Histochemical demonstration of mercury induced changes in rat neurons.

A histochemical method modified for ultrastructural studies of mercury induced changes is described. Rat neurons from areas known to be influenced by mercury are used as examples. The histochemical reaction, suggested to be caused by polymercury sulphide complexes, is localized to "dense bodies" where it is visible 14 days after initiation of peroral mercury treatment (20 mg HgCl2/l drinking water).     

Histochemical demonstration of copper in LEC rat liver

Livers of LEC rats were histochemically stained for copper according to the modified Timm's method, which includes trichloroacetic acid (TCA) treatment. TCA pretreatment was effective in removing zinc and iron, leaving copper as the major metal in the liver. Hepatocytes in 3-month-old rats were stained intensely by the modified Timm's method, both in frozen sections and in paraffin-embedded specimens. The centrilobular hepatocytes were usually stained, but positive cells were also randomly distributed in the hepatic lobes, showing a mosaic pattern. The staining was intensified in 8- compared to 3-month-old LEC rats. In contrast hepatocytes from LEA rats, the normal counterpart of LEC rats, were faintly stained for copper. Proliferating cholangioles found in older LEC rats were shown to lack copper deposition, and hepatocellular carcinoma showed less copper deposits than the hepatocytes surrounding the tumor. The copper staining was augmented in livers of LEC rats subjected to copper-loading, but was less intense in the livers treated with d-penicillamine. The staining intensity under the various experimental conditions showed good correlation with the copper concentration. Lysosomal deposition of copper in hepatocytes was demonstrated by electron microscopic analysis for copper. Thus the modified Timm's method was shown to produce valuable results in demonstrating copper in LEC rat livers, providing important information for an understanding of the mechanism of copper deposition and hepatic disease of the animal.     

Histochemical demonstration of exopeptidases in the rat visceral yolk-sac epithelium

The localization of exopeptidase activities was demonstrated histochemically (by simultaneous azo coupling) on the visceral endoderm of whole unfixed yolk sacs of rats (12.5-18.5 days of gestation). For comparison, the topochemistry of exopeptidases was studied by conventional section histochemistry of frozen yolk sacs. The study of unfixed visceral yolk-sac epithelium showed that different artificial peptidase substrates (Ala-, Met-, Phe-, Leu-, alpha-Asp-, alpha-Glu-, gamma-Glu, Tyr-, Val-, Ser-, Arg- and Gly-Pro-MNA) are hydrolysed in the apical-cell membranes (membrane-bound peptidases) and, in a number of cells, within the cytoplasmic matrix. Section histochemistry showed that peptidase activities were almost only directed against gamma-Glu- and Gly-Pro-MNA at the cell apices. It is concluded that most of the exopeptidase activities in the apical cell membrane of the visceral yolk-sac epithelium are only demonstrable in unfixed yolk sacs. These activities are of great importance for the supplying of the embryo with amino acids.     

Histochemical demonstration of exopeptidases in the rat visceral yolk-sac epithelium

Summary The localization of exopeptidase activities was demonstrated histochemically (by simultaneous azo coupling) on the visceral endoderm of whole unfixed yolk sacs of rats (12.5–18.5 days of gestation). For comparison, the topochemistry of exopeptidases was studied by conventional section histochemistry of frozen yolk sacs. The study of unfixed visceral yolk-sac epithelium showed that different artificial peptidase substrates (Ala-, Met-, Phe-, Leu-, -Asp-, -Glu-, -Glu, Tyr-, Val-, Ser, Arg- and Gly-Pro-MNA) are hydrolysed in the apical-cell membranes (membrane-bound peptidases) and, in a number of cells, within the cytoplasmic matrix. Section histochemistry showed that peptidase activities were almost only directed against -Glu-and Gly-Pro-MNA at the cell apices. It is concluded that most of the exopeptidase activities in the apiccal cell membrane of the visceral yolk-sac epithelium are only demonstrable in unfixed yolk sacs. These activities are of great importance for the supplying of the embryo with amino acids.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Histochemical demonstration of ATP: creatine phosphotransferase in rat skeletal muscle

Summary An attempt was made to locate the ATP: creatine phosphotransferase (creatine kinase, CKase) in rat skeletal muscle by a lead precipitation method. The muscle is not stained at all with creatine phosphate (CP), and only weakly with adenosine diphosphate (ADP) as substrate, while it hydrolyzes adenosine triphosphate (ATP) actively. Taking advantage of this fact, it is possible to demonstrate the CKase activity using both ADP and CP as substrate. The CKase activity thus obtained was located in various profiles of sarcoplasmic reticulum as well as in A bands, the staining being comparable to that obtained with ATP as substrate.A weak activity was found only in cisternal dilatations of sarcoplasmic reticulum when sections were incubated with ADP as substrate.     

Histochemical demonstration of zinc in rat epididymis using a sulphide-silver method

Summary We studied the histochemical distribution of zinc in rat epididymis using a sulphide-silver method. In the supranuclear cytoplasm of the principal cells that line the epididymis of rats, varying amounts of sulphide-silver-reactive zinc were visualized. In adult mating rats, significant amounts of zinc were found in the proximal portion of the epididymis, whereas in non-mating, mature and immature young rats, this heavy metal was most prominent in the distal portion of this organ. In all of the rats studied, zinc was sparsely distributed in the intermediate portion of the epididymis. From these results, it can be assumed that the zinc present in the epithelial lining of rat epididymis plays an important role in the maturation of spermatozoa. The present results represent a useful contribution to our understanding of the functional morphology of rat epididymis.Dedicated to Professor Dr. T.H.Schiebler on the occasion of his 65th birthday     

Histochemical demonstration of zinc in rat epididymis using a sulphide-silver method

We studied the histochemical distribution of zinc in rat epididymis using a sulphide-silver method. In the supranuclear cytoplasm of the principal cells that line the epididymis of rats, varying amounts of sulphide-silver-reactive zinc were visualized. In adult mating rats, significant amounts of zinc were found in the proximal portion of the epididymis, whereas in non-mating, mature and immature young rats, this heavy metal was most prominent in the distal portion of this organ. In all of the rats studied, zinc was sparsely distributed in the intermediate portion of the epididymis. From these results, it can be assumed that the zinc present in the epithelial lining of rat epididymis plays an important role in the maturation of spermatozoa. The present results represent a useful contribution to our understanding of the functional morphology of rat epididymis.     

Histochemical demonstration of copper in normal rat brain and spinal cord

Summary The high sensitivity of the magnesium-dithizonate silver-dithizonate (MDSD) staining procedure makes this method very suitable for the histochemical localization of copper in different regions of the central nervous system of adult rats. In the telencephalon (bulbus olfactorius, nucleus caudatus-putamen, septum pellucidum and are dentata), diencephalon (nucleus habenulae medialis, nuclei of the hypothalamus in the vicinity of the third ventricle, and corpus mamillare), mesencephalon (substantia nigra), cerebellum (mainly in the nodulus), pons (locus coeruleus, nucleus vestibularis), medulla oblongata (nucleus tractus solitarii) and spinal cord, the glial cells exhibit specific copper staining. The glial cells of some circumventricular organs (e.g. the subfornical organ) are also stained using the MDSD method. The significant staining observed in whitematter glial cells (e.g. in the corpus callosum, cerebellum and spinal cord) further indicates the very high sensitivity of this method. In glial cells of the same regions, the presence of copper can likewise be demonstrated using the modified sulphide silver method. On the basis of the present histochemical results, it is suggested that copper may play an important role in the normal physiological functioning of glial cells and also, via glia-neuron interactions, in neuronal processes.     

Histochemical demonstration of enterokinase

Summary A new simple method for the histochemical demonstration of enterokinase (enteropeptidase, EC 3.4.4.8) was elaborated. Unfixed cold microtome sections adherent to the slides are covered with agar-gel medium containing trypsinogen (0.5–1 mg per 1 ml), N-benzoyl-DL-arginine--naphthylamide hydrochloride (2–4 mM), and Fast Blue B Salt (0.5 mg per 1 ml) in 0.05 M Tris maleate buffer pH 6.5 with 0.05% CaCl₂. After the incubation in a wet chamber at 37° C the slides are chelated in 2% copper sulphate. The method enables a localization on the histological level. The evaluation of the overall staining obtained with this method in sections of the duodenum, jejunum and ileum of guinea pigs, rats, monkeys, dogs and humans reflects well the biochemical quantitative data on enterokinase activity obtained in homogenates of mucosal scrapings of the corresponding gut segments of the same animals.In all animals a proximodistal gradient was found. The highest activities were found in enterocytes covering apical parts of villi in the duodenum. According to the activities in the duodenum the investigated animals can be arranged in the following series: guinea pig (highest activity), monkey, man, rat and dog.     

Histochemical changes in the testes of lead induced experimental rats

The experiments were performed on mature male rats divided in five groups, one control and four experimental in which the animals received 1 mg, 2 mg, 4 mg and 6 mg/kg body weight lead acetate intraperitoneally respectively, over a period of 30 days. ALA-D and lead was estimated in the blood by the use of atomic absorption spectrophotometer and ATP-ase, AMP-ase, Alk-ase were histochemically localized. Significant increase in blood and testis of lead levels along with decrease of ALA-D levels were observed. Changes in the testicular tissue were encountered. Other details concerned with the damage of the testicular tissue are discussed.     

Silver enhancement of tissue mercury: demonstration of mercury in autometallographic silver grains from rat kidneys

The autometallographic silver enhancement method has been applied increasingly to detect trace amounts of mercury in preparations of biological tissue. It has, however, been difficult to establish the presence of a core of mercury within the silver grain by direct methods such as energy dispersive X-ray analysis. In the present work, a sample of autometallographic silver grains was prepared from kidneys of rats exposed to mercury in the drinking water. Frozen sections from the kidneys were silver-enhanced and subsequently all organic material was removed by enzymatic digestion. The remaining pellet of silver grains was analyzed by proton-induced X-ray emission (PIXE) and mercury was demonstrated in an amount of 0.1-0.5% compared to silver. In addition, it was demonstrated that two pools of catalytic mercury compounds exist, probably corresponding to sulfide- and selenium-bound mercury.     

Histochemical demonstration of neurotoxic esterase

We developed a histochemical method for localizing neurotoxic esterase (NTE), defined as the phenylvalerate (PV)-hydrolyzing esterase that is resistant to 40 microM paraoxon (A) but inactivated by paraoxon plus 50 microM mipafox (B). NTE is considered to be the target enzyme in the production of organophosphorus ester-induced delayed neurotoxicity (OPIDN). Cryostat sections were incubated in a medium containing alpha-naphthyl valerate and 6-benzamido-4-methoxy-m-toluidine diazonium chloride (fast violet B) after treatment with the above-mentioned inhibitors, leading to formation of an aqueous insoluble precipitate at sites of enzymatic activity. NTE activity was estimated as staining detectable in A but not in B. In the central nervous system (CNS) of chicken, NTE appeared to be present primarily in the somata of most neurons, but at sites indistinguishable from those of the other inhibitor-resistant and -sensitive alpha-naphthyl valerate-hydrolyzing esterases. It could not be distinguished in the CNS of cat, probably because it constitutes less than 3% of the total PV-hydrolyzing activity in the CNS of that species.     

Histochemical demonstration of heavy metals

Summary The three steps of the sulphide silver method have been examined: 1) Transformation of metals to metal sulphides; 2) Fixation and embedding or freezing of the tissue for sectioning; and 3) Deposition of metallic silver on the metal sulphides in a physical developer. Based on the results, a revised method is described and discussed. It is particularly important 1) To maintain a sufficient but low concentration of sulphide ions during the perfusion; 2) To avoid using oxidating or acid fixatives; 3) To ensure low temperatures while embedding in paraffin or during polymerization of Epon; and 4) to use a slow-acting physical developer. Examples of the metal sulphide pattern from various tissues are presented.     

Histochemical demonstration of phospholipase B (lysolecithinase) activity in rat tissues.

A method has been developed for the histochemical demonstration of phospholipase B (lysolecithinase) of rat tissues. The enzyme attacks lysolecithin with liberation of 1 mole of glycerylphosphorylcholine and 1 mole of fatty acid. The recommended procedure involves use of 6-10 micro frozen sections, fixed in cold calcium-formol and incubated at 37 degrees C in Tris buffered medium at pH 6.6 containing 2.2 X 10(-3) M lysolecithin and 1% cobalt acetate. The fatty acid liberated by enzymatic hydrolysis is trapped as a cobalt precipitate and is then converted to a black-brown precipitate by treatment with dilute ammonium sulfide in cold isotonic saline. Equivalent amounts of fatty acid and glycerylphosphorylcholine are recovered by extraction and analysis of the incubated sections and of the incubation medium, thus proving that lysolecithin hydrolysis occurs under the proposed reaction conditions. Staining is reduced by treating the sections with copper ions, mercury compounds, alcohols, acetone and by heating at 60 degrees C prior to incubation with substrate. Lowering of the pH of the incubation medium has similar effect. These findings are interpreted as evidence of the enzymatic nature of the reaction. Cells exhibiting a positive staining are found in the lamina propria of the intestinal villi and crypts, in the red pulp of the spleen and in the interstitial tissue of lung, liver and thymus. Similar elements are present in bone marrow smears and in leukocyte preparations obtained by peritoneal lavage. The morphologic and staining characteristics of these cells correspond to those of the eosinophilic leukocytes. Physical and chemical agents (x-irradiation, corticosteroids) which sharply decrease the number of eosinophils also reduce the number of cells shown histochemically to hydrolyze lysolecithin. A correspondent diminution of phospholipase B activity of homogenates of the same tissues can be shown in vitro. Differences in tissue distribution and chemical properties distinguish the phospholipase B from less specific esterases and lipases.