Abstract
In the present study, different histological staining methods specific for B, A and D cells of the pancreatic islets were applied. The effect of different factors, including fixation, pH, temperature and time, were examined. Thiosulfation of the aldehyde fuchsin procedure for B cells and the aqueous silver nitrate method of Grimelius method for A cells were found to be more specific and reproduceable than the other methods examined. Toning of sections with gold chloride preserved the silver grain for subsequent staining with aldehyde fuchsin and allowed the study of both A and B cells simultaneously in the same section, by light microscopy. Immunofluorescent and peroxidase labelled antibody methods were investigated. The immunoperoxidase method was found to be the method of choice for subsequent histological studies of pancreatic islets. In this method endogenous peroxide activity was blocked with a 0. 5% solution of hydrogen peroxidase in methanol. The method used incorporated blocking of non specific tissue binding sites with normal serum, incubation with high dilutions of antisera, and establishment of the optimal conditions for the histochemical reaction with the substrate. The method was modified for the localization of two hormone-producing cells in a single histological section. The first antigen was localized by the indirect method and the antisera were removed from the sections by elution, leaving the coloured reaction products identifying the antigen sites. The second antigen was localized similarly, using substrate that developed a reaction product of a different colour. The sequential technique was used identifying the first cell with 4Cl -1 -naphthol. The coloured reaction product was photographed and removed by placing the sections in xylene. The first antiserum was removed by elution. The sections were then stained for the second antiserum, using the same or different substrate, and rephotographed. The cellular composition of the pancreatic islets in the rat, mouse, rabbit, guinea pig and man were investigated using the immuno-cytochemical technique to demonstrate pancreatic islet hormones. It was shown that each species has its own specific topographic arrangement of cells within the islets. In addition to insulin and glucagon producing cells (B and A cells), islets of Langerhans contained other cell types which produce somatostatin (D or A1 cells) and pancreatic polypeptide (PP cells). In all species examined B cells were the most numerous cell type. Whereas B, A and D cells were found to be present in all islets throughout the pancreas, the PP cells were absent in some of the islets. Whenever the A cells were found, they were accompanied by D celIs. Gastric inhibitory polypeptide (GIP) (a member of the family of the gut hormones) was demonstrated in the gastrointestinal tract of different species, including the rat, mouse, rabbit, ferret and man. In all species examined the GIP immunoreactive material was also found to reside in the glucagon cells of the pancreas. The immunoreactive GIP was not observed within glucagon cells of man. Pancreatic islet tissue from lean and obese mice and Zucker rats of different ages were examined with histological and immunocytochemical techniques. In all obese animals hyperplasia of B cells was observed. No consistent changes were noted with glucagon, somatostatin or PP cells in obese normaglycemic animals. The number of glucagon cells were increased in obese hyperglycemic mice with 57BL/6J background. The quantitative changes were accompanied by qualitative ones, consisting in a displacement of the cell populations from their characteristic location in the islet. The number of GIP cells from different regions of gastrointestinal tract in obese mice (57BL/6J) and Zucker rats was counted and compared with their lean littermates. No significant difference was observed. The immunoreactive GIP within glucagon cells of the pancreas showed no difference between the obese animals when compared with their lean littermates. In an attempt to produce glucagon deficiency in rats, the effect of neutral red and cobalt chloride was studied and found to be ineffective. Rats immunized against glucagon were studied. The titre of antisera, the level of blood glucose, plasma FFA, triglyceride and insulin were examined. Glucose and arginine tolerance tests and cyto-logical examination of pancreatic islet cells were made. No significant differences were found between test and control animals. This was considered to be due to poor antisera titre. Immunocytochemical studies of a human pancreatic islet cell tumour were undertaken and a D cell tumour (somatostatinoma) was observed. This case had a different clinical presentation from those previously reported. It was concluded that the somatostatin secreted by the tumour cells are physiologically inactive. Finally the cellular composition of the pancreatic islets in two hypoglycemic patients was studied. One of them showed a normal islet system with an increase in the number of somatostatin and glucagon cells. The other case was found to have abnormal features of pancreatic islet cells. The islet cells were separated from each other and were scattered among the exocrine cells.