed by flow cytometry in individual pancreatic cells from rats with acute pancreatitis induced by pancreatic duct obstruction, from the earliest stages to 48 h after obstruction. Parallel morphological studies of the pancreas by electron microscopy and analysis of various parameters for the diagnosis of pancreatitis will allow research into the possible relationship between intracellular enzyme load and the severity of pancreatitis. Progressive increases in amylase activity in ascites and plasma, the volume of ascites, haematocrit, vacuolization, oedema and macrophage infiltration were observed between 1.5 h and 12 h after duct obstruction. A progressive increase in enzyme content was also observed in individual acinar cells at this stage. Interestingly, the larger increase was for trypsinogen, so that the trypsinogen/amylase ratio was significantly increased in all acinar cells by 12 h after duct obstruction. This represents a risk factor for the development of pancreatitis. Sections of pancreas taken from rats that had duct obstruction for 48 h showed massive dilatation and disorganization of the endoplasmic reticulum, focal apoptosis and necrosis. These severe alterations would affect enzyme synthesis, as reflected by the significant decrease in the intracellular enzyme load observed at this stage. However, not all acinar cells were affected equally by the damage induced by pancreatitis: R(1) cells appeared to be more sensitive than R(2) cells. In conclusion, intracellular accumulation of digestive enzymes occurs at early stages of pancreatitis, and this effect is proportionally greater for trypsinogen, a finding that could explain the degree of severity achieved in the course of pancreatitis.

Trypsinogen and amylase content has been analysed by flow cytometry in individual pancreatic cells from rats with acute pancreatitis induced by pancreatic duct obstruction, from the earliest stages to 48 h after obstruction. Parallel morphological studies of the pancreas by electron microscopy and analysis of various parameters for the diagnosis of pancreatitis will allow research into the possible relationship between intracellular enzyme load and the severity of pancreatitis. Progressive increases in amylase activity in ascites and plasma, the volume of ascites, haematocrit, vacuolization, oedema and macrophage infiltration were observed between 1.5 h and 12 h after duct obstruction. A progressive increase in enzyme content was also observed in individual acinar cells at this stage. Interestingly, the larger increase was for trypsinogen, so that the trypsinogen/amylase ratio was significantly increased in all acinar cells by 12 h after duct obstruction. This represents a risk factor for the development of pancreatitis. Sections of pancreas taken from rats that had duct obstruction for 48 h showed massive dilatation and disorganization of the endoplasmic reticulum, focal apoptosis and necrosis. These severe alterations would affect enzyme synthesis, as reflected by the significant decrease in the intracellular enzyme load observed at this stage. However, not all acinar cells were affected equally by the damage induced by pancreatitis: R(1) cells appeared to be more sensitive than R(2) cells. In conclusion, intracellular accumulation of digestive enzymes occurs at early stages of pancreatitis, and this effect is proportionally greater for trypsinogen, a finding that could explain the degree of severity achieved in the course of pancreatitis.

Cholecystokinin (CCK) has been suggested to be a contributory mediator in acute pancreatitis (AP). The aim of the present study was to assess the role of CCK in the development of oxidative stress at different stages of AP induced by pancreatic duct obstruction (PDO) in rats, using L364,718 (a potent CCK-receptor antagonist) to block CCK action. Intra-acinar oxygen free radical (OFR) generation was analysed by flow cytometry using dihydrorhodamine-123 as a fluorogenic dye. Parallel measurements of pancreatic levels of reduced glutathione (GSH) and of several parameters for the diagnosis of AP were performed in both untreated PDO rats and PDO rats receiving L364,718 (0.1 mg x 12 h(-1) x kg(-1)). Diagnosis parameters indicated a greater severity of AP in rats treated with the CCK antagonist. The increase in OFR generation observed in acinar cells up to 12 h after inducing AP was triggered at an earlier stages and reached higher values when L364,718 was administered. Accordingly, greater pancreatic GSH depletion was observed in rats with AP treated with the CCK antagonist. Two populations of acinar cells that were differentiated by flow cytometry, R1 and R2, showed similar behaviour with regard to OFR generation in PDO rats; however, R1 cells showed greater sensitivity to L364,718 administration, and thus OFR production was increased in R1 cells earlier than in R2 cells. In conclusion, CCK blockade anticipates and enhances the amount of OFR produced in acinar cells as a consequence of AP, thus leading to earlier development of and more severe disease. The detrimental effect of L364,718 in AP induced by PDO suggests that plasma CCK does not play a major role in the development of this AP model.