Vascular and leptomeningeal projections of the subcommissural organ in reptiles. Lectin-histochemical, immunocytochemical, and ultrastructural studies

In the snake, Natrix maura, and the turtle, Mauremys caspica, the basal processes of the ependymal cells of the subcommissural organ project toward the local blood vessels and the leptomeninges. These processes and their endings were studied using aldehyde-fuchsin (AF), periodic-acid Schiff (PAS), periodic-acid silver-methenamine (PA-SM), concanavalin A (ConA), wheat germ agglutinin (WGA), immunoperoxidase staining (employing an antiserum against bovine Reissner's fiber; AFRU), and conventional transmission electron microscopy. For the purposes of comparison, the ventricular cell pole was also analyzed. The secretory material located in the ventricular cell pole and that present in ependymal endings had only a few staining properties in common, i.e., affinity for AF, ConA, and AFRU at a dilution of 1:1000. On the other hand, PAS, PA-SM, WGA, and AFRU at a dilution of 1:200,000 stained the apical (ventricular) secretory material but not the secretory material of the ependymal processes. The histochemical features of the secretory material located in the terminals of ependymal processes, as well as the presence at these sites of numerous rough-endoplasmic-reticulum cisternae and secretory granules, suggest that secretory material may be synthesized in these terminals. The probable fate of this material, i.e., release to the perivascular and leptomeningeal spaces or transport to the ventricular cell pole, is discussed.     

Use of prostaglandin inhibitor, 2-(6-methoxy-2-naphthyl) propionic acid,with regard to morphological and enzymatic changes of gastric mucosa

2-(6-methoxy-2-naphthyl) propionic acid is introduced to treatment as non-steroid antiinflammatory drug (NSAID) under the trade-mark of Naprosyn. Inhibition of prostaglandin synthesis is regarded as the most likely mechanism of its action. In some patients, its side-effects include gastritis, reactivation of ulcerous niche, and upper gastrointestinal haemorrhage. The absence of complex studies addressed to the question of morphological and histochemical changes in gastric mucosa after oral administration of Naprosyn prompted our undertaking. In experimental animals, with varying doses (10 mg/kg and 50 mg/kg) and variously long administrations (1 week and 3 weeks), a trial has been reported here upon. In the frozen-sectioned preparations, the histochemical reaction for acid phosphatase activity, according to Gomori, was made. The paraffin sections were subjected to the HE staining, PAS staining according to McManus and with the Masson's method. Our results of the morphological and histochemical studies in rats support the clinical observations of mucosal destruction in stomach in patients after oral administration of Naprosyn.     

Histochemistry of the cuticle of the crab Menippe rumphii (Fabricius) (Crustacea: Brachyura) in relation to moulting

Histological and histochemical methods have been employed to study the formation and growth of the exoskeleton in relation to the moulting cycle of the crab Menippe rumphii (Fabricius). In the premoult condition the epidermal cells secrete a two-layered cuticle. Later these layers are widened by the secretions coming from the reserve cells, tegumental glands, and the Leydig cells. The fully formed cuticle of the intermoult crab is divisible into four layers, epicuticle, exocuticle, mesocuticle, and endocuticle.Histochemical observations on different cells have revealed that the tegumental glands secrete both neutral and acid mucopolysaccharides. The reserve cells are positive to PAS, BPB, Sudan Black B and Alizarin Red S techniques indicating the presence of carbohydrates, proteins, lipids, and mineral calcium. The Leydig cells are loaded with enzymes, including alkaline phosphatase, acid phosphatase, lipase, and phenoloxidase. Other histochemical tests have been employed to investigate the formation of different layers of the cuticle.     

Vascular and leptomeningeal projections of the subcommissural organ in reptiles

Summary In the snake, Natrix maura, and the turtle, Mauremys caspica, the basal processes of the ependymal cells of the subcommissural organ project toward the local blood vessels and the leptomeninges. These processes and their endings were studied using aldehyde-fuchsin (AF), periodicacid Schiff (PAS), periodic-acid silver-methenamine (PASM), concanavalin A (ConA), wheat germ agglutinin (WGA), immunoperoxidase staining (employing an antiserum against bovine Reissner's fiber; AFRU), and conventional transmission electron microscopy. For the purposes of comparison, the ventricular cell pole was also analyzed. The secretory material located in the ventricular cell pole and that present in ependymal endings had only a few staining properties in common, i.e., affinity for AF, ConA, and AFRU at a dilution of 1:1000. On the other hand, PAS, PA-SM, WGA, and AFRU at a dilution of 1:200 000 stained the apical (ventricular) secretory material but not the secretory material of the ependymal processes. The histochemical features of the secretory material located in the terminals of ependymal processes, as well as the presence at these sites of numerous rough-endoplasmic-reticulum cisternae and secretory granules, suggest that secretory material may by synthesized in these terminals. The probable fate of this material, i.e., release to the perivascular and leptomeningeal spaces or transport to the ventricular cell pole, is discussed.This work was partially supported by grants from the Stiftung Volkswagenwork, Federal Republic of Germany (1/38259), from the Dirección de Investigaciones, Universidad Austral de Chile (S-85-39), and from Fondo Nacional de Desarrollo Científico y Tecnológico, Chile (6027; all to E.M.R.)     

中华鳖肾脏的组织化学特性研究

利用光镜组织化学反应对中华鳖肾单位的结构和组织化学特性进行了详细的观察和分析。结果表明,中华鳖肾脏为分叶形的实质器官,肾小叶由被膜和实质组成,实质无髓质和皮质之分,但可以区分为外侧区和内侧区。外侧区嗜酸性,主要分布有近端小管和集合管。内侧区呈弱嗜酸性,肾小体、颈段、中间段和远端小管主要分布在内侧区。肾小球PAS反应呈阳性,但其琥珀酸脱氢酶(SDH)弱阳性,碱性磷酸酶(ALPase)、Na+/K+-ATPase和阿利新兰(AB)反应为阴性。足细胞酸性磷酸酶(ACPase)反应呈阳性。近端小管刷状缘嗜伊红,PAS反应以及ALPase、ACPase和Na+/K+-ATPase酶反应呈阳性,而SDH弱阳性。中间段、远端小管、集合管弱嗜酸性,SDH阳性。中间段Na+/K+-ATPase弱阳性。远端小管细胞侧面呈PAS阳性,腔面显示AB阳性。集合管胞质含有许多ACPase阳性颗粒,腔面呈PAS强阳性,AB阳性。甲苯胺兰(TB)染色可见集合管腔面有阳性颗粒,肾小管上皮含有亮、暗两种细胞。上述组化反应和分布结果表明,鳖的肾小管细胞类型较多,近端小管在原尿的重吸收中起主要作用,远端小管和集合管具有分泌黏液作用。中华鳖肾单位的结构与组化特性不仅与哺乳类和鸟类有一定差异,也与其他爬行动物不完全相同。       

基因修饰的小鼠肥大细胞瘤细胞的酶细胞化学变化

应用酶细胞化学方法对基因转导前后的Ｐ８１５小鼠肥大细胞瘤细胞进行酸性磷酸酶、非特异性酯酶及多糖的定位。研究结果显示,空载体转导的Ｐ８１５／ｎｅｏ细胞与其亲本细胞相比无显著改变,而ＩＬ２基因修饰的Ｐ８１５细胞的酸性磷酸酶及多糖反应明显增强,可能提示细胞内某些细胞器的形成以及细胞内多糖类合成功能的增强。     

Comparative histochemistry of the mucoproteic cells of the hypophysis from Rhamdia hilarii, Hypostomus punctatus, Prochilodus scrofa and Cyprinus carpio (Teleostei). Immunohistochemical identification of the gonadotropic cells

Adenohypophyseal cells showing positive histochemical reactions for mucosubstances were classified as type I-IV in Hypostomus (Plecostomus) punctatus (Loricariidae), Rhamdia hilarii (Pimelolidae), Prochilodus scrofa (Prochilodontidae) and Cyprinus carpio (Cyprinidae) according to cell shape, size, cytological characteristics and adenohypophyseal distribution. Cell types I and II are common to the four species, with each cell type showing very similar cytological and histochemical characteristics, in spite of different adenohypophyseal distribution of cell type II, according to the teleost species. Type I cells are globular basophils located in the proximal pars ditalis and are positive to PAS and Alcian blue pH 2.5 (AB) reactions, showing cytoplasmic vacuoles and changes in granule concentration in the mature phase of the gonadal cycle. The smaller type II cells are fusiform or oval basophils exhibiting a strong AB reaction but also reacting to PAS. Type III cells are located in the pars intermedia showing PAS-positive reaction. Considering different teleost species, these cells exhibit some variations specially in relation to cell size and shape which are not detected in mature male C. carpio. Otherwise cell type IV is only present in the rostral pars distalis of P. scrofa. They are weakly basophilic and negative to PAS, reacting strongly to AB. Only cell type I showed unequivocally positive immunohistochemical results with anti-salmon gonadotropin.     

Ultrastructural study on topographical variations of the ependyma in Bradypus tridactylus.

An electron-microscopic study of ependymal cells in 24 different areas of the encephalic ventricular system of Bradypus tridactylus was made, with a view to making a comparative analysis, of a topographical kind, of various cell components: cilia, microvilli, plasma membrane, cytoplasmic protrusions, types of cellular junctions, ribosomes, Golgi apparatus, endoplasmic reticulum, microfilaments, microtubules, mitochondria, granules and dense bodies. There were differences in the distribution of all these cytologic elements, according to the topography, being a complement to a similar work done by the same authors with light-microscopy. By means of a morphofunctional analysis they draw the conclusion that even if one can admit the passage of substances by an intracellular route, on the other hand few facts support the idea the intracellular absorption or secretion could be considered as important phenomena in a great number of the ependymal areas studied.     

Histochemical distribution of four types of enzymes and mucous cells in the gastrointestinal tract of reared half‐smooth tongue sole Cynoglossus semilaevis

The histochemical distribution of acid phosphatase (ACP), alkaline phosphatase (ALP), non‐specific esterase (NSE), peroxidase (POD) and mucous‐cell types was evaluated in the gastrointestinal tract of the half‐smooth tongue sole Cynoglossus semilaevis. The enzymes were detected in the entire stretch of the gastrointestinal tract. ACP activity was found in the supranuclear region of enterocytes and the lamina propria of the intestine, as well as the cytoplasm of epithelial cells of the stomach. The staining intensity of ACP in the anterior and posterior intestines was stronger than in the stomach. ALP activity was detected in the striated border of enterocytes and muscularis of the whole intestine, lamina propria and supranuclear cytoplasm of the enterocytes in the anterior intestine, as well as in the blood vessels of the stomach. The staining intensity for ALP in the anterior intestine was stronger than in the posterior segment and the latter was stronger than in the stomach. NSE activity was detected in the cytoplasm of the epithelial cells in the entire gastrointestinal tract, with the anterior intestine showing stronger intensity than the stomach. POD activity was located in the blood cells of the lamina propria of the gastrointestinal tract and the levels in the stomach were similar to the anterior and posterior intestines. Alcian blue (pH 2·5) periodic acid Schiff (AB‐PAS) histochemical results revealed three types of mucous cells in the gastrointestinal tract. Type I cells (PAS+AB‐) were observed among the gastric mucosa columnar cells in the stomach and enterocytes in the basal region of the villi and in the middle and top regions of the intestinal villi. Type II cells (PAS‐AB+) and type III cells (PAS+AB+) were not detected in the stomach but were distributed ubiquitously among enterocytes in the middle and top regions of the intestinal villi.     

Ultrastructure of the secretory cells of the submucosal glands in the human maxillary sinus

Tissue samples obtained from the lateral wall of the maxillary sinuses of five patients were examined by light microscopical, histochemical, and ultrastructural techniques. Submucosal glands were tubulo-alveolar mixed glands. The acini consisted of either all serous or all mucous cells, or a mixture of both. Serous granules were stained by toluidine blue, or by hematoxylin and eosin (H and E), but showed little or no reaction with periodic acid-Schiff (PAS) or Alcian blue. Mucous granules were pale in toluidine blue or H and E preparations, and consisted primarily of acid mucosubstances, as demonstrated by their staining reaction with PAS and Alcian blue. At the electron microscope level, the serous granules were either homogeneously dense, or showed a substructure consisting of at least two layers of distinctly different electron-opacity. Typical mucous droplets consisted of a fibrillar network dispersed in a translucent matrix. A second secretory product was present in the mucous cells in the form of elongated, membrane-bounded structures containing numerous parallel filaments, which measured about 55 Å in diameter. The mucous droplets and the filamentous bodies appear to arise from the opposite faces of the Golgi complex in the mucous cells. The filamentous bodies showed a pronounced tendency to fuse with the mucous droplets. All acini were surrounded by a well-defined myoepithelial layer and contained intercellular nerve terminals.     

Enzyme histochemical changes in retinal ganglion cells and the optic nerve after goldfish optic nerve lesion. A regenerative and hypertrophic phenomena study

After sectioning of the goldfish optic nerve a number of enzyme histochemical changes are observed in the hypertrophied retinal ganglion cells and in the optic nerve. Between one and eighteen days postoperatively an increase in the amount of acid phosphatase reaction product is noted. The enhanced activity decreased to normal first in the optic nerve, followed by the optic tract and tectum. Four days postoperatively higher levels of activity were noted in the hypertrophic retinal ganglion cells for the enzymes NADH tetrazolium reductase, cytochrome oxidase, glutamate dehydrogenase and lactate dehydrogenase. The same enzymes also showed an activity increase in the lesioned optic nerve after four to ten days postoperatively, beginning at the cut and gradually spreading towards the optic tectum. Between fifteen and eighteen days the activity dropped to normal in the hypertrophic retinal ganglion cells, while in the lesioned nerve raised levels of reaction products could be seen till days thirty-five and/or forty-five. It was concluded that the degeneration of the optic pathway is marked by the increase of acid phosphatase activity, whereas the process of regeneration is characterized by an increase of NADH tetrazolium reductase, cytochrome oxidase, glutamate dehydrogenase and lactate dehydrogenase activities. The possible functional implications of these enzymes in the regenerative phenomena are discussed.     

Enzyme cytochemical and immunocytochemical studies of flask cells in the amphibian epidermis

The localization of oxidoreductases and transport enzymes in flask cells of the amphibian epidermis was studied at the light-microscopic level. In these cells, the deposition of cytochemical reaction products was very similar to that found in fish epidermal ionocytes, thus demonstrating histochemical similarities between these two types of cells. The present histochemical results revealed high levels of activity of alkaline phosphatase (ALPase), potassium-dependent nitrophenylphosphatase (K+-p-NPPase) and carbonic-anhydrase isozymes (CA-I and CA-II) in the apical region of the flask cells, indicating that enzyme zonation may be the main site of the ion pumping.     

A micromorphological and histoenzymological study on the third ventricular ependyma of the teleost Clarias batrachus (L.).

The circum ventricular region of C. batrachus is highly vascular and the ependymal cells appear differently when stained with haematoxylin, silver impregnation and Golgi-Cox techniques. The ventricule has PAS and AF positive material and some ependymal cells themselves are PAS positive. Few AF positive peptidergic and several AF negative small neurons have liquor contacting terminals. Golgi-Cox preparations reveal a variety of forms among the tanycytes. Their basal processes which are barbed or studded with varicosities, usually end on blood vessels and other neuronal elements. These basal processes themselves are often seen in direct morphological contact. Smaller silver positive cells without basal processes are also evident. Some tanycytes have apical processes resembling broadened endfeet. Few neurosecretory tracts are Golgi-Cox positive and can be differentiated from the tanycytic processes by their smooth surface. Varying degrees of ascorbic acid activity are noticed inside the ventricle, among the tanycytes and in the neurons of the NLT. Some of the latter neurons have liquor contacting terminals as well. The ChE activity noticed in some parts of the ependyma and in some NLT cells suggest their probable differential cholinergic control. Presence of SDH, NADPH and NADH diaphorases and cytochrome oxidase in varying quantities in the ependymal cells suggests that they are metabolically active. Presence of MAO positive tracts bridging the subependyma and ventricle suggests the degradation of monoamines at these sites. The presence of various enzymes and the morphological relationship of the tanycytes described in this species are comparable to those of the mammals. It is significant as this species is reported to have a median eminence morphologically resembling the tetrapods.     

Histochemical and biochemical studies on the localization and activity of lysosomal enzymes in fracture callus

Summary Quantitative biochemical studies on the activities of four lysosomal hydrolases during different stages of fracture healing in the rat were performed, and the results obtained were integrated with those of histochemical observations relating to changes in the localization of acid phosphatase in the same tissue.The findings showed presence of all the four lysosomal enzymes assayed in the callus; during early callus formation the enzyme activities calculated on a DNA basis increased up to about 12 days after the fracture. The enzyme activities appeared to be roughly reflected histochemically by the acid phosphatase staining. The increasing activity during early callus formation seemed to depend on the presence of numerous macrophage-like cells in the tissue containing many large lysosomes. A decrease in enzyme activity was found after day 12. Comparison with the histochemical and ultrastructural findings suggested that this decrease was due to a reduction in the number of macrophage-like cells and a concomitant increase in osteogenic cells with a lower enzyme content.     

Characterization of glycoconjugates in the secretory epithelium of the equine ampulla ductus deferentis

The present work was undertaken to determine the glycoconjugates secreted by the epithelium of the equine ampulla ductus deferentis, using conventional (PAS, AB pH 2.5, AB pH 1.0) and lectin histochemical procedures in conjunction with enzymatic digestion and chemical treatment. The presence of abundant apical cytoplasmic blebs suggests that the equine ampulla secretes its products mainly in an apocrine manner. Glandular cells secrete neutral and acidic sialylated glycoconjugates as revealed by conventional histochemical procedures. Lectin histochemistry helped us to discover the following histological positive sites: the mucosal cells, the glandular epithelial cells, the apical cytoplasmic blebs and the basal cells. The ampullary secretions contained both glycoproteic material (revealed by Con-A-, LCA-, GSA-II-, WGA-, RCA-I- positivity) and sialomucins (evidenced by the reactivity of GSA-II, SBA, PNA and RCA-I after sialidase digestion) having different functional roles. The mucosal cells reacted with Con-A, LCA, and also with sialidase/GSA-II-, SBA-, PNA-, and RCA-I sequences, contributing to the chemical heterogeneity of ampullary secretions. DBA lectin was a specific marker for basal cells. The results obtained were compared with our previous findings regarding the differences in the lectin binding pattern of the plasma-membrane of equine sperm collected from epididymal cauda and the ampulla ductis deferentis. Our results support other studies that indicate that ampullary secretions are involved in altering the plasma-membrane glycoconjugates of spermatozoa, contributing to their maturation.     

The subcommissural organ of the frog Rana perezi is innervated by nerve fibres containing GABA

The innervation of the frog subcommissural organ was studied by light-microscopic and ultrastructural immunocytochemistry using antisera against serotonin, noradrenaline, dopamine, gamma-aminobutyric acid (GABA), glutamic acid decarboxylase, different GABA receptor subunits and bovine Reissner's fibre material (AFRU). In the proximity of the organ, serotonin- and noradrenaline-containing fibres were rare whereas dopamine-immunoreactive fibres were more numerous. Many GABA- and glutamic acid decarboxylase-containing nerve fibres were found at the basal portion of the ependymal cells of the subcommissural organ. Under the electron microscope, these GABA-immunolabelled nerve endings appeared to establish axoglandular synapses with secretory ependymal cells of the subcommissural organ. In addition, the secretory ependymal cells expressed high amounts of the beta2-subunit of the GABA(A) receptor. Since GABA-immunoreactive neurons were present in the frog pineal organ proper and apparently contributed axons to the pineal tract, we suggest that at least part of the GABAergic fibres innervating the frog subcommissural organ could originate from the pineal organ.     

Enzyme cytochemical and immunocytochemical studies of flask cells in the amphibian epidermis

Summary The localization of oxidoreductases and transport enzymes in flask cells of the amphibian epidermis was studied at the light-microscopic level. In these cells, the deposition of cytochemical reaction products was very similar to that found in fish epidermal ionocytes, thus demonstrating histochemical similarities between these two types of cells. The present histochemical results revealed high levels of activity of alkaline phosphatase (ALPase), potassium-dependent nitrophenylphosphatase (K⁺-p-NPPase) and carbonic-anhydrase isozymes (CA-I and CA-II) in the apical region of the flask cells, indicating that enzyme zonation may be the main site of the ion pumping.     

A histochemical study on the adrenal components of the teleost Cirrhinus mrigala (Ham.)

The adrenal components of C. mrigala are embedded in the pronephric cephalic kidney around the post cardinal vein. The cortical cells responded positively to the lipids, ascorbic acid, delta 5-3 beta-HSD, G-6-PD, MAO, acid and alkaline phosphatase tests. The presence of intense MAO activity may suggest the possible involvement of monoamines in the adrenocortical function. Localization of lipids and delta 5-3 beta-HSD show the sites of corticosteroid synthesis. In the chromaffin cells, MAO, acid and alkaline phosphatase activity was moderate whereas they gave a strong reaction to ascorbic acid test in comparison to the cortical cells. Noradrenaline (NA) and adrenaline (A) storing cells were differentiated adopting glutaraldehyde silver, dichromate and iodate techniques. NA and A storing cells are almost totally depleted of their contents after reserpine treatment. The histochemical response of the adrenal gland of this species is largely comparable to that of higher vertebrates.     

Isolation and thin-layer chromatographic identification of several peptides with NH2-terminal tryptophan and their histochemical demonstration in the ACTH cells of the rat hypophysis

Methods for the isolation and thin-layer chromatographic identification of amino-terminal tryptophyl-peptides presumably responsible for histochemical tryptophyl-peptide reactions in the ACTH cells of the rat hypophysis are described. In the hypophyseal extract several tryptophylpeptide bands--depending on the homogenization solution--were demonstrated on thin-layer chromatograms. Tryptophyl-peptides were demonstrated from their fluorescence induced 1) with glyoxylic acid (glyoxylic acid introduced into the homogenization solution), 2) by exposure of the chromatographic plates to combined formaldehyde and chloral vapour or 3) by exposure to combined formaldehyde and acetyl chloride vapour. A positive PAS reaction was demonstrated in some tryptophyl-peptide bands. Thus, some tryptophylpeptides seem to contribute to the observed PAS positivity of the ACTH cells.     

Cytochemical demonstration of phosphatases in membrane-recycling structures of endodermal cells

We applied cytochemical procedures to demonstrate the presence of acid and alkaline phosphatase in the visceral yolk-sac endoderm of rats using frozen, aldehyde-fixed tissue with cerium as the capture agent. This procedure allowed more detailed topochemical localization than was possible using unfrozen tissue or with lead as the capture agent. Acid phosphatase was found to be present in lysosomes as well as in a small number of apical canaliculi, which are thought to be recycling structures of the cell membranes in endodermal cells. Reaction products of alkaline phosphatase were observed on the outer surface of apical, lateral, and basal cell membranes. In addition, some apical vacuoles contained alkaline phosphatase, and more apical canaliculi were positive for alkaline phosphatase than for acid phosphatase. However, most of the apical canaliculi were negative for both enzymes. It is suggested that acid and alkaline phosphatase are taken up by different numbers of apical canaliculi during the detachment of apical canaliculi from lysosomes and resorption vacuoles.