Histochemical analyses of anti-microbial substances in canine perianal skin with special reference to glandular structures.

Circumanal glands are prominent features of the canine perianal skin, which are often located near to the sebaceous glands and apocrine glands. As the functional relevance of circumanal glands is yet unknown, we studied the localisation of sialic acids and anti-microbial substances (lysozyme, immunoglobulin A, lactoferrin, ß-defensin) in these glandular structures by lectin histochemistry and immunohistochemistry. The glands exhibited a number of sialic acids that were linked to α2-6Gal/GalNAc and α2-3Galß1-4GlcNAc. Additionally, lysozyme, lactoferrin and ß-defensin could be demonstrated in the three types of skin glands, whereas IgA was only detectable in the apocrine glands. The results of the study suggest the specific significance of the circumanal glands. Independent of a certain endocrine role, their products may mainly function as protective agents to preserve the integrity of the anal region, considering that sialic acids and anti-microbial substances are important in defence mechanisms.

Histochemical localization of sialic acids and antimicrobial substances in eccrine glands of porcine snout skin.

The distribution of sialic acids and antimicrobial products (lysozyme, IgA, lactoferrin, ß-defensin 2) as well as Rab3D in the eccrine glands of porcine snout skin was studied by sialoglycoconjugate histochemistry and immunohistochemistry. The secretory epithelium consisted of two types of secretory cells: dark and clear cells. The dark cells exhibited considerable amounts of sialoglycoconjugates, which included O-acetylated sialic acids, whereas sialic acids in the sequence Siaα2-3Gal1-4GlcNAc were confined to some of the dark cells. All antimicrobial substances and Rab3D were demonstrated to be also mainly present in some of the dark cells. Additionally, in the cytological and cytochemical features, the different characteristics were observed among the dark cells. The results obtained are discussed with regard to the functional significance of the eccrine glands. The secretory products elaborated by this gland type may function as protective agents in order to preserve the skin integrity of the snout region, considering that sialic acids and antimicrobial substances are important in general defense mechanisms.

Immunohistochemical aspects of anti-microbial properties in goat submandibular glands.

Salivary glands are known as the principal source of anti-microbial substances, which are considered to be essential components of saliva. The distribution of anti-microbial substances in the submandibular glands of Japanese miniature (Shiba) goat was studied using immunohistochemical methods as performed by physical development procedures. In the goat, anti-microbial substances such as lysozyme, IgA, lactoferrin and ß-defensin were demonstrated to be immunolocalized in the submandibular glands, especially in the serous demilunes and duct cells. The results obtained are discussed with regard to the specific functions of the saliva. The presence and secretion of anti-microbial substances suggest that they participate in the maintenance of oral health among the elements of saliva.

Histochemical properties of sialic acids and antimicrobial substances in canine anal glands.

The functional properties of sialic acids appear to be manifold. Additionally, antimicrobial substances serve as a non-specific defense against microorganisms. In this study, therefore, the localization of sialic acids and antimicrobial substances in the anal glands of dog was studied by sialoglycoconjugate histochemistry and immunohistochemistry. The secretory epithelium, luminal secretions and excretory ducts exhibited high levels of sialoglycoconjugates that terminated in Siaα2-6Gal/GalNAc or Siaα2-3Gal1-4GlcNAc. Additionally, O-acetylated sialic acids were detectable in these glandular structures. Antimicrobial substances, such as lysozyme, immunoglobulin A (IgA), lactoferrin and the peptide group of ß-defensins, were also demonstrated as products of the anal glands. The results obtained are discussed with regard to the functional significance of the anal glands. These secretory products may create a defensive barrier against microbial invasion at the anal mucosa.

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.

Localization of hyaluronic acid in the seminal vesicles of the miniature pig.

We studied the detailed localization of hyaluronic acid in the seminal vesicles of the miniature pig, using hyaluronic acid-binding protein as a specific histochemical probe at the ultrastructural level. According to the results, the basolateral surface of the plasma membrane of the glandular epithelial cells, was found to contain hyaluronan. However, abundantly present was hyaluronan in the subepithelial connective tissue, in particular, in the extracellular matrix surrounding the fibroblasts, smooth muscle cells, small blood vessels and capillaries. The substance was also observed in the surface coat of the plasma membrane of the fibroblasts, but not in that of the smooth muscle cells. The findings suggest that hyaluronan in the seminal vesicles of the miniature pig is synthesized onto the surface coat of the plasma membrane of the fibroblasts, is contributed to the extracellular matrix, and consequently concentrates in the subepithelial connective tissue. The substance may particularly be involved in a variety of cellular functions to maintain morphological organization as well as to regulate physiological homeostasis in the reproductive organ of this species, rather than participate in sperm functions.

Morphological, histochemical and immunohistochemical characterization of secretory production of the ciliary glands in the porcine eyelid.

In addition to performing general histology and cytology of the ciliary glands of the miniature pig, we studied the localization of glycoconjugates and beta-defensins in these glands with the use of carbohydrate histochemical and immunohistochemical methods. The secretory cells of the glands were equipped with non-homogeneous secretory granules, a well-developed Golgi apparatus and rough endoplasmic reticulum. The secretory epithelium and luminal secretion of the glands contained large amounts of neutral and acidic glycoconjugates with various saccharide residues (alpha-L-Fuc, beta-D-Gal, alpha-D-GalNAc and sialic acid). The sebaceous glands and tarsal glands also exhibited positive reactions to most of the histochemical methods. Additionally, the antimicrobial peptide group of beta-defensins was demonstrated to be products of the ciliary glands, as well as the sebaceous glands and tarsal glands. The results obtained are discussed with regard to the specific function of the ciliary glandular secretions. These secretory products may be related to the moistening and general protection of the skin surface of the eyelid and ocular surface.

Anatomicopathological study of vascular and biliary systems using cast samples of Fasciola-infected bovine livers.

In 117 livers with fascioliasis, this study was focused on the number of Fasciola, the number and intrahepatic localization of affected hepatic ducts and bile ducts, and the degree of fibrosis in the hepatic segments and bile ducts. The degree of pathological changes in bile ducts caused by fascioliasis was classified into five levels. The site of Fasciola habitation was most often the hepatic ducts of the porta hepatis: it was the left hepatic duct in 101 livers and the right hepatic duct in 88 livers. Casts were prepared by infusing synthetic resin into the hepatic arterial, portal, hepatic venous and biliary systems of 15 bovine livers with fascioliasis and then examined. In the left lobe, quadrate lobe, and caudate process where atrophic fibrosis was noted, the bile ducts became rod-shaped by losing branches, and the samples resembled dead branches of liver. Portal branches were thinned or completely terminated with marked fibrosis. Fine and irregular newly formed bile ducts not parallel with portal branches were observed in livers with markedly chronic fascioliasis. Distal portal branches in the right lobe, caudate lobe, and papillary process showed hypertrophic proliferative changes. The arterial system was generally well developed in thickened walls of bile ducts and formed vascular beds, and surrounded the bile ducts as tubes. In livers with severe fibrosis, capillaries were markedly developed and resembled glass cotton.

Cytochemical localization of glycoconjugates in the secretory epithelial cells lining the prostate gland of the miniature pig.

The aim of the study was to emphasize several specific functional aspects of the mammalian prostate gland. In this connection, the subcellular localization and characterization of glycoconjugates in the secretory epithelial cells were examined that line the prostate gland of the miniature pig, using cytochemistry, including lectin-gold methods. The results verified a diversified pattern of glycoconjugates in the structures of the secretory cells. The secretory granules and the free surface coat of the plasma membrane contained several sugar moieties such as alpha-D-Man, alpha-D-Glc, alpha-L-Fuc, beta-D-Gal, GalNAc, GlcNAc and NeuAc (sialic acid), whereas the Golgi apparatus exhibited GlcNAc and sialic acid residues. In addition, the cisterns of the endoplasmic reticulum showed alpha-D-Man and alpha-D-Glc moieties. It seems likely that the secretory glycoconjugates demonstrated contribute to favourable conditions for fertilization.

Three-dimensional vasculature of the bovine liver.

To clarify anatomical distribution of Fasciola infection, the vascular and ductal architectures of the liver were studied by means of corrosion cast technique using synthetic resin. The arteria hepatica propria (AP) passes as the arteria gastroduodenalis (AG); AP becomes the left trunk after the porta hepatis; AP passes on the right side of vena porta communis (VPC) and projects AG while curving in a U-shape below the portal vein. Hepatic veins located between the vena hepatica media (HM) and vena hepatica dextra (HD) varied widely among specimens and were irregular, including the vena hepatica dorso-lateralis sinistra (Hds), vena hepatica dorso-lateralis dextra (Hdd), vena hepatica lobi caudati (Hlc), venae hepaticae processus caudati (Hpc), venae hepaticae processus papillaris (Hpp), and the hepatic vein to the dorsal intermediate part, which directly or indirectly drained into the vena cava caudalis. The courses of the bovine hepatic veins were markedly diverse, and anastomoses between vena hepatica sinistra (HS) and Hds were observed in about a half of the livers. The portal vein entered the liver as VPC slightly above the centre of the right lobe on the visceral surface. The intermediate or transverse part [pars transversa trunci sinistri (PTS)] of truncus sinister (TS), which extends from the entry of the portal vein into the left lobe of the liver, was slightly arched downward [pars umbilicalis trunci sinistri (PUS)]. The portal vein further arched from the distal end of TS to the umbilical vein and ran towards the inter-lobar incision between the left lobe and quadrate lobe. Based on these branches, hepatic segments were determined as 13 or 14 areas. A total of 15 bile ducts were derived from various lobes. The hepatic duct was about 2.6-6 cm long from the confluence of the right and left hepatic ducts to the division of the cystic duct and the common hepatic duct.

Ultracytochemical demonstration of glycoproteins in the eccrine glands of the digital pads of the North American raccoon (Procyon lotor).

In the eccrine glands of the digital pads of the North American raccoon (Procyon lotor), ultrastructural localization of glycoproteins with various saccharide residues was studied using preferably lectin cytochemical methods. Secretory granules observed in the dark cells exhibited glycoproteins with different terminal sugars (alpha-D-mannose, beta-D-galactose, beta-N-acetyl-D-glucosamine and sialic acid). The cytoplasm of the clear cells contained numerous glycogen particles. Several sugars were also detectable in the surface coat of the plasma membrane of the secretory cells. The results obtained could be helpful in understanding secretion production and cell related secretion functions of the eccrine glands of the raccoon digital foot pads.

Glycoconjugate histochemistry of the seminal vesicles of the Japanese miniature (Shiba) goat.

The present study localizes and characterizes complex glycoconjugates in the seminal vesicles of the Japanese Shiba goat, using several carbohydrate histochemical procedures, including lectin techniques at light and electron microscopic levels. Glandular epithelial cells and luminal secretions were shown to contain neutral and acidic glycoconjugates with various saccharide residues, such as alpha-D-Man, alpha-D-Glc, alpha-L-Fuc, beta-D-Gal, GalNAc, GlcNAc, and NeuAc (sialic acid). The terminal oligosaccharide chains of sialoglycoconjugates present in the seminal vesicles were NeuAc alpha 2-3Gal beta 1-3GalNAc and NeuAc alpha 2-3Gal beta 1-4GlcNAc. In addition, in lysosomes of the glandular epithelial cells alpha-D-Man, alpha-D-Glc, GlcNAc and NeuAc (sialic acid) residues could be detected, the secretory vesicles contained alpha-L-Fuc, and the endoplasmic reticulum exhibited alpha-D-Man and alpha-D-Glc residues. The complex glycoconjugates with various sugar residues found in the seminal vesicles of the goat may be involved in various fertilization-related events.

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.

The application of three-dimensional internal structure microscopy in the observation of mare ovary.

The ovary of the mare has a unique structure which differs totally from that of other mammals. However, because of its relatively large size, conventional histological techniques were unsuitable for the observation of the internal structure of the whole ovary. Three-dimensional internal structure microscopy (3D-ISM) consists of a cryotome-CCD camera-laser disc recorder-PC-based control system coupled with a graphic workstation. The internal structure of the ovary is observed by processing over more than 1,000 stored images of serially sliced surfaces of each frozen equine ovary. The 3D reconstruction was done using the full-coloured, volume-rendering method. The relationship between the localization of medulla, cortex and ovulation fossa was clarified. The ovulation fossa is localized in the centre of the ovary and is surrounded by a broad ovarian cortex. A trace of ovulation was observed only at the ovulation fossa. Medulla are localized in narrow peripheral areas. The phenomenon of the competition to occupy the cortical area ahead of the ovulation fossa by developing secondary follicles was visualized. Spatial localization of various sized follicles was identified from 3D-reconstructed images. In this study, it has been clarified that application or this novel computerized technique can clarify the anatomical arrangements of the equine ovary and the complex mechanism of equine follicular development.

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.

Establishment of a swine monocyte cell line.

A swine monocyte cell line was established from peripheral blood sample collected from a healthy adult male pig. The cloned cells grow actively in forming monolayers in both glass and plastic cell culture flasks with the growth medium reported previously (Kadoi, 2000) at 36.5 degrees C incubation. The plating efficiency is more than 95%. Densely grown cells in flasks show an epithelioid morphology. The fundamental properties of the cells were examined for cytological definition as monocytes. A positive property detected was guinea pig complement receptor, porcine IgG receptor, non-specific esterase, and acid phosphatase. A significant phagocytic activity proved by the inoculation of Saccharomyces cerevisiae is also one of the characteristics observed in the LPS-activated cells.

Ultracytochemistry of glycosaminoglycans in the canine knee synovium.

The accurate localization and nature of glycosaminoglycans (GAGs) in the canine knee synovium were studied by ultracytochemical methods that involved high or low iron diamine-thiocarbohydrazide-silver proteinate (physical development) staining in combination with enzyme digestion control procedures. The results obtained indicated that heparan sulfates and hyaluronan were present mainly in the plasma membrane of the B (fibroblast-like) cells. In contrast, the plasma membrane of the A (macrophage-like) cells showed negative reactions after the histochemical examination. Dermatan sulfates, chondroitin sulfates (A and/or C) and hyaluronan were localized in the extracellular matrix of the synovial intima, whereby dermatan sulfates were confined to the fibrous component, whereas chondroitin sulfates and hyaluronan were found in the interfibrous matrix. Heparan sulfate was the only notable GAG molecular species localized in the basement membrane of the capillary wall. It is obvious that differences in the quality and localization of glycosaminoglycans in the canine synovial tissue are of specific interest in understanding normal functions as well as pathological alterations of the knee synovium in mammals.

Histochemical analyses of glycosaminoglycans in the synovial membrane of the canine knee joint.

The accurate localization and nature of glycosaminoglycans in the synovial membrane of the canine knee joint were examined histochemically by means of the selective sensitized diamine procedures based upon high and low iron diamine stainings in combination with enzyme digestions. Using these methods, it was possible to clearly and easily detect exceedingly small amounts of glycosaminoglycans in synovial tissues, which cannot be visualized by methods employed to date. The sensitized high iron diamine (S-HID) procedure resulted in positive reactions of varying intensities in the intercellular matrix of synovial intima, and in the extracellular matrix and small capillary walls of the superficial layer in the synovial subintima, and also reacted vividly in the extracellular matrix and blood vessel walls of the deeper layer in the synovial subintima. In particular, the sensitized low iron diamine (S-LID) procedure resulted in positive reactions of the extracellular matrix in the synovial subintimal layers. The S-HID and S-LID procedures combined with the enzyme digestions proved that glycosaminoglycan molecular species such as chondroitin sulfate A/C, dermatan sulfate, heparan sulfate and hyaluronic acid are present in various concentrations in the synovial membrane of the canine knee joint. The present results were discussed with reference to the histophysiological and pathophysiological functions of glycosaminoglycans in the synovium of domestic mammals.

Cytological and lectin histochemical characterization of secretion production and secretion composition in the tubular glands of the canine anal sacs.

The study reports on secretion production and composition in the tubular glands of the canine anal sacs. For this purpose, light and electron microscopical (TEM, SEM) as well as several histochemical methods for the demonstration of lysosomal acidity, lipofuscin, and complex carbohydrates were used. The glandular tubules exhibited a pseudostratified epithelium with secretory cells of a different shape as related to secretion production activity, and regionally varying amounts of basal cells. Flat, cuboidal or columnar cells with or without apocrine-like protrusions were assembled in one glandular endpiece, although grouping of these cell types often occurred. Active secretory cells were columnar with many cytoplasmic vesicles and a typically merocrine and/or micro-apocrine exocytosis of vesicle contents. Additionally, many lysosomes of different sizes could be found, whereby in aged cells giant secondary lysosomes (autophagolysosomes, about 7 microm in diameter) occupied the major cell part. These giant lysosomes were shed by an apocrine-like process forming a final bottleneck stage of the upper cell part, and consisted of ceroid-type lipofuscin. The general carbohydrate histochemical and the lectin histochemical methods revealed that the secretion produced was composed of strongly concentrated neutral glycoproteins with the following saccharide residues: alpha-D-mannose, beta-D-galactose, beta-N-acetyl-D-glucosamine, alpha-L-fucose and N-acetyl-neuraminic acid (sialic acid); the luminal secretion contained only beta-D-galactose and, especially, N-acetyl-neuraminic acid. This luminal secretion showed a spatially orientated maturation beginning in terminal tubular regions and finishing near the excretory duct, independent of the different secretory cell types. The results obtained demonstrated highly active secretion production, with a regional variation in the glandular tubule, and at least three different modes of secretion by the secretory cells, whereby the shedding of giant lipofuscin granules seems to be very specific. The high amounts of sialic acids in the glycoproteins found may influence the rheological properties of the secretion by their water-binding capacities.

Immunohistochemical localization of inhibin and steroidogenic enzymes in the ovary of common tree shrew (Tupaia glis) and northern smooth-tailed tree shrew (Dendrogale murina).

To study the ovarian function of the Order Scandentia, the localization of inhibin and steroidogenic enzymes (3 beta-hydroxysteroid dehydrogenase/isomerase and aromatase) in the ovaries of common tree shrew (Tupaia glis) and northern smooth-tailed tree shrew (Dendrogale murina) was immunohistochemically analysed. As in the results reported for other mammals, inhibin alpha-chain was localized in the follicular epithelium of secondary or Graafian follicles in the two species. The localization of aromatase in the ovary of these two species, however, was different. In the common tree shrew, the aromatase was localized in the thecal cells, whilst in other mammals it is localized in the granulosa cells. These results indicate that in the ovary of the common tree shrew, the oestradiol may be synthesized in the thecal cells.