Silver stains demonstrating neuroendocrine cells

During the preimmunohistochemical era, silver stains were an important part of the staining arsenal for identifying certain tissue structures and cell types in tissue sections. Some of them were useful for demonstrating endocrine cells, especially in the gastrointestinal tract. Until the late 1950s, silver stains, particularly those identifying endocrine cells, were accompanied by a number of technical difficulties resulting from uncontrolled staining factors. In the 1960s, new silver stains were developed for endocrine cell types and these stains gave reproducible results. One of the “older” silver stains and two of the “newer” ones are emphasized in this presentation, namely the Masson, the Grimelius and the Sevier-Munger techniques. The Masson stain demonstrates the enterochromaffin (EC, serotonin) cells, the Grimelius stain is a broad endocrine cell marker, and the Sevier-Munger technique demonstrates EC and EC-like cells and the C-cells of the thyroid. Especially in the preimmunohistochemical era, these staining methods often were used for histopathological diagnosis, particularly the Grimelius technique. The silver stains were developed empirically, and with few exceptions the chemical background is not known. Staining protocols are included.     

Endocrine cells of the gastric mucosa of Rana temporaria L

The endocrine cells of the gastric mucosa of Rana temporaria have been studied according to the ultrastructure, the staining properties of the granules with Masson Fontana's and Grimelius' silver methods, silver impregnation of Davenport on deplasticised semithin sections and immunocytochemical techniques. Seven different types of endocrine cells have been described. Six were regarded as belonging to known types: G, A, EC, ECL, D and P cells. One type was considered as unclassifiable.     

Silver stains demonstrating neuroendocrine cells

During the preimmunohistochemical era, silver stains were an important part of the staining arsenal for identifying certain tissue structures and cell types in tissue sections. Some of them were useful for demonstrating endocrine cells, especially in the gastrointestinal tract. Until the late 1950s, silver stains, particularly those identifying endocrine cells, were accompanied by a number of technical difficulties resulting from uncontrolled staining factors. In the 1960s, new silver stains were developed for endocrine cell types and these stains gave reproducible results. One of the “older” silver stains and two of the “newer” ones are emphasized in this presentation, namely the Masson, the Grimelius and the Sevier-Munger techniques. The Masson stain demonstrates the enterochromaffin (EC, serotonin) cells, the Grimelius stain is a broad endocrine cell marker, and the Sevier-Munger technique demonstrates EC and EC-like cells and the C-cells of the thyroid. Especially in the preimmunohistochemical era, these staining methods often were used for histopathological diagnosis, particularly the Grimelius technique. The silver stains were developed empirically, and with few exceptions the chemical background is not known. Staining protocols are included.     

On argyrophil reactions of endocrine cells in the human fetal pancreas

Summary The endocrine cells in the pancreas of five human fetuses with gestational ages of 18–20 weeks were examined by light and electron microscopy with special regard to argyrophil reactions. B-cells and typical A and D-cells were easily identified electron microscopically on the basis of their typical secretory granules. In the Grimelius argyrophil silver stain, a concentration of silver grains over the less electron dense peripheral mantle of the A-cell secretory granules was observed by electron microscopy. In the Hellerström and Hellman modification of the argyrophil Davenport alcoholic silver stain, silver grains were concentrated over the internal structures of the D-cell secretory granules. With this stain an accumulation of silver grains was also seen at the surface of the A-cell secretory granules. The argyrophil reaction of the A-granules was less pronounced than in the D-cells. In addition to B-cells and A- and D-cells, two other types of endocrine cell were observed by electron microscopy. These cells were argyrophil with the silver impregnation method of Grimelius. The electron microscopic findings at least partly explain the frequent overlapping between the two staining methods observed at the light microscope level.This study was supported by the Swedish Medical Research Council (Project No. 102)     

Endocrine cells of the human gastric mucosa

Summary By light and electron microscopy investigation of the human gastric mucosa five types of ultrastructurally different endocrine cells have been detected: 5-hydroxytryptamine storing enterochromaffin (EC) cells, gastrin storing G cells, and functionally undefined ECL, D and D₁ cells. By direct application of Masson's argentaffin reaction as well as of Sevier-Munger's and Grimelius' argyrophil method to electron microscopy specimens, selective deposition of silver grains upon the endocrine granules of such cells was obtained. In particular, only EC cell granules reacted to the argentaffin method, granules of both EC and ECL cells heavily reacted to Sevier-Munger's technique, granules of EC, ECL, G and D₁ cells reacted to Grimelius' technique, while D cell granules failed to react either to argentaffin or argyrophil methods. By the application of the same silver methods to paraffin sections as well as by other selective staining methods for endocrine granules (5-hydroxytryptamine techniques, lead-haematoxylin, HCl-basic dye method), at least four of the above cell types were also identified under light microscope. This opens the way for extensive studies of such cells in conventional histologie specimens.This investigation was supported in part by grant N.70.01022.04 from the Italian Consiglio Nazionale delle Ricerche.     

Development of the diffuse endocrine system in the chicken proventriculus

Summary The development of endocrine cells in the chicken proventriculus has been investigated using light-and electron-microscopy in conjunction with silver and immunocytochemical techniques. The first morphologically detectable endocrine cells were found in 5-day-old embryos by electron microscopy. From the 9th to the 13th day, endocrine cells in contact with the lumen of the organ could be detected both by electron and light (silver impregnation) microscopy. The number of open-type endocrine cells progressively decreased and the number of closed-type increased after this stage. Until the 16th day, endocrine cells were located exclusively in the luminal epithelium, but afterwards they appeared in progressively greater numbers in the compound glands. After hatching, long cytoplasmic processes could be seen in the endocrine cells. Immunoreactivities to regulatory substances appeared in the following order: serotonin (day-14), avian pancreatic polypeptide, glucagon and somatostatin (day-16), bombesin and neurotensin (day-18), and finally, met-enkephalin (day-21).     

鲢,鳙,银鲫和团头鲂肠道内分泌细胞中3种肽激素的免疫组化研究

应用ＡＢＣ免疫组化技术,用抑胃多肽、５－羟色胺和内啡肽３种哺乳动物抗血清对鲢、鳙、银鲫和团头鲂的肠粘膜中内分泌细胞进行了检测。结果４种鱼的肠粘膜中均有抑胃多肽免疫反应内分泌细胞存在,但未发现５－羟色胺和内啡肽的免疫反应。抑胃多肽免疫反应内分泌细胞主要分布在前肠前段,并单个地散布在肠褶顶部上皮细胞与杯状细胞之间,多呈长梭形。本文比较了不同食性鱼类和其他动物肠道抑胃多肽免疫反应内分泌细胞的分布规律,讨论了用５－羟色胺和内啡肽免疫染色的结果。     

The fine structure of the stomach mucosa of the llama (Llama guanacoe)

Summary The epithelium of the fundic region mucosa of the hind stomach in the Llama guanacoe has been studied using morphological and histochemical methods. Morphology suggests that solute and water absorption may occur in the epithelium of the surface and of the foveolae, although this absorption can not be estimated because of the extensive secretion of the gastric glands. The same cells of the surface and foveolar epithelium show numerous secretory granules. The glands reveal neck cells, chief cells, a large number of oxyntic cells, four types of endocrine cells (A-like, ECL, D and EC), brush cells and wandering cells. PAS and Alcian blue reactions for light microscopy suggest a secretion of neutral and acidic mucosubstances in the surface and foveolar epithelium, of neutral mucosubstances only in the neck cells. Periodic acid-thiocarbohydrazide silver proteinate (PA-TCH-SP) reaction for electron microscopy confirms the presence of neutral mucosubstances within the secretory granules of the surface, foveolar and neck epithelial cells. In all these cells, the reaction product is also evident within sacculi and vesicles of the maturing surface of the Golgi apparatus. A positive PA-TCH-SP reaction also occurs on the membrane (and not on the contents) of the Golgi apparatus (maturing surface) and of the secretory granules of the chief cells as well as on the membrane of the Golgi apparatus and of apical vesicles and tubules of the oxyntic cells. In addition, silver granules slightly enhance the electron density of the contents of the secretory granules in the endocrine cells. Morphological and histochemical findings are discussed and compared with results described by others for monogastric mammals.     

Ultrastructure and immunocytochemistry of endocrine cells in the midgut of the desert locust,Schistocerca gregaria (Forskal)

Summary The endocrine cells of the midgut epithelium of the desert locust are found dispersed among the digestive cells and are similar to those of the vertebrate gut. According to their reactivity to silver impregnation techniques and the ultrastructural features of the secretory granules (shape, electron-density, size, and structure) 10 types of endocrine cell have been identified, of which seven are located in the main segment of the midgut or in the enteric caeca, and the other three seem to be present only in the ampullae through which the Malpighian tubules drain into the gut. The endocrine cells have a slender cytoplasmic process that reaches the gut lumen, a feature that supports the receptosecretory nature postulated for this cellular type in insects as well as vertebrates. Antisera directed against mammalian gastrin, CCK, insulin, pancreatic polypeptide and bombesin reacted with some of the endocrine cells. This is the first time that insulin- and bombesin-like immunoreactive cells have been described in the midgut of an insect.     

Light and electron microscopic identification of several types of endocrine cells in the gastrointestinal mucosa of the cat

Summary The following histological methods, previously proved to be useful in selective light microscopic detection of endocrine cells, were applied to the cat gastrointestinal mucosa: for the identification of biogenic amines, diazonium, ammoniacal silver and xanthydrol methods; for granules identification, methyl green-red acid dyes, toluidine blue, HCl-basic dye, lead-haematoxylin, phosphotungstic haematein and argyrophil methods. Results were compared with those of an extensive electron microscopic investigation.Five types of endocrine cells were identified in the gastric mucosa. Three types were found in the pyloric mucosa: the previously described 5-hydroxytryptamine-producing enterochromaffin cell, the gastrin producing G cell and a cell with an unknown function, labelled in this paper the X cell. Four types were found in the fundic mucosa: enterochromaffin cells (rarely observed), enterochromaffin-like cells secreting a 5-hydroxyindole but showing some ultrastructural and staining differences from true enterochromaffin cells (numerously present), A-like cells (few), resembling A cells of the pancreatic islets, and X cells, resembling those in the pyloric mucosa.In the intestinal mucosa, at least three endocrine cell types were distinguished in its duodenal part: enterochromaffin cells and two types of polypeptide-producing cells — some with smaller granules (S cells) and others with larger granules (L cells). Only two types were found in the mucosa of terminal ileum: enterochromaffin cells and numerously-occurring cells with large granules resembling in part duodenal L cells. The possibility of a relationship between S and L cells and the production respectively of the intestinal hormones secretin and cholecystokinin-pancreozymin was discussed.This investigation was supported by a grant N. 115/1139/0/4715 of the Italian Consiglio Nazionale delle Ricerche.     

A mixed pattern of endocrine cells in metaplastic Barrett's oesophagus. Evidence that the epithelium derives from a pluripotential stem cell

In order to characterize the differentiation of endocrine cells present in Barrett's oesophagus and to determine if they express a single or multiple hormonal pattern, endoscopic biopsies were taken from both the lesion and the fundus of 45 patients and studied at the light microscopical level. Conventional histology revealed three different epithelial patterns: gastric atrophic fundic, intestinal and junctional. A mixture of these patterns was present in 28 cases (62%) and the single type was identified in 17 cases (38%). The use of three silver staining methods and antibodies to human chromogranins allowed us to identify numerous endocrine cells in all but 1 case. Eleven sera against all the most common hormones stored in the endocrine cells of the gut were used to identify the main products of the cells. The following immunoreactivities were identified: 5-hydroxytryptamine (5-HT) (in 75% of the studied cases), somatostatin (87%), motilin (31%), pancreatic polypeptide (PP) (20%), glucose-dependent insulinotropic polypeptide (20%), gastrin (15%), glucagon (15%), peptide tyrosine tyrosine (13%), secretin (7%) and neurotensin (2%). No cholecystokinin-immunoreactive cells were identified. Our results indicated that, in Barrett's epithelium, both gastric and intestinal endocrine cells differentiate, in accordance with the variability of differentiation in the non-endocrine cells present in the different types of columnar epithelium. These findings provide support for the conclusion that Barrett's epithelium arises from a pluripotential stem cell capable of both gastric and intestinal differentiation.     

Double simultaneous staining of B and argyrophil cells of the pancreatic islets: a sequential thiosulphation-aldehyde fuchsin and Grimelius silver procedure

The thiosulphation-aldehyde fuchsin (TAF) method for the insulin-producing B cells can be followed by the Grimelius silver impregnation for the argyrophil cells. This double staining is useful to study, in normal and pathological tissues, the spatial distribution of the two main endocrine cell populations of the pancreatic islets. A treatment with potassium ferrocyanide has been found to enhance the argyrophilia of A cells.     

Development of the endocrine pancreas during larval phases of Rana temporaria

Summary The pancreatic endocrine component was studied at different stages of development in the tadpoles of Rana temporaria. The material was embedded in Epon, and serial semithin and thin sections were made in order to correlate ultrastructural features and tinctorial traits of the endocrine cells. Serial semithin sections were also stained with the peroxidase-antiperoxidase immunocytochemical method and with silver impregnations for argyrophilia and argentaffinity. In early larvae (legless tadpoles), A and B cells are present. Both can be found within ducts and exocrine tissue or, more frequently, in cellular clusters among the ducts and acini. These primitive islets are solid structures, surrounded but not penetrated by capillaries. Mitoses were observed in A and B cells. In the following phase (tadpoles with hindlegs), D and pancreatic polypeptide-immunoreactive cells are also present, as well as numerous endocrine cells scattered among exocrine tissue. There is also a change in the vascular-insular pattern: capillaries not only surround but also penetrate the endocrine group. The structure of the endocrine pancreas in older tadpoles is similar. Tinctorial traits and ultrastructural features of endocrine cells are described, and the origin of primitive islets is discussed.     

Basis for the specificity of anti-5-HT-like antisera in immunocytochemistry applied to the central nervous system

Summary The neuropeptide tyrosine precursor (pre-pro-NPY) messenger RNA (mRNA) has been localised in formaldchyde-fixed human phaeochromocytoma tissue using a sensitive in situ hybridisation procedure and a novel single-stranded cDNA probe. The reaction product was revealed by avidin-biotin-peroxidase complex and streptavidin-gold complex with silver enhancement. This technique may be applied for the determination of biosynthetic activity of endocrine and neuronal cell bodies. This is largely due to its rapidity by comparison with conventional autoradiographic procedures, to the permanence of the reaction product and to the sensitivity of the visualisation steps.     

An histological and immunocytochemical study of the neuroendocrine cells in the intestine of Podarcis hispanica Steindachner, 1870 (Lacertidae)

Summary Numerous endocrine cells can be observed in the gut of the lizard Podarcis hispanica after application of the Grimelius silver nitrate technique. The argyrophilic endocrine cells are usually tall and thin in the small intestine but short, basal, and round in the large intestine. Eleven types of immunoreactive endocrine cells have been identified by immunocytochemical methods. Numerous serotonin-, caerulein/gastrin/cholecystokinin octapeptide-and peptide tyrosine-tyrosine-immunoreactive cells; a moderate number of pancreatic polypeptide-, neurotensin-, somatostatin-, glucagon-like peptide-1-and glucagon-immunoreactive cells, and few cholecystokinin N-terminal-and bombesin-immunoreactive cells were found in the epithelium of the small intestine. Coexistence of glucagon with GLP-1 or PP/PYY has been observed in some cells. In the large intestine a small number of serotonin-, peptide tyrosine-tyrosine-, pancreatic polypeptide-, neurotensin-, somatostatin-and glucagon-like peptide-1-immunoreactive cells were detected. Vasoactive intestinal peptide immunoreactivity was found in nerve fibers of the muscular layer. Substance P-immunoreactive nerve fibers were detected in lamina propria, submucosa and muscular layer. Chromogranin A-immunoreactive cells were observed throughout the intestine, although in lower numbers than argyrophilic cells.     

Endocrine cells in the cardial glands of the esophagus in man

Distribution of endocrine cells in composition of the secretory epithelium of the cardial glands of the human esophagus in both sex and at various age has been investigated. In spiral paraffin slices the endocrine cells have been revealed by means of different silver impregnation methods (after Grimelius, Masson--Hamperl, Sevier--Munger), Sevke technique, ferry-ferrocyanide method. Some cells have been revealed, which according to the specific signs of their granule staining resemble very much G-, EC-, ECL-cells of the stomach. They can be triangular, flatten or polygonal and are stained in the cardial gland epithelium as single diffuse cells, or as groups of cells. Staining of the slices with aldehyde-fuchsin in various modifications reveals dark cells with dark-violet granules and lighter cells with acidophilic granules. Sometimes among these cells certain cells with light-violet cytoplasm are revealed. All these cells can be arranged both in composition of the secretory epithelium of the glands and in conglomerates of cells, resembling pancreatic islands. According to their tinctorial properties they resemble A-, B-, D-cells of these islands.     

A mixed pattern of endocrine cells in metaplastic Barrett's oesophagus

Summary In order to characterize the differentiation of endocrine cells present in barrett's oesophagus and to determine if they express a single or multiple hormonal pattern, endoscopic biopsies were taken from both the lesion and the fundus of 45 patients and studied at the light microscopical level. Conventional histology revealed three different epithelial patterns: gastric atrophic fundic, intestinal and junctional. A mixture of these patterns was present in 28 cases (62%) and the single type was identified in 17 cases (38%). The use of three silver staining methods and antibodies to human chromogranins allowed us to identify numerous endocrine cells in all but 1 case. Eleven sera against all the most common hormones stored in the endocrine cells of the gut were used to identify the main products of the cells. The following immunoreactivities were identified: 5-hydroxytryptamine (5-HT) (in 75% of the studied cases), somatostatin (87%), motilin (31%), pancreatic polypeptide (PP) (20%), glucose-dependent insulinotropic polypeptide (20%), gastrin (15%), glucagon (15%), peptide tyrosine tyrosine (13%), secretin (7%) and neurotensin (2%). No cholecystokinin-immunoreactive cells were identified. Our results indicated that, in Barrett's epithelium, both gastric and intestinal endocrine cells differentiate, in accordance with the variability of differentiation in the non-endocrine cells present in the different types of columnar epithelium. These findings provide support for the conclusion that Barrett's epithelium arises from a pluripotential stem cell capable of both gastric and intestinal differentiation.     

Preliminary evaluation of neuroendocrine cells in the respiratory tract in rats with experimental uremia

The goal of this study was to investigate the influence of experimetally induced chronic renal failure on endocrine cells in the respiratory tract in rats. After 30 days of uremia, the fragments of rat lungs were collected. Paraffin sections were stained using H+E, silver impregnation and immunohistochemistry with specific antibodies against calcitonin (CT), synaptophysin (SY), somatostatin (ST), and neuron-specific enolase (NSE). A large number of endocrine cells with a strong calcitonin immunoreactivity were observed in the respiratory tract of rats with experimental uremia, as compared with the control group. Other immunoreactions were weakened.     

Ultrastructural identification of a new cell type — the N-cell as the source of neurotensin in the gut mucosa

The neurotensin-cell is identified immunohistochemically and ultrastructurally by differential counting of endocrine cells in the gut of a primate (Tupaia belangeri). Utilizing light microscopy, the EC-cells are identified by the Masson-Fontana silver stain; with the same method the neurotensin cells are not stained. The other endocrine cells have been quantified in the small intestine using the peroxidase-antiperoxidase stain with antisera against glucagon, somatostatin, cholecystokinin, gastrin, secretin, pancreatic polypeptide, gastric inhibitory peptide and neurotensin. In the ileal mucosa of Tupaia, the most frequent endocrine cell is the EC-cell followed by the glucagonoid cell, (L-cell). The immunoreactive neurotensin cell represents the third most frequent endocrine cell in this region. On the ultrastructural level, this third most frequent endocrine cell is a heretofore undescribed cell, the N-cell, containing electron dense secretory granules measuring 335 +/- 87 nm in diameter.     

Light and electron microscopic study on the endocrine cells of the pancreas in a marine teleost,Fugu rubripes rubripes

Summary The pancreatic endocrine tissue of Fugu rubripes rubripes consists of numerous round principal islets (Brockmann bodies) of various sizes scattered around the gall-bladder. The endocrine cells are divided into A-, B-, D-, and F_f-cells. Each cell type was identified by comparing thick and thin sections in both light and electron microscopy. Aldehyde-fuchsin positive B-cells contain numerous round secretory granules (average diameter 300 nm) each of which has a round compact core of moderate density; a narrow space exists between this core and the limiting membrane. Grimelius' silver positive A cells contain round secretory granules (average diameter 360 nm) with a hexagonal or tetragonal crystalline core (average diameter 170 nm) of high density; the silver grains preferentially appear in the space between the limiting membrane and the core. The crystalline core of each -granule often contains an appendix-like structure of variable shape. D cells blackened by the silver impregnation method of Hellman and Hellerström (1960) have round secretory granules (average diameter 320 nm) filled with a flocculent material of low density. The fourth cell type (F_f-cell) has a clear cytoplasm after differential staining for light microscopy. By electron microscopy, this cell has elongated fusiform secretory granules (520 nm average length × 230 nm average width) filled with numerous filaments arranged in parallel with the longitudinal axis. Figures suggesting granule formation in the sacs of the Golgi apparatus were obtained in all of islet cell types. Equivalents of emiocytotic release of secretory granules were encountered in the A and F_f cells.