A mesoscopic technique for the study of the development of the peripheral system in rat foetuses

In order to study the morphogenesis of the nervous system in the rat an acetylcholinesterase in toto method for staining nervous tissue in rat foetuses was developed. Procedure: Rat foetuses of 14-22 days are fixed "en bloc" for 24 hours in a cold sucrose-formol solution. Fixed specimens are rinsed for 2 days in cold 0.22 M sucrose in a sodiumcacodylate buffer (pH 7.2). The specimens are cut (mid-)sagittally with the aid of a razor-blade, and incubated in a medium of acetylthiocholine iodide in acetate buffer (pH 5.0). Then, dehydration in glycerine/water mixtures of increasing glycerine content follows. The specimens may be stored in pure glycerine or embedded in epoxy-resin blocks and can be studied under a binocular dissecting microscope. In using this in toto staining method both the continuity of the central and peripheral parts of the nervous system as well as details up to the level of individual perikarya and motor endplates are preserved. With this mesoscopic method the three-dimensional architecture of the peripheral nervous system and its topological relations to other structures can be studies in one specimen. The exact procedure and the results as well as a method for embedding specimens in epoxy-resin blocks for teaching purposes are described. The advantages of this mesoscopic staining method for foetuses are discussed.     

Fine structure of intramembranous particle aggregates in ADH-treated frog urinary bladder and skin: Influence of glutaraldehyde and N-ethyl maleimide

Summary The fine structure of ADH-induced intramembrane particle aggregates has been studied in different tissues and under different experimental conditions. Particle aggregates similar to those previously observed in the amphibian urinary bladder and in the mammalian collecting duct were also found in the frog skin, another ADH target tissue. In the frog urinary bladder, typical aggregates were observed in the absence of glutaraldehyde fixation. Two experimental approaches were used a) the absence of both fixative and cryoprotectant treatments and b) the absence of only glutaraldehyde treatment. In the latter case the reversal of hydrosmotic action was prevented by exposing the preparations to N-ethyl maleimide. In specimens of frog urinary bladder conventionally fixed with glutaraldehyde, two fracture levels could be observed in the aggregates, suggesting that the aggregated particles span an appreciable part of the membrane thickness.J. Chevalier is a career investigator from the Institut National de la Santé et de la Recherche Médicale, INSERM U 48, France     

Migration of gall stones.

The factors influencing the migration of gall stones are ill understood. Altogether 331 patients undergoing cholecystectomy were studied prospectively. The diameters of the cystic and common bile ducts and of stones in the gall bladder and bile ducts were measured. Increasing pressure was applied to the freshly excised gall bladder in an attempt to evacuate stones through the cystic duct. Stones passed in 33 (60.0%) of patients with choledocholithiasis, 45 (67.2%) of patients with pancreatitis, and 7 (3.2%) of patients without either pancreatitis or choledocholithiasis. Stones migrated in 6 (3.0%) who had a normal cystic duct diameter (less than or equal to 4 mm) and in 46 (32.5%) with a duct over 4 mm diameter. Common bile duct stones were often larger than the diameter of the cystic duct and when reintroduced into the gall bladder would not migrate. The passage of debris (less than or equal to 1 mm) through the cystic duct bore no relation to the presence or absence of choledocholithiasis or a dilated cystic duct. Small stones (1-4 mm diameter) must migrate to initiate and facilitate further migration; some must increase in size in the common bile duct. Increased biliary pressure consequently dilates the duct system retrogradely, allowing larger stones to follow. Patients at risk of stone migration and thereby pancreatitis and jaundice have large ducts that can be detected by ultrasound assessment.     

New species of myxosporidia (Myxozoa: Myxosporea) of fish from the northwestern Sea of Japan

Three new species of mixosporidia of the genera, Myxidium and Myxoproteus, from the gall bladder and urinary bladder of fishes from Japan Sea: Myxidium licodae sp. n., M. rarum sp. n., and Myxoproteus ovale sp. n.     

Staining Procedures for Ultrathin Sections of Tissues Embedded in Polyester Resin

Tissues were fixed for 30 min In cold (0-2° C) 1% OsO₄ (Palade) buffered at pH 7.7, to which 0.1% MgCl₂ was added. Dehydration was in a graded ethanol series (containing 0.5% MgCl₂) at 0-2° C, and terminated with 2 changes of absolute ethanol. Tissues were then transferred by a graded series to anhydrous acetone. Infiltration of the tissue with Vestopal-W (a polyester resin), is gradual with the aid of graded solutions of Vestopal-W in acetone. The infiltrated tissue is encapsulated and initial polymerization is done under ultraviolet light at room temperature for 8-16 hr. This is followed by final hardening at 60° C for 36-48 hr. Sections (0.2-1 μ) were cut, dried on slides, placed in acetone for 1 min and then treated by either of the following staining procedures: (1) Thionin-azure-fuchsin staining: Flood the preparation with 0.2% aqueous thionin and heat to 60-80° C for 3 min; if the preparation begins to dry, add stain. Rinse in distilled water. Flood the slide with 0.2% azure B in phosphate buffer at pH 9. Heat to 60-80° C for 3 min; do not permit the preparation to dry. Rinse in distilled water. Dip the slide in MacCallum's variant of Goodpasture's carbol-fuchsin stain for 1-2 sec. Rinse in distilled water. Check the preparation microscopically for intensity of the fuchsin stain. Repeat dips as may be needed to obtain the desired intensity. Rinse in distilled water. Dehydrate quickly in 95% and absolute alcohol; clear in 2 changes of xylene and cover in Permount or similar synthetic resin. (2) Thionin-azure counterstain for the periodic acid-Schiff reaction: Oxidize the tissue in 0.5% periodic acid for 15 min and transfer to Schiff's leucofuchsin solution for 30 min. Counterstain with 0.5% aqueous thionin for 3 min; wash in distilled water; stain in 0.2% azure B in phosphate buffer at pH 5.5; wash in distilled water; dehydrate; clear and cover as in the first method. For temporary preparations let dry after absolute alcohol and apply a drop of immersion oil directly on the section.     

Methods for the Study of Leaf Anatomy in Palms

Large size, hardness, combinations of thick-walled fibers and sclereids with thin-walled parenchyma cells, and the occurrence of silica, calcium oxalate, and tannins make anatomical preparations of palm leaves difficult. Samples for anatomical study should encompass one-half a pinna or a comparable portion from palmate and entire leaves including the midrib, all large ribs, and the margin. Similar pieces from herbarium specimens are reconstituted in glycerin alcohol, aerosol OT and distilled water (10:3:90). All samples are fixed in formol-acetic-alcohol (FAA) but stored in glycerin alcohol to minimize hardening. Transverse and longitudinal sections IS microns thick, epidermal macerations, and pieces for clearing and for scanning electron microscopy are prepared from the FAA fixed material. Samples for electroscanning are gradually changed to 100% acetone, critical point dried with CO₂, and coated with 100-300 angstroms of gold. Leaf material for microtomy is treated with hydrofluoric acid, embedded in Paraplast, and sectioned at 15 microns at a temperature of 7.2 C. Paraplast sections are floated directly on a modification of Sass' Adhesive III, mounted unstained or stained in safranin and fast green, and observed in polarized light. Epidermal peels are prepared by soaking pieces 5 mm square for 12-24 hours in undiluted bleach. Pieces for observation of transverse veins are cleared by treatment in 5% sodium hydroxide in a 60 C oven, washed rapidly in three changes of distilled water, and placed in one-third strength commercial bleach until clear. The same procedures can be used to prepare reproductive material for anatomical observations, but time schedules must be increased for larger specimens.     

Effect of calcium on intracellular pH

The effect of intracellular calcium on intracellular pH in the turtle urinary bladder was examined with phosphorus nuclear magnetic resonance. The turtle urinary bladder is capable of acidification in vitro and urinary acidification by this membrane is inhibited by an increase in intracellular calcium. Since calcium is capable of altering intracellular pH, it remains unclear whether the inhibition of urinary acidification is the result of an increase in intracellular pH. In the present study, intracellular calcium was increased by the cholinergic agent, carbachol, the ionophore A23187 and replacement of extracellular Na by sucrose. All agents decreased intracellular pH in the turtle bladder, thus suggesting that inhibition of urinary acidification by these agents is not due to an increase in intracellular pH.     

Tannic acid-iron alum reactions: stain of choice for macroscopic sections of brain to be embedded in plastic.

As a macroscopic stain for gross brain sections to be embedded in plastic, tannic acid-iron alum is superior to the generally recommended LeMasurier's variation of the Berlin blue technique because of its greater permanency in plastic. However, as originally adopted for use with brain tissue by Mulligan, the intense black staining of gray matter is too dark for plastic embedded specimens. A modification of this method designed to overcome this difficulty is described. Staining procedure: Wash formalin-fixed brain slices overnight in running water. Wash in distilled water, 2 changes, 30 minutes each. Place slices individually in Mulligan's solution at a temperature of 60-65 C for 4 minutes. Rinse in ice water for 10 seconds. Mordant in 0.4% tannic acid in distilled water for 1 minute. Wash in running tap water for 1 minute. Develop in 0.08% ferric ammonium sulfate in distilled water until gray matter is light gray, about 10-15 seconds. Wash in lukewarm running water for 1 hour, then gently hand-rub whitish film from myelinated surfaces. Store briefly in 3% formalin or 25% glycerine if necessary depending on plastic embedding procedure to be followed.     

Tannic Acid-Iron Alum Reaction: Stain of Choice for Macroscopic Sections of Brain to be Embedded in Plastic

As a macroscopic stain for gross brain sections to be embedded in plastic, tannic acid-iron alum is superior to the generally recommended LeMasurier's variation of the Berlin blue technique because of its greater permanency in plastic. However, as originally adopted for use with brain tissue by Mulligan, the intense black staining of gray matter is too dark for plastic embedded specimens. A modification of this method designed to overmme this difficulty is described. Staining procedure: Wash formalin-fixed brain slices overnight in running water. Wash in distilled water, 2 changes, 30 minutes each. Place slices individually in Mulligan's solution at a temperature of 60-65 C for 4 minutes. Rinse in ice water for 10 seconds. Mordant in 0.4% tannic acid in distilled water for 1 minute. Wash in running tap water for 1 minute. Develop in 0.08% ferric ammonium sulfate in distilled water until gray matter is light gray, about 10-15 seconds. Wash in lukewarm running water for 1 hour, then gently hand-rub whitish film from myelinated surfaces. Store briefly in 3% formalin or 25% glycerine if necessary depending on plastic embedding procedure to be followed.     

Immunocytochemistry of the acellular slime mold Physarum polycephalum. V. Cryosectioning now allows analysis of non-extracted plasmodia of any and all stages

The spatial distribution of cytoplasmic actin in endoplasmic drops as well as in plasmodial strands can be demonstrated in cryosections by fluorescently labelled phallotoxins and actin antibodies. Our results on cryosections show an identical fibrillar actin distribution as revealed in semithin sections after conventional fixation and embedding. Thus, it is now possible to apply immunocytochemical analysis to any and all plasmodial stages with or without prior fixation and without using extraction procedures. Consequentially the loss of soluble compounds during processing is avoided. The most protective pretreatment of the living specimens before freezing is a 15 min incubation in 1.5 M sucrose containing 50 mM KCl, 10 mM EGTA and 10 mM PIPES buffer, pH 7.0, at 4 degrees C.     

Water pH and urinary excretion in silver catfish Rhamdia quelen

The effect of acute exposure to different water pH levels on urinary excretion and plasma ion levels in silver catfish Rhamdia quelen was analysed. Fish were exposed to pH 4·0, 5·0, 7·5, 8·0, and 9·0 for 4 days and urine was collected. Other specimens were also exposed to the experimental pH for 24 h and blood was sampled. Urine flow rate, urine and plasma pH showed a significant trend to increase with the increase of water pH. Urinary Na⁺ excretion rate also increased and ammonia urinary excretion rate decreased with the increase of water pH. There was a significant trend to decrease volume, ammonia, Cl⁻ and Na⁺ urinary excretion rate with increasing mass in fish exposed to all pH levels studied. Plasma ammonia levels showed a slight decrease in fish exposed to water pH from 4·0 to 8·0, but those exposed to water pH 9·0 presented the highest ammonia levels. Most plasma ions and urinary excretion changes observed in silver catfish exposed to acidic or alkaline water were similar to those already detected in rainbow trout Oncorhynchus mykiss . In addition, the kidney and urinary bladder might participate on acid–base balance in silver catfish, since urine pH changed according to plasma pH.     

Microvideography of gas bladder inflation in larval walleye

High-resolution microvideograph observations supported the hypothesis that first filling of the gas bladder of larval walleye Stizostedion vitreum is accomplished by the fish penetrating the air-water interface to gulp air, then transmitting a swallowed air bubble through the gut and the pneumatic duct to the gas bladder. The snout of the larva penetrated the water surface with extension of most of the mouth into the air for only 1.5 s. An air bubble ( c. 100 μm) in the foregut was broken up into progressively smaller bubbles (10–15 μm), presumably by the combined effect of surfactant derived from the gall bladder and mechanical (peristaltic) action of the gut. These smaller bubbles seemed to be aligned in the pneumatic duct before being forced to the gas bladder by pressures generated from peristalsis.     

Scanning electron microscopy: Identification of mast cells by indirect cathodoluminescence

Summary The epithelial tissues of the rabbit gall bladder reacted for acid mucosaccharides were studied with the electron microscope. A series of acid mucosaccharide-containing ultrastructures of the gall bladder epithelium were observed in specimens treated with dialyzed iron, colloidal thorium and ruthenium red. In the epithelium stained with dialyzed iron, reactive ultrastructures are not only extra- but intracellular; the surface coat of the plasma membrane, pinocytotic vesicles, granules of secretion and certain elements of the Golgi apparatus. In the epithelial tissues stained by colloidal thorium or ruthenium red, the surface coat of the plasma membrane is the only ultrastructure which is reacted positively for the acid mucosaccharide stains. The present images of ultrastructural elements containing acid mucosaccharides are taken to indicate a multiple function of the substances in rabbit gall bladder epithelium and are well correlated with the results of previous light and electron microscopic studies on the gall bladder epithelium of various vertebrate species.     

Ampicillin Levels in Human Bile in the Presence of Biliary Tract Disease

Ampicillin levels were measured in the serum and in the bile from both the gall bladder and the common bile duct in patients undergoing surgery for biliary tract diseases. In patients with radiologically non-functioning gall bladders ampicillin was either not present or its concentration was lower than normal. Therapeutic levels were present in the common bile duct of all patients except those with obstruction of the common bile duct. Hence ampicillin fails appreciably to penetrate the obstructed viscus in obstructive biliary tract disease, and it is unlikely to be effective in treating infection associated with this.     

Biochemical mechanisms and effects of Mimosa pudica (Linn) on experimental urolithiasis in rats

Urolithiasis, following implantation of Zn discs in urinary bladder (foreign body insertion technique), was examined in albino rats of either sex. Marked variation was observed between sex, regarding the formation of bladder stones. Ethylene glycol (1%) mixed in drinking water for 4 weeks, was unable to augment Zn disc-induced stone deposition. Chemical nature of stones was identified as of magnesium ammonium phosphate type. Neither urinary pH nor infection in the urinary bladder/tract affected chemical nature and quantity of stone formed. There was no significant influence of electrolytes or metabolic products on the uroliths. No correlation could be drawn between the quality and quantity of uroliths formed and the urinary electrolytes concentration. M. Pudica was not effective in either preventing stone deposition or dissolving preformed stones.     

五氯苯酚(PCP)在鱼体胆囊内积累的研究

鱼体吸收水中的异生物质能迅速、大量地分配主胆囊。胆囊的功能好像一个强烈的生物浓集器,杂交鲫暴露在含PCP水溶液中48h,胆囊对五氯苯酚(PCP)的富集系数(BCF)高达11365.胆囊积累和排除PCP是随水温升高而增快的。草鱼胆汁内积累的PCP90%以上为结合态,并随暴露时间延长而增加,48h残留达到904mg/kg;BCF为6027.草鱼暴露在自来水和东湖水中48h,用气相色谱分析胆汁内的有机物分别为24和25个。这一结果说明了鱼胆汁可能用于监测水中某些异生物质。       

Effect of various antimicrotubular drugs on the osmotic water flow through the wall of frog's urinary bladder]

The action of antimicrotubular drugs (colchicine, vinblastine and copper) on the osmotic water flow through the wall of the urinary bladder of Rana temporaria has been studied. The osmotic gradient was made by five- or tenfold dilution of the internal Ringer solution. The water flow was estimated gravimetrically. The water flow was induced by pituitrin (50 milliunits/ml), cyclic AMP (cAMP, 0.5-10(-3) M) and nystatine (3.5-10(-5) M). Pituitrin and cAMP and all the antimicrotubular drugs were added from the serosal surface of the bladder. Nystatine was introduced with the help of a fixed polyethylene tube. Preincubation with colchicine lasted 4 hours and that with vinblastine and copper (CuSO4), 1 hour. The drug concentrations varied between 10(-5)--10(-4) M. All the drugs studied showed a significant inhibitory effect toward pituitrin. The action of cAMP on the water flow was seen inhibited in the presence of colchicine and copper. The nystatine induced water flow was supressed by copper, colchicine being in this case inactive. A conclusion is drawn that the inhibition of cAMP formation does not cause a decreased pituitrine effect in the presence of antimicrotubular drugs. It has been assumed that the microtubules may be involved in the directed water flow within the cell.     

Proteins of guinea-pig bile: selective resorption in the gall bladder

We have examined and compared the proteins present in guinea-pig bile as collected either from the common hepatic duct or from the gall bladder. Guinea-pig bile, collected from the common bile duct, has a rather low concentration of protein. Detailed examination shows that the concentrations of actively transported proteins such as immunoglobulin A and haptoglobin.haemoglobin complexes are markedly lower than in rats although the concentrations of proteins which, like albumin, leak non-specifically into bile are similar in the two species. We also find that the protein composition of guinea-pig bile is extensively and selectively modified by resorp-tion of protein in the gall bladder.     

The Ultrastructural Route of Fluid Transport in Rabbit Gall Bladder

The route of fluid transport across the wall of the rabbit gall bladder has been examined by combined physiological and morphological techniques. Fluid transport was either made maximal or was inhibited by one of six physiological methods (metabolic inhibition with cyanide-iodoacetate, addition of ouabain, application of adverse osmotic gradients, low temperature, replacement of Cl by SO₄, or replacement of NaCl by sucrose). Then the organ was rapidly fixed and subsequently embedded, sectioned, and examined by light and electron microscopy. The structure of the gall bladder is presented with the aid of electron micrographs, and changes in structure are described and quantitated. The most significant morphological feature seems to be long, narrow, complex channels between adjacent epithelial cells; these spaces are closed by tight junctions at the luminal surface of the epithelium but are open at the basal surface. They are dilated when maximal fluid transport occurs, but are collapsed under all the conditions which inhibit transport. Additional observations and experiments make it possible to conclude that this dilation is the result of fluid transport through the spaces. Evidently NaCl is constantly pumped from the epithelial cells into the spaces, making them hypertonic, so that water follows osmotically. It is suggested that these spaces may represent a "standing-gradient flow system," in which osmotic equilibration takes place progressively along the length of a long channel.