Stainable Substance Research Articles

Localization of megalin in rat vestibular dark cells and endolymphatic sac epithelial cells

Conclusion: Megalin immunoreactivity was observed in kidney proximal tubule cells, vestibular dark cells, and epithelial cells of the endolymphatic sac. Endocytic mechanisms appear to differ between the endolymphatic sac and proximal tubule cells. We speculate that megalin is secreted by a certain type of cell into the endolymphatic space, and is then absorbed from the endolymphatic space by another type of cell to maintain endolymphatic sac homeostasis. Objectives: We previously detected megalin immunoreactivity in the rat cochlear duct. Megalin may be involved in endocytosis in the vestibular organ and endolymphatic sac. To examine this possibility, we extended our immunocytochemical investigation to the rat inner ear cells with special attention to vestibular dark cells and endolymphatic sac. Materials and methods: We observed immunoreactivity of megalin under light and electron microscopy. The primary antibody was rabbit polyclonal antibody that had been raised against rat immunoaffinity-purified megalin. Results: The luminal membrane and subapical area of dark cells in the semicircular canal were immunolabeled. The stainable substance in the endolymphatic space was strongly stained. The cytoplasm of epithelial cells was also stained in various patterns.

Artifacts in Biological Confocal and Non-linear Microscopy

Conventional histological sectioning and staining techniques produce tissue sections, by large, independent from each other (e.g. the staining intensity depends on the total stainable substance within the section, but not influence by the section above or below it). Although the hardness of the structure in a given section may influence the quality of the following sections, never the less, each physical section can be generally regarded as an independent sampling event. However, the image intensity and relative contrast in an optical section obtained from confocal or non-linear optical microscopy varies depending on the position of the section where it was obtained. Therefore, in sampling terms, optical sections are dependent sampling events. Artifacts caused by the geometry and optical properties (e.g. absorption, scattering, refractive index) of objects within a specimen can interfere the interpretation of the result and hinder the usefulness of three-dimension reconstruction. Figure 1 demonstrates intensity variation in confocal and multi-photon micrographs due to the geometry of the specimen. Depends on the absorption coefficient and path length of the illumination and the fluorescecent light, a solid spherical object produced a 3D data set, when reconstructed, resemble an inverted Chinese rice bowl. Figure 2 shows a 3D reconstruction of an Arabidopsis thaliana mesophyll protoplast; note only half of the spherical cell facing the illumination is imaged as shown in the cut-off view in FIG 3.

Embryonic and postnatal accumulation of homogeneous substance in the endolymphatic sac in the guinea pig

Objective: The endolymphatic sac (ES) of vertebrates contains varying amounts of a homogeneous substance (HS) that stains deeply with basic aniline dyes. Histochemically, HS is characterized as a carbohydrate–protein complex, being both neutral and acidic in nature. In the present study, deposition of HS in the ES was studied in the guinea pig from the 3rd week of gestation to 104 weeks postnatally in order to find out if HS accumulates with age, at which point during embryonic development this substance appears, if its presence is correlated to the sense of hearing and if the amount of substance in the left versus right ear of one and the same animal is correlated to any degree. Methods: Sixty-nine endolymphatic sacs were evaluated in 38 guinea pigs. The ES specimens were sectioned for light and transmission electron microscopy and the amount of HS filling was categorized in four groups: none, low, medium and a high level of substance. Results: The substance was not discerned until after 7 weeks of gestation, when it filled only a minor part of the distal ES lumen. At 9 weeks gestation the nature of the substance altered, becoming homogeneous, as visualized by osmium–toluidine blue staining and approximately filling the distal half of the luminal space. In the postnatal period, 65% of ES specimens were filled with HS to the intermediate or proximal ES, whereas only 6.5% of the ES specimens were devoid of the substance. The extent of filling of the ES in the prenatal temporal bones was significantly less than postnatally ( P<0.0001, χ 2-test). The extent of postnatal filling was not correlated with age. Left and right ears were closely correlated in one and the same animal. Phagocytic cells were often found at the border between clear endolymph and stainable substance. Conclusion: The appearance of HS seemed to coincide temporally with the onset of hearing during the prenatal period indicating that it could play a part in normal inner ear functioning in the guinea pig. The close correlation regarding the level of the HS in the left and right ear, both pre- and postnatally could reflect a general symmetry in endolymph pressure–volume conditions within the inner ear fluid systems, as well as in the environmental hydrostatic pressure in the posterior cranial fossa.

Presence of Glycosaminoglycans in the Endolymphatic Sac

Earlier morphological investigations have revealed that the endolymph, which is present in the endolymphatic sac (ES) seems to differ from that found elsewhere in the labyrinth, in that it contains a stainable substance. Histochemical investigations indicate that this substance is rich in glycosaminoglycans (GAGs). It has been speculated that the stainable substance might play a role in regulation of fluid and ions and also, possibly, the pressure within the endolymphatic sac compartment. Endolymphatic sac specimens obtained from adult guinea pigs, rats, mice and in vitro cultured fetal inner ears were incubated with monoclonal antibodies against epitopes, and after enzymatic digestion revealed five different GAGs: hyaluronan, chondroitin-4-sulphate + dermatan sulphate, chondroitin-6-sulphate and keratan sulphate. In order to verify the specificity of these antibodies, otocysts from fetal mice were incubated in the same way. These cartilaginous specimens are known to contain GAGs in abundance and served as positive controls. The results indicate that the hyaluronan visualized by the monoclonal antibody is present to a large extent in the lumen of the ES and in the epithelial cells. Keratan sulphate and chondroitin-4-sulphate 4- dermatan sulphate are present within the epithelial lining, in the subepithelial tissue, and to some minor extent in the lumen, while chondroitin-6-sulphate does not show any specific staining.

Effect of glycerol on the guinea pig inner ear after removal of the endolymphatic sac.

The effects of glycerol were investigated in guinea pigs in which the endolymphatic sac (ES) and duct (ED) had been obliterated unilaterally. Twenty-four hours after obliteration of the right ES and ED, glycerol was injected intraperitoneally. One to two hours after administration of glycerol, collapse or folding of Reissner's membrane was observed in the operated cochlea. No remarkable change was found in the nonoperated ear. The lumen of the ES of the nonoperated ear collapsed with intraluminal stainable substance within 1 h. The volume of the stainable substance increased with time and could still be seen 4 h after the injection of glycerol. Twenty-four hours after the injection of glycerol, the configuration of the ES was normal again. In addition, the animals showed marked unilateral (operated side) vestibular dysfunction after injection of glycerol. These findings suggest that the ES also has a secretory capacity and plays an important role in inner ear fluid volume and pressure regulation.

Formation and degradation of lipid bodies found in the riboflavin-producing fungus Ashbya gossypii

Microdroplets in the hyphae of Ashbya gossypii were found to become stained by Nile red. Purification of the stainable substance showed that the yellow fluorescent bodies consisted of triacylglycerol. During growth on glucose as the carbon source 8%–12% of the mycelial dry weight was found to be neutral lipid. When glucose declined in the medium, the content decreased to 3%–4% and the respiration quotient shifted to 0.6 indicating a reserve function of the fat. The fatty acid composition of the storage lipid was found to be strongly influenced by the carbon source. Mycelia cultivated on glucose contained 5% linoleic acid and 20% palmitoleic acid in their neutral fat while linoleic acid made up 54% and palmitoleic acid was not detectable (< 0.1%) in soybean-oil-grown mycelia. When plant oil was given as the sole carbon source, the fatty acid composition of the storage lipid showed a high similarity to the fed fat. 14C-labelled free oleic acid added to a culture growing on soybean oil was immediately incorporated into the fungal lipid. A pulse of 0.9 g/l free palmitoleic acid, fed during growth on olive oil, increased the content of this particular fatty acid in the fungal triacylglycerol from 0.8% to 9.6%. In addition, a liberation of free fatty acid and diacylglyceride was found in the culture supernatant when pure triolein was given as the sole carbon source. Obviously, the fungus cleaved the lipid serving as the carbon source extracellularly and used the liberated fatty acids for its storage lipid formation.

Histological Changes in Endolymphatic Sac after Interruption of Endolymphatic Duct in Guinea Pigs

Several investigators have recently reported that experimental conditions in a mouse model which affect the pressure/volume ratio in the endolymphatic space increase the amount of a stainable substance in the endolymphatic sac (ES) lumen. They suggest that this stainable substance may play an important role in the regulation of pressure/volume homeostasis. The guinea pig is often used as an animal model of experimentally induced endolymphatic hydrops. In this study we interrupted the endolymphatic duct selectively in guinea pigs. The histology of the ES was studied by light microscopy at various time intervals after the operation. The amount of the stainable substance in the ES lumen on the operated side was increased for a few weeks postoperatively. This finding suggests that in the guinea pig model also the stainable substance in the ES lumen is involved in the regulation of pressure/volume homeostasis in the endolymphatic space.

Immunohistochemical Study on Localization of Tenascin in the Cervical Region of Rat Spinal Cord During Development.

Tenascin (TN) is transiently expressed in some organs and tumors at various developmental stages. It has also been reported that tenascin was a major extracellular matrix component in a developing nervous system, and played an important role in neurite outgrowth and pattern formation of the cerebral and cerebellar cortices.In the present study, we examined, under light and electron microscopes, the localization of TN in the cervical region of rat spinal cord during the late prenatal and early postnatal stages by means of conventional and immunohistochemical methods. In the specimens stained with hematoxylin-eosin and thionin, the following results were obtained: the structure of spinal cord was developing at the postnatal stages and attained a definite form around the 5th day after birth. Nissl bodies in neurons were maturing during this period and the processes of neurons elongated toward white matter. Concomitantly, neuroglia were also increased in the white matter.In accordance with it, luxol fast blue (LFB) and sudan black B stainable substances appeared in the white matter, especially ventral and lateral funiculi around the 5th day of age.The results of immuno-light microscopical observation showed that TN-labeling was observed only in the gray matter of the spinal cord at the 18th day of gestation, but both in gray and white matters from the 20th day of gestation to the 5th day after birth. The most intense TN-labeling was detected in the boundary of gray and white matter in the ventral and lateral funicili at the 2nd to 5th day after birth. As the development proceeded, up to the 2nd week after birth, TN-labeling was mainly distributed in the white matter except for the ventral region of dorsal funiculus and almost completely disappeared from the gray matter. Here, it was also noticed that the ventral region of dorsal funiculus became stainable with LFB or sudan black B between the 2nd and 3rd week after birth and at the same stage of development, TN-labeling became detectable in this region.At the electron microscopical level, TN-labeling was detected in the intercellular spaces of the gray matter during the 1st to 10th day of age. That is, TN was expressed associating with the cytoplasmic membrane and processes of neurons and neuroglia. TN-labeling was also observed along the cytoplasmic membrane of myelinating oligodendrocytes and in the cytoplasm of neuroglia, especially oligodendrocytes in the white matter at the 10th day to the 2nd week after birth. Clear reaction products for TN could not be detected in the synaptic region.From this evidence, it is concluded that TN mediates the interaction of neuron and neuroglia during late prenatal and early postanatal stages and plays an important role in elongation of neuronal processes and migration of neuroglia as well as the myelination in the developing rat spinal cord.

Experimental interruption of the endolymphatic duct and its effect on the DC potential in the endolymphatic sac.

The endolymphatic sac (ES) of the guinea pig was isolated from the remainder of the inner ear by means of surgical interruption of the endolymphatic duct (ED). The DC potential in the ES lumen and the morphology of the ES were studied using glass micro-electrodes and a light microscope at various time intervals after the interruption of ED. The DC potential did not significantly change 1 h postoperatively, compared to findings in the non-operated ear, but a significant decrease of the DC potential was observed after 1, 3 and 7 days postoperatively. A histologically-stainable substance in the ES lumen was enhanced on the operated side. The isolated ES shows a disturbance of mechanism(s) maintaining the DC potential and there is a secretion of a stainable substance into its lumen.

Glycoconjugates in the human fetal endolymphatic sac as detected by lectins

The distribution of glycoconjugates in the 11 to 16 weeks old human fetal endolymphatic sac (ES) was analyzed using six biotinylated lectins; Wheat germ agglutinin (WGA), Abrus precatorius agglutinin (APA), Ulex europaeus agglutinin I (UEA-I), Ricinus communis agglutinin 120 (RCA120), Helix pomatia agglutinin (HPA), Concanavalin A (ConA). In the 11 week old human fetus, fluorescent reactions with WGA, APA, RCA120 and ConA were detected in the ES. There was almost no reaction with HPA and UEA-I. In the 14 week old human fetus, however fluorescent reactions with HPA and UEA-I appeared. This result suggests that the presence of glycoconjugates changes during the maturation process of the ES. Glycoconjugates detected with HPA were related to the epithelial cell elements of the ES epithelium. The reaction with UEA-I suggests that the stainable substance present in the ES lumen may be secreted locally by the ES itself.

The endolymphatic sac and inner ear homeostasis. I: Effect of glycerol on the endolymphatic sac with or without colchicine pretreatment

The combined effects of glycerol and colchicine on the endolymphatic sac were investigated in mice. Glycerol induced signs of secretion from the epithelium with formation of secretory granules in the light epithelial cells. Other characteristics of the epithelial lining were also changed resulting in an increased widening of the lateral intercellular spaces, a partial collapse of the lumen and with a deposition of a stainable substance within the lumen. This reaction lasted from 30 min to 24 h following the injection. Pretreatment with colchicine was found to decrease or inhibit the glycerol-induced secretion of macromolecules into the sac. The lumen collapsed but frequently there was no presence of stainable substance. Animals treated with both glycerol and colchicine showed marked signs of inner ear malfunction which could indicate that the secretory activity in the sac might be closely related to the regulation of inner ear fluid homeostasis and that functional disturbances in this system may lead to disorders of inner ear function.

Effects of hyperosmolar substances on the endolymphatic sac.

The murine endolymphatic sac (ES) was studied 15 min to 8 hours after intravenous glycerol administration. Initially the ES showed varying degrees of obliteration and this was mostly pronounced at 15-60 min after the injection. After 2 hours the normal volume was regained and after 4 hours the lumen was dilated to 160% of its normal volume. After 6-8 hours the ES had almost regained its normal appearance. The epithelial lining showed an increase in the number of granular cells which, after two hours, reached a peak of 15.8% (p less than 0.01) compared to normal controls which showed 6.1% granular cells of total cell population in the ES. The increase of granular cells was accompanied by filling of the ES lumen with a stainable substance. The epithelial reaction may serve the purpose of counteracting decreases in endolymph pressure either in the ES or in the entire labyrinth.

Uptake of Radioactive Sulphur in the Endolymphatic Sac:An Autoradiographic Study

Autoradiographic uptake of labelled sulphur (S35) in the endolymphatic sac was analysed after performing surgical labyrinthectomy and after systemic administration of glycerol and ethacrynic acid. Accumulation of radioactive substance was observed after surgical labyrinthectomy and associated with a deposition of stainable substance in the endolymphatic sac lumen. Increased activity was noted in the epithelial layer after administration of ethacrynic acid, whereas glycerol did not increase radioactivity in the endolymphatic sac despite the deposition of stainable substance therein. This fact was believed to depend on a too short time interval between the injection and the sacrifice of the animals, as well as a weaker concentration of the sulphated compound within the endolymphatic sac. The results suggest that the endolymphatic sac may secrete sulphur-containing substances presumably associated with a deposition of a stainable substance in the endolymphatic sac.

Cytochemical staining characteristics of peripheral nodes of Ranvier in hexacarbon intoxication.

Changes in the distribution of stainable gap substance and subaxolemmal density at peripheral nodes of Ranvier in hexacarbon-intoxicated rats have been studied by light and electron microscopy. Cupric ion binding to the nodal gap substance was seen in normal nodes as a discrete annulus by the formation with ferrocyanide of Hatchett's Brown reaction product. Staining of osmicated fibres with ferrocyanide ions alone gave a deposit of black reaction product at the subaxolemmal region at nodes of Ranvier. Paranodal distension by neurofilamentous masses and separation of the terminal myelin loops in the early phase of paranodal dilatation produced no change in the distribution of the two kinds of stainable material. Paranodal myelin retraction with increases both in nodal gap width and nodal axon diameter resulted in displacement and attenuation of both stained regions. Axonal protrusion at the nodal region tended only to displace the stained gap substance, but sometimes it resulted in its attenuation. Occasionally loss of subaxolemmal staining was found. The possible functional relevance of these abnormal findings is discussed in relation to changes in conduction in affected nerves.

The cysts in C cell complexes of dog thyroids studied by immunoperoxidase staining and autoradiography.

The C cell complexes in the dog thyroid are the remnants of ultimobranchial bodies which retain fetal characteristics, and contain cyst structures showing various sizes, shapes and liminal contents. This study deals with these cysts with regard to their morphology, immunoperoxidase reactions to anti-calcitonin, anti-19S thyroglobulin and anti-C-thyroglobulin antisera, and ability to incorporate radioiodine. Small cysts lined by densely packed small cells are devoid of a stainable substance in their lumina and show no reaction to any of the antisera. They are regarded as immature structures. The large cysts, covered mainly by a single layer of cuboidal cells, store varying amounts of secretory products in their lumina. The secretory products showing colloid-like, flocculent, or granular features are intensely stained with the anti-19S thyroglobulin and anti-C-thyroglobulin antisera. However, they scarcely accumulate silver grains after injection of Na125I. C cells are distributed in the cyst epithelium but the cystic contents reveal no immunoreaction for calcitonin. In conclusion, the present study indicates that the cyst structures in C cell complexes synthesize and store a thyroglobulin-like glycoprotein but are not directly involved in thyroid hormone synthesis.

Electron microscopy of cell surfaces of Paramecium caudatum stained with ruthenium red

The cell surface of Paramecium caudatum, syngen 3, was examined by electron microscopy using ruthenium red (RR) staining. The RR-positive surface coat is of uniform thickness and is found on the entire surface of paramecia, including the gullet area. The surface coat is also observed on the membranes in the tightly united region of conjugating cells. Measurements of the thickness of the surface coat in six stocks of P. caudatum demonstrate a significant difference between complementary mating types: cells of mating type VI have a thicker layer than those of mating type V. No detectable differences in morphology of the surface coat are observed between mating reactive and unreactive cells. Observations in detached cilia indicated that changes in the morphology of the surface coat provoked by the detachment procedure have no effect on mating reactivity. RR stainable substances are detected on both sides of ciliary membranes.

Studies on the Aleurone Layer I. Oonventional and Fluorescence Microscopy of the Cell Wall With Emphasis on Phenol-Carbohydrate Complexes in Wheat

In the ungerminated seed, the cell walls of the aleurone layer of wheat (Triticum aestivum cv. Heron) are strongly autofluorescent and are stained by aniline blue, resorcinol blue, toluidine blue, and the periodic acid-Schiff's reaction. Thin-layer chromatography of acid or alkaline extracts and microspectrofluorimetry indicate that the fluorescent component of the wall is ferulic acid. During germina-tion, the ferulic acid disappears from the aleurone cell wall in a specific pattern and its loss is accompanied by loss of stainable substance except in the innermost layer of the wall. These facts suggest that the aleurone cell wall contains a ferulic acid-carbohydrate complex which renders it relatively resistant to the action of the hydro-lases which digest the endosperm.

Skin and hair regeneration after calciphylaxis. Histologic changes during formation and shedding of the calciphylactic carapace of the rat.

Detailed histologic analysis of the stages of localized calciphylaxis in rat skin suggests that the calcified carapace does not contain the complete skin and that sufficient connective tissue and epithelium remains behind to serve as a source of regeneration. The regenerate is incomplete and has many features of scar tissue. Elastic fibers are not restored. The number of hairs is greatly diminished. It could not be ascertained whether all of the hairs growing later come from surviving hair roots, or if true neogenesis plays a part. Other details of interest are the greatly increased amount of calcium in growing hairs and the absence of alcian blue stainable substances in mast cell granules following calciphylaxis.

Modifications in the neurosecretory system of the fish, Misgurnus fossilis, indiced by lasting inanition.

The reaction of the hypothalamic neurosecretory system to prolonged starvation was examined in the fish,Misgurnus fossilis L. Two and four months of starvation resulted in storage of neurosecretory substance in the perikarya of the nucleus preopticus and in the preoptico-hypophysial tract. Further starvation, prolonged to five or seven months, caused degenerative changes in the nucleus preopticus. However, there were no drastic changes in the amount of stainable substance in the neurohypophysis. The examination of control animals revealed that the nucleus preopticus undergoes seasonal changes which differ from those brought about by starvation.

Cytological observations on a duck hybrid: Anas clypeata × Anas penelope

The chromosomes of the two parental forms and those of the hybrid do not show any detectable numerical or large structural differences. The pairing in the hybrid is frequently abnormal in that both free ends (highly charged with stainable substances) of many chromosomes show strong pairing, while midsegments repel each other. This partial failure of paring suggests that in the two parental forms gene mutations rather than chromosomal structural changes are responsible for incipient divergence.It is suggested that the stainable substance of the chromosomes acts as a carrier mechanism ensuring orderly pairing, segregation and numerical constancy of bivalents in the hybrid where the partner chromosomes have become partly non-homologous.The distinction between long and short chromosomes is typical for the avian karyotype, its evolutionary significance is discussed.