Electron Microscopic Histochemical Methods Research Articles

Cytochemistry of sialoglycoconjugates, lysozyme, and β‐defensin in eccrine glands of porcine snout skin as studied by electron microscopy

In most mammals except for humanoid primates, eccrine glands are confined to the skin of a series of specific body regions. Sialic acids and antimicrobial substances exhibit various functional properties and serve as a component of nonspecific defense against micro-organisms, respectively. In this study, the distribution of these moieties was studied by electron microscopic histochemical methods. The eccrine glandular acini consisted of two types of dark cells as well as clear cells. The secretory granules and Golgi apparatus of both types of dark cells contained sialic acid residues linked to α2-6Gal/GalNAc. On the other hand, sialoglycoconjugates with Siα2-3Galβ1-4GlcNAc sequence were confined to those of the Type II dark cells. In addition, lysozyme and β-defensin were mainly detected in the secretory granules of the Type II dark cells. These secretory products may create a defensive barrier against microbial invasion and play an essential role in preservation of the integrity of porcine snout skin as a sensory organ.

Histochemistry of Sialoglycoconjugates in Goat Submandibular Glands

The functional properties of sialic acids appear to be manifold. Therefore, they are considered as essential components of saliva. In this study, the localization of sialoglycoconjugates in the submandibular glands of Japanese miniature (Shiba) goat was examined by light and electron microscopic histochemical methods. The submandibular glands exhibited a large amount of sialic acids. Additionally, sialic acids with O-acetyl substitutions were detectable in the mucous acinar cells and serous demilunar cells. According to lectin histochemical methods, the mucous and serous cells mainly contained the Siaα2-6Gal/GalNAc sequence. These sialoglycoconjugates generated by the submandibular glands may specifically participate in the maintenance of the viscoelastic properties of saliva, protection of oral tissues and prevention of pathogenic microbial attacks. Therefore, our results suggest that they are essential components of saliva to maintain oral health.

Histochemical analysis of glycoconjugates in the eccrine glands of the raccoon digital pads.

The distribution and selectivity of complex carbohydrates in the eccrine glands of the digital pads in the North American raccoon (Procyon lotor) were studied using light and electron microscopic histochemical methods, particularly lectin histochemistry. In the eccrine glands, the dark cells exhibited neutral and acidic glycoconjugates with different saccharide residues (alpha-L-fucose, beta-D-galactose, beta-N-acetyl-D-glucosamine and N-acetyl-neuraminic acid); the clear cells contained numerous glycogen particles and showed a distinct reaction of alpha-L-fucose. The presence of complex carbohydrates with various terminal sugars was evident in the excretory duct cells. In addition, beta-D-galactose and N-acetyl-neuraminic acid residues were mainly observed in the luminal secretion. The glycoconjugates produced by the eccrine glands of the raccoon digital pads may protect the epidermis against physical damage or microbial contamination. In this way, the normal functioning of the sensory apparatus of the foot pads is ensured.

Histochemistry of complex carbohydrates in the ceruminous glands of the goat.

The localization and chemical nature of complex carbohydrates in the ceruminous glands of the Japanese miniature (Shiba) goat were studied using light and electron microscopic histochemical methods, particularly lectin histochemistry. The epithelial cells and luminal secretion of the caprine ceruminous glands contained large amounts of neutral and smaller amounts of acidic glycoconjugates with different terminal sugars (alpha- d-mannose, alpha-L-fucose, alpha-N-acetyl-D-galactosamine, beta-D-galactose, beta-N-acetyl-D-glucosamine, and N-acetyl-neuraminic acid). Several sugars (alpha-L-fucose, beta-D-galactose, beta-N-acetyl-D-glucosamine, and N-acetyl-neuraminic acid) were also detectable in the secretion of the sebaceous glands. The results obtained are discussed with regard to the specific function of the glandular secretion mixture. The complex glycoconjugates found in the ceruminous gland secretion may control viscoelasticity of and bacterial proliferation within the cerumen in order to protect the external auditory canal against physical damage or microbial attacks.

Vacuole formation in the endothelium of rat extremity vessels depends on fixation techniques and vessel type

Applying immersion fixation for electron microscopy, huge clear endothelial membrane-bound vacuoles of 0.1–3 μm diameter were noted in the extremity veins of Sprague–Dawley rats. Histological and electron microscopic histochemical methods were applied to determine whether they were the product of programmed cell death or any other kind of cell damage. Image analyzer was used to measure the total area of the vacuoles in the endothelium cells. Neither lipid content nor acidic phosphatase activity could be identified in the vacuoles. In saphenous and brachial veins, the vacuoles occupied 20.6±2.21% and 18±2.45% of the endothelium, respectively. Venous endothelium of two different strains of rat also contained the vacuoles. No such structures appeared in extremity arteries. Long-term tilting did not influence vacuolization. Using in vivo whole body fixation, only pinocytotic and dense microvesicles, but no vacuoles were noted. In conclusion, the clear vacuolar structures represent neither lipid inclusions nor secondary lysosomes. The method of tissue fixation is critical when venous endothelial vesicles are investigated. It is presumed that the vacuoles originated from intra- or intercellular microstructures, and that in case of the collapsible vein segments, their size is increased under the pathological—hypoxic and low-pressure—conditions of in vitro fixation.

Parasympathetic control of the heart. III. Neuropeptide Y-immunoreactive nerve terminals synapse on three populations of negative chronotropic vagal preganglionic neurons.

The vagal postganglionic control of cardiac rate is mediated by two intracardiac ganglia, i.e., the sinoatrial (SA) and posterior atrial (PA) ganglia. Nothing is known about the vagal preganglionic neurons (VPNs) that innervate the PA ganglion or about the neurochemical anatomy of central afferents that innervate these VPNs. These issues were examined using light microscopic retrograde labeling methods and dual-labeling electron microscopic histochemical and immunocytochemical methods. VPNs projecting to the PA ganglion are found in a narrow column exclusively in the ventrolateral nucleus ambiguus (NA-VL). These neurons are relatively large (37.6 +/- 2.7 microm by 21.3 +/- 3.4 microm) with abundant cytoplasm and intracellular organelles, rare somatic and dendritic spines, round uninvaginated nuclei, and myelinated axons. Previous physiological data indicated that microinjections of neuropeptide Y (NPY) into the NA-VL cause negative chronotropic effects. The present morphological data demonstrate that NPY-immunoreactive nerve terminals formed 18 +/- 4% of the axodendritic or axosomatic synapses and close appositions on VPNs projecting to the PA ganglion. Three approximately equal populations of VPNs in the NA-VL were retrogradely labeled from the SA and PA ganglia. One population each projects to the SA ganglion, the PA ganglion, or to both the SA and PA ganglia. Therefore, there are both shared and independent pathways involved in the vagal preganglionic controls of cardiac rate. These data are consistent with the hypothesis that the central and peripheral parasympathetic controls of cardiac rate are coordinated by multiple potentially redundant and/or interacting pathways and mechanisms.

Localization of epidermal hyaluronan in the foot pads of the North American raccoon (Procyon lotor)

The localization of the epidermal hyaluronan in the digital foot pads of the North American raccoon (Procyon lotor) was examined by light and electron microscopic histochemical methods. The thick epidermis contained five epidermal layers composed of typical keratinocytes. In the stratum basale and stratum spinosum, hyaluronic acid was clearly detectable at cytoplasmic processes, in close vicinity to the surface coat of the plasma membrane, and/or in the intercellular space between the basal and spinous cells. The results suggest that epidermal hyaluronan that fills the intercellular space may particularly control water maintenance in the epidermis of the raccoon digital pads to guarantee the optimal mechanoreceptive function of the very specific and sensitive sensoric subepidermal equipment in this mammalian species.

Histochemical analysis of glycoconjugates in the ceruminous glands of the North American raccoon ( Procyon lotor)

The distribution and selectivities of glycoconjugates in the ceruminous glands of the North American raccoon (Procyon lotor) were studied by light and electron microscopic histochemical methods, particularly lectin histochemistry. In the modified apocrine glands present, the apocrine secretion mode was combined with exocytosis, whereby the secretory epithelium and the luminal secretion of the ceruminous glands exhibited considerable amounts of complex carbohydrates with various terminal sugars (alpha-D-mannose, beta-D-galactose, alpha-L-fucose, alpha-N-acetyl-galactosamine, beta-N-acetyl-D-glucosamine, N-acetyl-neuraminic acid). Alpha-L-fucose and N-acetyl-neuraminic acid were distinctly prominent in secretory granules or within the free surface coat of the plasma membrane of the glandular cells, as well as in the luminal secretion. Several free sugars (alpha-D-mannose, alpha-L-fucose, beta-D-galactose, beta-N-acetyl-D-glucosamine) were also detectable in the secretion of associated sebaceous glands. The ceruminous gland secretion may control viscoelasticity and/or bacterial degradation of the glandular secretion mixture to improve the protection of the external auditory canal against physical damage or microbial contamination.

Lectin histochemistry of glycoconjugates in the feline hair follicle and hair

The distribution of glycoconjugate in the feline hair follicle and hair was studied by light and electron microscopic histochemical methods. The hair apparatus was found to contain considerable amounts of complex carbohydrates with different saccharide residues (alpha-D-mannose, beta-D-glucose, alpha-L-fucose, beta-N-acetyl-D-glucosamine). Variations of those were detected in the plasma membrane of the hair follicle cells during the course of their differentiation and keratinization, namely, alph-D-glucose, alpha-L-fucose and beta-N-acetyl-D-glucosamine in the suprabulbar and bulbar regions. The reaction level of sialic acid residues in the plasma membrane decreased in some cell layers during the course of differentiation. The results obtained from the present study indicated that interaction between saccharide residues of neutral carbohydrates and sialyl groups during the anagen phase might contribute to cell keratinization in hair follicles and hairs. It is discussed whether the existence of glycogen in outer root sheath cells might enable these cells to provide other hair apparatus cells with energy when necessary. Moreover, it became obvious from variations in sialyl residue distribution that cell differentiation processes terminate first of all in Huxley's and Henle's layers within the suprabulbar region of the hair follicle, as followed by the hair cortex.

Histochemistry of carbohydrates as performed by physical development procedures.

The histochemistry of carbohydrates demonstrated by means of physical development procedures has been reviewed in terms of the use and reliability of the procedures, physical developers, practice of the procedures, a fundamental series of light and electron microscopic methods and certain other promising aspects of this area of histochemistry. A line of fundamental light- and electron-microscopic histochemical methods for carbohydrates using physical development procedures such as periodic acid-thiocarbohydrazide-silver protein-physical development (PA-TCH-SP-PD), high- or low-iron diamine (HID or LID)-TCH-SP-PD and lectin-gold (LT-G)-PD and related methods has been found to be more efficient, compared with those without physical development procedures. Since a series of other promising histochemical methods for carbohydrates using physical development procedures have been derived or are now being introduced, these procedures could be regarded as an unusually potent vehicle for effectively advancing carbohydrate histochemistry in both light and electron microscopy.

Acetylcholinesterase in immature thalamic neurons: Relation to afferentation, development, regulation and cellular distribution

The transient appearance of intense acetylcholinesterase reactivity in some immature, noncholinergic neurons has not been adequately explained. In this study two questions were investigated that relate to several possible roles for acetylcholinesterase. First, what factors influence the onset and maintenance of reactivity? Second, what are the temporal and spatial features of the cellular expression in relation to stages of neuronal development? Using light- and electron-microscopic histochemical methods, the non-cholinergic ventrobasal complex in thalamus of the immature rat was examined. Ultrastructural observations on fetal ventrobasal complex demonstrated that the onset of acetylcholinesterase reactivity precedes ingrowth of most extrinsic afferents. These inputs are, therefore, unlikely to provide the signal for onset. In transplants and expiants, acetylcholinesterase persisted in ventrobasal complex neurons independent of their principal afferents. However, afferentation can affect reactivity. The patterned variation in intensity, characteristic of infant ventrobasal complex, was dramatically altered by unilateral interruption of its afferentation. The changes in intensity patterning could reflect changes in acetylcholinesterase metabolism, since postnatal treatment with an irreversible inhibitor (diisofluorophosphate) in vivo demonstrated resynthesis of acetylcholinesterase. The period of peak intensity of acetylcholinesterase reactivity normally began abruptly at 18 days of gestation ±12 h and continued until 4–6 days postnatally. This period follows neurogenesis and migration, but precedes active synaptogenesis. It coincides with outgrowth and initial contacting of cell processes in the ventrobasal complex. The timing complements the ultrastructural finding that acetylcholinesterase-dependent reaction product most commonly is localized to small patches of surface membrane, where distal processes contact each other, non-synaptically. Together these data suggest three points. First, that the expression of acetylcholinesterase in the immature ventrobasal complex neuron is probably under active metabolic control, responsive to both intrinsic and environmental factors. Second, that acetylcholinesterase expression is unlikely to result from a transient cholinergic input. Third, that the temporal and spatial characteristics of histochemical reactivity enable exclusion of several previously suggested explanations for the occurrence of acetylcholinesterase in the ventrobasal complex. At the same time, these data provide a basis for a first-order hypothesis: acetylcholinesterase plays a role in the formation of early intercellular relationships and/or process outgrowth. Possible mechanisms for these roles are discussed that would be consistent with the present data.

A quantitative study of anterograde and retrograde axonal transport of exogenous proteins in olfactory nerve C-fibers

The pike olfactory nerve which consists of a homogeneous population of C-fibers of 0.25 μm diameter or less was used to study quantitatively both anterograde and retrograde axoplasmic transport of wheat germ agglutinin and horseradish peroxidase. It was found that even in these extremely thin axons anterograde and retrograde transport takes place. Activity distribution profiles (transport profiles) for retrograde transport were established and found to be similar to the typical profiles of anterograde transport as they consisted of a small rapidly moving peak and a saddle region followed by the bulk of the material which moved more slowly. Horseradish peroxidase activity profiles were obtained both after injection into the synaptic region and after injection into the perikaryal region. From these transport profiles a maximal velocity of 25 mm/d (19°C) for the leading peak and of about 7 mm/d for the slower component could be determined. There is no significant difference between the velocities for anterograde and retrograde transport. In the case of wheat germ agglutinin, only injection into the synaptic region resulted in typical transport profiles (retrograde transport) with a peak and saddle region. The maximum velocities of retrograde transport were about the same as for horseradish peroxidase [26 mm/d and 7 mm/d (19°C)]. The electron microscopic analysis of horseradish peroxidase revealed that after injection into the olfactory bulb it was taken up into the neurons where it was found mainly in multivesicular bodies (0.5 μm diameter). In longitudinal sections of the nerve similar but slightly more elongated organelles (diameter 0.25 μm, length 0.4 μm) were found in those segments in which the slowly moving bulk of the peroxidase activity was located. The number of these organelles decreased with distance from the site of injection. The horseradish peroxidase transported within the leading peak could not be assigned to specific structures although several electron microscopic-histochemical methods were applied. It was concluded that anterograde and retrograde transport occur simultaneously in these axons, and that, therefore, even the large organelles, each of which almost fills the axon, must be able to pass each other. This would necessitate that the axons are able to transiently enlarge their diameter considerably.

Morphological changes in rat striatal boutons after chronic methamphetamine and haloperidol treatment

Dopaminergic (DA) synaptic boutons were identified in rat striatum with an electron microscopic histochemical method. In rats which developed behavioral hypersensitivity after treatment with methamphetamine (MAP) for about 2 weeks, significantly fewer DA boutons were found. This effect was specific to DA boutons without mitochondria and was not seen in boutons with mitochondria. The density of granular synaptic vesicles in DA boutons, however, did not change significantly. These morphological changes would represent a high capability of nerve cells to reorganize synaptic connections under altered chemical environments, but they could not be related uniquely to behavioral hypersensitivity, as similar effects were observed in rats treated with haloperidol which did not develop behavioral hypersensitivity. Peculiarly, the effects of MAP treatment on both behavior and DA boutons were prevented by combined administration of haloperidol.

The pathogenesis of Dupuytren's contracture: Contractile mechanisms of the myofibroblasts

The role of myofibroblasts in the pathogenesis of Dupuytren's contracture was investigated by light and electron microscopic histochemical methods. Dupuytren's myofibroblasts contain an intracellular contractile mechanism that is driven by the dephosphorylation of adenosine triphosphate. Our study of calcium adenosinetriphosphatase (ATPase) activities verifies that the site of this energy system is on the myofilaments of the myofibroblasts. The degree of ATPase activity, as determined by cell counts, appeared to correlate with the residual contracture as predicted by the Legge and McFarlane Outcome Standard Formula. Further, alcian blue staining on the ultrastructural level indicates that the myofibroblasts are associated with each other and with surrounding collagen by a glycosaminoglycan matrix 300 to 1000 A thick. Collagen fibrils are attached by a similar matrix comprised of 100 A thick fibrils. The dynamic cellular architecture of the multiple adjacent myofibroblasts with their connections to surrounding collagen may be partially responsible for the residual clinical deformities seen in this disease.

Subcelluläre lokalisation des calciums in den neurosekretorischen axonterminalen der Neurohypophyse der ratte nach erregung durch die verabreichung von hypertonischer kochsalzlösung

The calcium accumulating capacity of neurohypophysial axon terminals stimulated by the intravenous injection of hypertonic saline has been studied by using light and electron microscopic histochemical methods and X-ray microanalysis. In the stimulated neurosecretory terminals Ca++ containing granules appeared in the mitochondria. Smaller granules of similar morphological appearance occurred in the smooth endoplasmic reticulum as well. It is suggested that the mitochondria and the smooth endoplasmic reticulum are intimately involved in the regulation of the intraaxonal free calcium level during the process of vasopressin secretion.

Autonomic innervation of the urogenital system: adrenergic and cholinergic elements

The major organs of the male urogenital (UG) system have been examined in various mammals, including man, using light and electron microscopic (EM) histochemical methods. For the light microscopic study, the urinary bladder, the vas deferens and the penis (corpora cavernosa and corpus spongiosum) were studied in the rat, cat, dog, monkey and man using a glyoxylic acid (GA) method modified for peripheral adrenergic nerve fibers, and a thiocholine method for acetylcholinesterase (AChE). Fine structural analysis was done on the vasa of rat, cat, monkey and man, and on the bladder and penis of cat, dog, monkey and man. Tissue was fixed in glutaraldehyde (GMO) as a control or in glutaraldehyde-dichromate (GDC) for the specific localization of norepinephrine (NE). All organs studied demonstrated numerous adrenergic nerve fibers throughout the muscular layers, in the connective tissue, and in the adventitia of most blood vessels. These fibers had a brilliant fluorescence when visualized with the GA method, and demonstrated many varicosities with small (400–600 A) and/or large (800–1200 A) granular vesicles in both control and GDC-fixed tissue examined with the EM. Evaluation of the vesicles with the analytical electron microscope (AEM) verified that those in the GDC-fixed tissue were chrome-positive, and, therefore, NE-containing. In the vas and penis, acetylcholinesterase(AChE)-positive nerve fibers were encountered less frequently at the light microscopic level than adrenergic fibers, and few typical cholinergic varicosities were seen in these organs with the EM. In the bladder, cholinergic nerves were seen with about the same frequency as adrenergic fibers in both light microscopic and EM preparations. Also observed frequently in each of the viscera were varicosities with large to very large (800–2000 A) granular vesicles of the kind presently hypothesized to be peptidergic or purinergic. Few varicosities of the type considered sensory, with large (800–1200 A) clear vesicles and numerous mitochondria, were observed in this tissue. Evidence from this study suggests that mammalian UG organs are innervated extensively by adrenergic nerves, and, excepting the bladder, have a limited cholinergic innervation; in the bladder, numerous fibers of each type can be found. In addition, another type of nerve fiber, perhaps peptidergic or purinergic, is found in large numbers in each of the organs studied and thus may represent a significant effector of autonomie regulation.

Changes in ultrastructure and subcellular mercury distribution in rat liver after separate and combined administrations of mercuric mercury and selenite

AbstractThe ultrastructural changes and the subcellular mercury distribution in the liver of rats, which were injected with HgCl2 and/or Na2SeO3, were studied using electron microscopy and electron microscopic histochemical methods. Mercuric chloride (10 μmol per kg body weight) caused heavy cell damage both in hepatic parenchymal cells and in reticuloendothelial cells. Selenite (10 μmol per kg body weight) simultaneously administered had little influence upon the morphological feature of hepatic injury caused by mercuric chloride. However, the density of fine gold grains, which display the mercury deposits, appeared to be higher in the livers of rats treated with mercury plus selenite than in those administered mercury alone. In both groups of rats (those which received mercury alone and those which received mercury plus selenite), mercury was predominantly deposited in Kupffer cells and localized in lysosomal dense bodies. Also, in hepatic parenchymal cells, mercury was mainly deposited in the lysosomal dense bodies neighbouring bile canaliculus, although the level of mercury estimated by the density of gold grains seemed to be lower than that in Kupffer cells.

Die Veränderungen der Butyrylcholinesterase-Aktivität der fenestrierten Capillaren in der Area postrema während der postnatalen Entwicklung

Ontogenetic changes in butyrylcholinesterase (BuChE) activity of fenestrated capillaries in the rat Area postrema lying outside of the blood-brain barrier were investigated by means of light and electron microscopic histochemical methods. Marked enzyme activity could be demonstrated in the capillary endothelial cells of the neonatal rat. The intensity of the light microscopically detectable BuChE activity in the capillaries decreased gradually during the postnatal ontogenesis, but the capacity of the enzyme synthesis of the endothelium remained unchanged in the adult rat, too. Our results do not indicate any direct correlation between BuChE positivity of capillaries and the existence of the blood-brain barrier.

The histochemistry of complex carbohydrates in the epithelium lining the ventral prostate of the rat.

In the epithelium lining the ventral prostate of the rat, complex carbohydrate-containing structures have been studied by means of both light and electron microscopic histochemical methods. According to light microscopy, the free surface and granules of different sizes in the distal cytoplasm of the epithelial cells were found to exhibit positive reactions for complex carbohydrates with 1,2-glycol and acidic groups and sialic acid residues. In addition, secretory substances within the glandular lumen were shown to exhibit positive reactions for similar groups and saccharide residues of complex carbohydrates. In electron microscopy, the surface coat of the plasma membrane, certain elements of the Golgi apparatus and lysosomal dense bodies were found to exhibit positive reactions for glycoproteins with 1,2-glycol groupings. The histophysiological significances of the carbohydrate-containing structures have been discussed with special reference to the known physiological functions of the prostate in the rat.

Histochemische analyse der vielfältigen wirkungen von organophosphaten ( Wofatox, DFP) bei ratten

In vivo effects of Wofatox and DFP has been studied by light and electron microscopic histochemical methods in the central nervous system, in cardiac and striated muscles of rats. Singleintraperitoneal injection of Wofatox and DFP LD50 resulted in complete inhibition of the histochemically detectable acetyIcholinesterase (AChE) activity in the central nervous system. Various striated muscles exhibit different degrees of sensitivity towards inhibitors, neither can the enzyme activity of some motor end plates be completely inhibited. In addition to AChE inhibition in striated muscles, organophosphorous compounds induce also subacute myopathic changes. In the case of a lethal intoxication, ultra-structural alterations suggesting myocardial lesion can be observed.