Influence of the CCK-antagonist loxiglumide on bile-induced experimental pancreatitis.

The present study investigates the effect of CCK-receptor blockade on taurocholate-induced pancreatitis in rats using the potent antagonist loxiglumide. Intraperitoneal administration (50 mg/kg) of loxiglumide began 3 h before, or 10 min or 3 h after induction of pancreatitis. Mean survival times of the experimental groups were 31.2, 23.6, and 20.5 h, respectively, compared to 18.2 h for controls. Survival for 24 h after induction of pancreatitis was significantly improved when the antagonist was given 3 h before, but not in the time periods after induction. After 72 h, survival time was not significantly altered in any of the groups. Furthermore, amylase and lipase levels quantified 10 h after induction of pancreatitis in ascites, blood, or tissue did not indicate a significant difference, nor was improvement in survival seen when the CCK-antagonist was tested in rats receiving a basal treatment with intravenous volume substitution, peritoneal lavage, and protease inhibition. We conclude that CCK-receptor blockade does not improve the final outcome of bile-induced pancreatitis in the rat, even if treatment is started before induction of pancreatitis.

Camostate (FOY-305) improves the therapeutic effect of peritoneal lavage on taurocholate induced pancreatitis.

The effect of peritoneal lavage with the addition of camostate to the lavage fluid on the outcome of taurocholate pancreatitis in rats was studied. Camostate is a low molecular weight protease inhibitor which has been developed recently. Peritoneal lavage was performed for 12 hours and camostate was added to the lavage fluid in five concentrations. At 0.1 mg/ml the survival rate increased significantly (11 of 20 v controls 4 of 20, p less than 0.05); the maximal effect was observed at 0.2 mg/ml, and no effect was seen at 0.01 and 0.05 mg/ml. Adverse effects occurred at 0.5 mg/ml. The addition of 0.1 mg/ml camostate significantly reduced the acidosis in arterial blood: mean (SD) pH 7.30 (0.035) v controls 7.23 (0.054) (n = 9, p less than 0.01) and arterial base excess: -15.4 (1.26) mmol/l v controls: -17.4 (2.51) mmol/l (n = 9, p less than 0.05). There was no difference, however, in plasma amylase activity and in the histological degree of specific tissue damage to the pancreas. A combination of intravenous camostate and lavage with the addition of camostate to the lavage fluid yielded a significantly improved survival compared with treatment with intravenous camostate alone (10 out of 16 animals v intravenous camostate alone, two out of 16, p greater than 0.01). We conclude that lavage with camostate significantly improves the prognosis of severe necrotising pancreatitis in rats.

LR-White and LR-Gold resins for postembedding immunofluorescence staining of laminin in mouse kidney.

This work describes a method for the immunolocalization of laminin on 1 micron-thick tissue sections using a postembedding immunofluorescence technique. Embedding of unfixed or formaldehyde-fixed mouse renal cortex in either of the acrylic resins LR-White or LR-Gold permitted reliable postembedding immunofluorescence staining for laminin. LR-White was heat-cured at 50 degrees C whereas LR-Gold was polymerized at -25 degrees C. A stronger immunostaining for laminin was obtained from tissue embedded in polymerized LR-Gold compared with the staining from tissue embedded in LR-White. Prerequisites for adequate postembedding immunostaining are the partial dehydration of the tissue (maximum ethanol concentration, 70%) and pepsin treatment of the tissue sections prior to performing the immunostaining reactions.

Localization of fucosyl moieties in the mouse renal cortex by lectin histochemistry using the fucose binding lectins LTA and UEA I and by autoradiography using 3H-labelled fucose.

The binding patterns of the two fucose binding lectins, Lotus tetragonolobus (LTA) and Ulex europeus I (UEA I) were investigated using fluorescence lectin histochemistry on the unfixed renal cortex of the mouse (NMRI) embedded in LR-Gold. The fluorescence staining results were compared with the autoradiographic localization of the incorporation of radioactive fucose into the renal cortex. For this study the turnover of incorporated 3H-fucose in the renal cortex was investigated 30 min, 2 h and 8 h after application. The localization of the radioactive fucose within the renal cortex corresponded well to the labelling pattern observed for lecting histochemistry using LTA. In contrast, with UEA I, no binding sites for this lectin could be observed. The results of our investigation clearly showed that fucosyl moieties in the renal cortex of the NMRI mouse are recognized by the fucose binding lecting LTA, but not by UEA I and that postembedding fluorescence histochemistry with LTA on the LR-Gold embedded kidney is a suitable technique for the localization of fucosyl moieties at the light microscopical level.

Endocrine pancreatic function during atrophy of the exocrine gland.

Serum levels of glucose, insulin, and gastric inhibitory polypeptide (GIP) in response to intraduodenal and intravenous glucose loads have been examined in rats with exocrine pancreatic atrophy induced by feeding them a copper-deficient diet supplemented with D-penicillamine for 10-12 weeks. Whereas pancreatic weight and protein and trypsin content were drastically reduced as compared with pair-fed controls, insulin content was not significantly different between experimental (1.10 U) and control (1.40 U) rats. The intravenous glucose infusion was given in a dose (1.2 g/kg/h) simulating the glucose concentrations observed in response to an intraduodenal glucose load of 2.0 g/kg body weight. Both basal and stimulated insulin concentrations were lower in experimental animals as compared with controls. However, if the relative insulin response is considered, insulin secretion was almost identical in experimental and control animals. Both groups released significantly more insulin after intraduodenal glucose load than after intravenous glucose application. It is concluded that the entero-insular axis is intact in rats with exocrine pancreatic atrophy.

Light and electron microscopical postembedding lectin histochemistry for WGA-binding sites in the renal cortex of the mouse embedded in polyhydroxy aromatic resins LR-White and LR-Gold.

This work describes a technique which permits study of the postembedding lectin histochemistry for WGA-binding sites at the light and electron microscopical level on the same resin embedded tissue without removing or etching of the resin. Unfixed kidney pieces or kidney pieces fixed in 4% formaldehyde were embedded in the hydrophilic polyhydroxy aromatic resins LR-Gold and LR-White, following dehydration in up to 70% ethanol, 90% ethanol or 100% ethanol. LR-Gold was cryopolymerised at -25 degrees C using the light sensitive initiator benzil, whereas LR-White was heat-cured at +50 degrees C. The localisation of WGA-binding sites at the light microscopical level was investigated using FITC-labelled WGA. The ultrastructural localisation of WGA-binding sites was performed using 15 nm gold-labelled WGA. The best fluorescence staining results were obtained on fixed or unfixed tissue dehydrated in up to 70% ethanol and embedded in LR-Gold. At the ultrastructural level, the best staining results for WGA-binding sites were seen on tissue sections, dehydrated in up to 90% ethanol prior to embedding in LR-Gold.

Methodological dependence in the ultrastructural immunolocalization of laminin in tubular basement membranes of the mouse kidney.

The ultrastructural localization of the basement membrane glycoprotein laminin was investigated in basement membranes of proximal tubules of the mouse kidney. The localization of laminin was determined using two different immunoperoxidase and one immunogold preembedding technique and one immunogold postembedding technique on unfixed and formaldehyde fixed tissue. Strong differences in the immunolocalization for laminin were found in the lamina densa of the tubular basement membrane using different techniques. After preembedding immunostaining for laminin using IgG--PO as secondary antibody, a positive reaction for the lamina densa was found in the formaldehyde fixed as well as in the unfixed kidney. After preembedding immunostaining for laminin using Protein-A--PO, staining of the 1. densa was seen in the unfixed, but not in the fixed kidney. It was striking that no clear immunoreaction in the 1. densa of the tubular basement membrane was seen in either the fixed or unfixed tissue after preembedding immunostaining for laminin using protein A-gold. With a direct postembedding immunogold technique laminin was localized only in the 1. fibroreticularis and the 1. rara but not in the 1. densa of basement membranes of proximal tubules of the unfixed and the fixed kidney.