Identification of collagen, elastic, elaunin and oxytalan fibres in ganglia of the myenteric plexus of the human oesophagus

Summary The connective tissue associated with the myenteric plexus of the human oesophagus was studied by light and electron microscopy. Collagen fibres were identified by picrosirius staining with polarization microscopy and from their fine structural morphology. A capsule of connective tissue invests the ganglia while septa of connective tissue separate groups of ganglion neurons, surrounding each individual ganglion neuron and each nerve bundle. Collagen fibrils surround the ganglia, each ganglion neuron and each nerve bundle. The fibrils are disposed in various orientatons forming networks. Elastic, elaunin and oxytalan fibres were identified by their staining characteristics and fine structural morphology. The bulk of the ganglion sheath consists of coarse elastic fibres and elaunin fibres. Elaunin and oxytalan fibres form the intraganglionic network. Oxytalan, elaunin and elastic fibres appear to be located in areas related to different stresses and deformation to which the ganglia of the myenteric plexus are exposed during the contraction of the esophageal wall. The ganglia of the myenteric plexus of the human oesophagus show structural organisation of the connective tissue component similar to that seen in sympathetic and parasympathetic ganglia.     

Omega-exonuclease activity in basophils of certain animal species

Blood films of dogs, cats, cattle, horses, goats, pigs, rhesus monkeys, gibbon and alpaca and bone marrow smears of pigs, dogs and cats were stained to demonstrate omega-exonuclease, a marker enzyme for basophils in humans. Omega-exonuclease activity was found only in basophils of the dog, cat, rhesus monkey and gibbon. Positively stained basophils were readily discerned even when rare.     

Microscopic anatomy of the visceral fasciae

The term ‘visceral fascia’ is a general term used to describe the fascia lying immediately beneath the mesothelium of the serosa, together with that immediately surrounding the viscera, but there are many types of visceral fasciae. The aim of this paper was to identify the features they have in common and their specialisations. The visceral fascia of the abdomen (corresponding to the connective tissue lying immediately beneath the mesothelium of the parietal peritoneum), thorax (corresponding to the connective tissue lying immediately beneath the mesothelium of the parietal pleura), lung (corresponding to the connective tissue under the mesothelium of the visceral pleura), liver (corresponding to the connective tissue under the mesothelium of the visceral peritoneum), kidney (corresponding to the Gerota fascia), the oesophagus (corresponding to its adventitia) and heart (corresponding to the fibrous layer of the pericardial sac) from eight fresh cadavers were sampled and analysed with histological and immunohistochemical stains to evaluate collagen and elastic components and innervation. Although the visceral fasciae make up a well‐defined layer of connective tissue, the thickness, percentage of elastic fibres and innervation vary among the different viscera. In particular, the fascia of the lung has a mean thickness of 134 μm (± 21), that of heart 792 μm (± 132), oesophagus 105 μm (± 10), liver 131 μm (± 18), Gerota fascia 1009 μm (± 105) and the visceral fascia of the abdomen 987 μm (± 90). The greatest number of elastic fibres (9.79%) was found in the adventitia of the oesophagus. The connective layers lying immediately outside the mesothelium of the pleura and peritoneum also have many elastic fibres (4.98% and 4.52%, respectively), whereas the pericardium and Gerota fascia have few (0.27% and 1.38%). In the pleura, peritoneum and adventitia of the oesophagus, elastic fibres form a well‐defined layer, corresponding to the elastic lamina, while in the other cases they are thinner and scattered in the connective tissue. Collagen fibres also show precise spatial organisation, being arranged in several layers. In each layer, all the fibrous bundles are parallel with each other, but change direction among layers. Loose connective tissue rich in elastic fibres is found between contiguous fibrous layers. Unmyelinated nerve fibres were found in all samples, but myelinated fibres were only found in some fasciae, such as those of the liver and heart, and the visceral fascia of the abdomen. According to these findings, we propose distinguishing the visceral fasciae into two large groups. The first group includes all the fasciae closely related to the individual organ and giving shape to it, supporting the parenchyma; these are thin, elastic and very well innervated. The second group comprises all the fibrous sheets forming the compartments for the organs and also connecting the internal organs to the musculoskeletal system. These fasciae are thick, less elastic and less innervated, but they contain larger and myelinated nerves. We propose to call the first type of fasciae ‘investing fasciae’, and the second type ‘insertional fasciae’.     

Immunofluorescence studies with a specific antiserum to the microfibrillar protein of elastic fibres. Location in elastic and non-elastic connective tissues.

The microfibrillar protein (MFP) of foetal bovine ligamentum nuchae elastic fibres was prepared and used to produce a monospecific antiserum. Indirect immunofluorescence studies employing the specific antiserum demonstrated its selectivity for MFP associated with elastic fibres. The antiserum was demonstrated to react with vascular elastic tissue, perivascular connective tissue and reticular basement membranes in a variety of tissues. This evidence suggests that the MFP (or an immunologically related protein) is not confined to elastic fibres but is widely distributed in connective tissue being associated with both elastin and collagen fibres.     

Connections between Adrenergic Nerves and other Tissue Components in the Eye

The connections between adrenergic nerve fibres and other ocular structures were studied in normal embryonic material (man, dogs, cats, guinea-pigs, and rats) as well as with a special vessel injection technique (adult rats, guinea-pigs, and rabbits). It was established that adrenergic fibres are a normal constituent of the cornea. The adrenergic nerves were more numerous in the embryo than in the adult, and also occurred within the embryonic corneal epithelium. These intraepithelial fibres disappear shortly after birth. Adrenergic fibres running in the connective tissue without connection to vessels were further found in the iris, the limbus region, the chamber angle (of the guinea-pig predominantly) and in the chorioid. It cannot be excluded that these fibres innervate some connective tissue component. In the sphincter pupillae, only a few adrenergic fibres were connected to the vessels, such as was the case also in the ciliary muscle of the guinea-pig. Under the ciliary epithelium of the ciliary body and the ciliary processes there was a thick and dense plexus of adrenergic fibres. Only a restricted number of them was associated with the vessels. The “capillaries” of the ciliary processes were remarkable in that they seemed to possess adrenergic fibres. No adrenergic innervation to the melanophores was apparent.     

An unusual squamous cell carcinoma in a sheep: a case report

Squamous cell carcinoma (SCC) is a tumour consisting of squamous epithelial cells. This tumour is common in horses, cows, cats and dogs, but relatively uncommon in sheep, goats and pigs. In this study, we discuss gross, radiological and histopathological features of an oral SCC in a female sheep. A 3-year-old ewe with a progressive mass located in the intraoral cavity and with a history of reduced appetite, weight loss, salivation and halitosis was referred to the veterinary clinic. At clinical examination, a mass was located in the floor of the mouth, its surfaces showed signs of ulceration and haemorrhage. Lateral radiograph of the mandible region near the tumour showed bone destructive changes. Biopsy specimen was taken and histopathologic examination showed moderately differentiated oral SCC.     

Orcein-picroindigocarmine--a new multiple stain

A new "orcein-picroindigocarmine staining", a colour combination of orcein, indigo carmine, and picric acid, was developed for histological applications. The new technique was tested on different human tissues. Colours ranging from red to brown, yellow, green and blue were observed in paraffine sections of tissues stained by this method. Nuclear structures in all tissues were stained dark brown to dark blue. Squamous epithelium was stained light brown with varying shades of blue in upper horny layers, whereas the ciliated epithelium was tinged blue grey. When connective tissue was stained, collagen fibrils appeared strongly blue next to elastic fibres, which took on a rust brown tinge; cellular components were all coloured brown. The matrix of hyaline cartilage was stained in different shades of blue, with the chondrocytes rust brown. Sections of bone components appeared dark blue to dark green. Skeletal muscle cells were coloured yellow and green with blue collagenous septa. The new staining is useful for distinguishing connective tissue components such as elastic fibres and collagen fibrils. It also demonstrates chondrocytes in favourable contrast to the cartilage matrix. The technique produces aesthetic staining colouring that could supplement histological investigations and provide an alternative to other staining materials.     

Vertebrate hosts of Aedes aegypti and Aedes mediovittatus (Diptera: Culicidae) in rural Puerto Rico

The distribution of Aedes (Stegomyia) aegypti (L.), the main vector of dengue viruses (DENV) worldwide, overlaps with Aedes (Gymnometopa) mediovittatus (Coquillett), the Caribbean treehole mosquito, in urban, suburban, and rural areas. Ae. mediovittatus is a competent vector of DENV with high rates of vertical DENV transmission in the laboratory. This study determined whether Ae. mediovittatus feeds on humans and compared its feeding patterns with co-occurring Ae. aegypti in two rural communities of Puerto Rico. Adult mosquitoes were captured for three consecutive days every week from July 2009 to May 2010 using BG-Sentinel traps with skin lures that were placed in the front yard of houses in both communities. Three methods were used to identify the 756 bloodmeals obtained in this study: a multiplex polymerase chain reaction (PCR) for humans and dogs targeting cytochrome b; a PCR targeting the 16S rRNA; and a nested PCR targeting cytochrome b. Ae. mediovittatus fed mostly on humans (45-52%) and dogs (28-32%) but also on cats, cows, horses, rats, pigs, goats, sheep, and chickens. Ae. aegypti fed mostly on humans (76-79%) and dogs (18-21%) but also on cats, horses, and chickens. Our results indicate that Ae. mediovittatus may have a relatively high rate of vector-human contact, which might facilitate virus transmission or harborage in rural areas of Puerto Rico.     

Immunohistochemical detection of estrogen receptor alpha in articular chondrocytes from cows, pigs and humans: in situ and in vitro results.

Clinical observations suggest that estrogens are involved in the pathogenesis of postmenopausal osteoarthritis, but only little is known about the influence of these hormones on articular cartilage cells. The effect of estradiol is mediated by estrogen receptors alpha and beta. The goal of the present study was to search for estrogen receptor alpha in articular tissue from cows, pigs and humans by immunohistochemistry to form a basis for in vitro studies. In addition, we also tried to detect estrogen receptor alpha in cultivated articular chondrocytes from cows and bulls under certain culture conditions. Estrogen receptor alpha is detected by the use of antibody 13H2 in articular chondrocytes from cows, bulls, pigs and humans. Chondrocytes are physiologically exposed to reduced oxygen tension. In isolated articular chondrocytes from cows and bulls incubated either with 21% O2 or with 5% O2 positive cells were also found. These positive results therefore encourage testing the influence of estradiol on cultivated articular cartilage cells in these species under different culture conditions.     

Differential distribution of the fibres of the collagenous and elastic systems and of glycosaminoglycans in the rat pubic joint

The pubic joint of male and female rats was studied at the light- and electron microscopical levels using methods that selectively disclose the extracellular matrix fibres and glycosaminoglycans. The interpubic tissue showed no difference between sexes (including pregnant and intrapartum females). The medial ends of the pubic bones were covered by articular caps of hyaline cartilage that blended in the midline. The whole articular cartilage was covered dorsally and ventrally (as well as craneally and caudally) by a typical perichondrium. The differential distribution of the fibres of the collagenous and elastic systems in the pubic joint agreed with the results reported in the literature for other rat cartilages. Collagen fibres, composed mainly of type-I collagen, were localised to the fibrous perichondrium and bone. Type-II collagen was localised to the central nucleus of hyaline cartilage, whereas reticulin fibres (rich in type-III collagen) were found in the adventitial loose connective tissue adherent to the most superficial layer of the perichondrium. The central nucleus of hyaline cartilage possessed the two types of elastic-related fibres: elaunin fibres were localised mainly to the chondrogenic layer of the perichondrium, whereas oxytalan fibres were found in the matrix that surrounded the chondrocytes. The bulk of the glycosaminoglycans present in the pubic joint cartilage corresponded to hyaluronic acid and chondroitin sulphate. The propriety of classification of the rat pubic joint as a true synchondrosis (instead of symphysis), and the fact that the unaltered pelvis of the rat seems to be adequate for normal parturition, are discussed.     

The thickness, shape and arrangement of elastic fibres within the nuchal ligament from various animal species

As shown by statistical evaluation carried out within various animal species (e.g. ruminants, equids, carnivores and proboscidates) the thickness of elastic fibres of the nuchal ligament is a specific character, i.e. unrelated to the somatic size of taxonomically different specimens. On the whole, ruminants are characterized by thicker elastic fibres than those from equids and carnivores. Moreover, within ruminants a correlation between the thickness of elastic fibres and body size has consistently been found. However, this condition occurs only when comparison between zoologically related species is drawn. During postnatal growth clearcut structural changes were brought to light, represented by the following phenomena simultaneously taking place: a) increase in thickness and lenght of preexisting elastic fibres; b) a progressive increase in the number of fibre splittings as well as of collaterals given off by individual fibres along their extension; c) neoformation of elastic fibres, and their addition to preexisting ones. In the bovines, already at six months of postnatal life, the elastic fibres of larger caliber have attained their full size.     

The elastic network of articular cartilage: an immunohistochemical study of elastin fibres and microfibrils

The elastic network of articular cartilage was investigated by immunohistochemistry using specific antibodies to elastin and fibrillin‐1. Articular cartilage was dissected from defined regions of bovine metacarpophalangeal joints. Elastin fibres and microfibrils were dual‐immunostained by labelling with distinct fluorescent dyes. A conventional fluorescence microscope combined with a polarized light filter was used to study the organization and degree of colocalization of elastin fibres, microfibrils and of the collagen network. We observed an elaborately organized elastic network. In the uppermost superficial zone, where few cells were present, elastin fibres and microfibrils formed a dense three dimensional network showing some degree of colocalization. The thickness and organization of this elastic network varied dramatically from region to region and was most extensive in the metacarpal palmar region. In the middle and deep zones, very few elastin fibres were observed but microfibrils formed a network in the inter‐territorial matrix and dense network around the cells. Our finding of a three dimensional network of dense, well organized elastin fibres and microfibrils in the surface zone of the articular cartilage matrix, and a dense network of microfibrils around the cells deeper into the tissue suggests the elastic network could play both a mechanical and a biological role in articular cartilage.     

Bones in the heart skeleton of the otter (Lutra lutra)

In most mammalian species the cardiac skeleton is composed of coarse collagen fibres, fibrocartilage, and pieces of hyaline cartilage. Bone, the os cordis, is a regular constituent of the ruminant heart. The cardiac skeleton of the otter ( Lutra lutra ) has not previously been described. The skeleton in 30 otter hearts was studied by x-ray analysis and light microscopy. Serial sections were cut parallel to the atrioventricular plane and histochemical staining methods were performed to identify connective tissue fibres, glycosaminoglycans, mineral deposits, and bone. Age and sex of the animals under investigation were considered. The otter heart skeleton was composed of coarse collagen fibres with intercalated pieces of fibrous and/or hyaline cartilage, calcified cartilage, and lamellar bone with red or white marrow. Pieces of hyaline cartilage were not clearly defined: a perichondrial layer was missing and coarse connective tissue continuously transformed into fibrous and hyaline cartilage. In both sexes the amount of cartilage and bone were found to increase with age.
Our results establish the presence of bony material in the heart skeleton of the otter, a small mammalian species. This finding indicates that differentiation of bone is not exclusively related to the size of the organ. Increasing amounts of calcified cartilage and bone correlated with increasing age.     

Vascularization and cartilage mineralization of the thyroid cartilage of Munich minipigs and domestic pigs

Thyroid cartilages of Munich minipigs and domestic pigs were investigated by polychrome sequential labeling, radiography, intravascular injections, histologic examination and scanning electron microscopy in order to gain further insight into the process of vascularization and cartilage mineralization. The relationship between vascularization and cartilage mineralization has only been studied in chondroepiphyses of long bones. Vessels branch off the perichondrial vascular network and enter parts of the thyroid cartilage with a large transverse diameter. Cartilage canals, which are perichondral invaginations, contain an arteriole, a venule, a capillary network and connective tissue. The capillaries form a glomerulus-like structure deep in the matrix of the cartilage. Neighbouring cartilage canals do not display any anastomoses. Cartilage mineralization occurs in large areas of the thyroid cartilage. It is only found in the interterritorial extracellular matrix. Mineralization of the cartilage is evident in areas supplied with cartilage canals as well as in non-supplied areas. Mineralized interterritorial matrix is composed of circular structures of different sizes fusing to form plaques. In scanning electron microscopy circular structures appear as globules. It is possible to visualize the dynamic process of cartilage mineralization with polychrome sequential labeling; it proceeds up to 4 μm per week. Distribution of cartilage canals reveals their nutritional role for the cartilage. According to investigations in chondroepiphyses, cartilage mineralization starts adjacent to the glomerular end of cartilage canals. In contrast, no correlation between cartilage vascularization and the beginning of cartilage mineralization of the thyroid cartilage of Munich minipigs and of domestic pigs has been found.     

Vascularization and cartilage mineralization of the thyroid cartilage of Munich minipigs and domestic pigs

Thyroid cartilages of Munich minipigs and domestic pigs were investigated by polychrome sequential labeling, radiography, intravascular injections, histologic examination and scanning electron microscopy in order to gain further insight into the process of vascularization and cartilage mineralization. The relationship between vascularization and cartilage mineralization has only been studied in chondroepiphyses of long bones. Vessels branch off the perichondrial vascular network and enter parts of the thyroid cartilage with a large transverse diameter. Cartilage canals, which are perichondral invaginations, contain an arteriole, a venule, a capillary network and connective tissue. The capillaries form a glomerulus-like structure deep in the matrix of the cartilage. Neighbouring cartilage canals do not display any anastomoses. Cartilage mineralization occurs in large areas of the thyroid cartilage. It is only found in the interterritorial extracellular matrix. Mineralization of the cartilage is evident in areas supplied with cartilage canals as well as in non-supplied areas. Mineralized interterritorial matrix is composed of circular structures of different sizes fusing to form plaques. In scanning electron microscopy circular structures appear as globules. It is possible to visualize the dynamic process of cartilage mineralization with polychrome sequential labeling; it proceeds up to 4 microm per week. Distribution of cartilage canals reveals their nutritional role for the cartilage. According to investigations in chondroepiphyses, cartilage mineralization starts adjacent to the glomerular end of cartilage canals. In contrast, no correlation between cartilage vascularization and the beginning of cartilage mineralization of the thyroid cartilage of Munich minipigs and of domestic pigs has been found.     

Bioengineering of elastic cartilage with aggregated porcine and human auricular chondrocytes and hydrogels containing alginate, collagen, and kappa-elastin.

Transplantation of isolated chondrocytes has long been acknowledged as a potential method for rebuilding small defects in damaged or deformed cartilages. Recent advances in tissue engineering permit us to focus on production of larger amounts of cartilaginous tissue, such as might be needed for reconstructive surgery of the entire auricle. In this report we describe modification of the basic techniques that lead to production of a large amount of elastic cartilage originated from porcine and human isolated chondrocytes. Small fragments of auricular cartilage were harvested from children undergoing ear reconstruction for microtia or extirpation of preauricular tags and from ears of juvenile pigs. Enzymatically isolated elastic chondrocytes were then agitated in suspension to form the chondronlike aggregates, which were further embedded in molded hydrogel constructs made of alginate and type I collagen augmented with kappa-elastin. The constructs were then implanted in nude mice and harvested 4 and 12 weeks after heterotransplantation. The resulting neocartilage closely resembled native auricular cartilage at the gross, microscopic, and ultrastructural levels. Immunohistochemistry and electron microscopy additionally confirmed that the newly produced cartilage contained the major components of the elastic cartilage-specific matrix, including collagen type II, proteoglycans, and well-assembled elastic fibers.     

Species distribution of coagulase-positive staphylococci in animals.

A total of 268 isolates of coagulase-positive staphylococci from a variety of animal species, including dogs, horses, cats, monkeys, goats, and cows, were assigned to species on the basis of the API Staph- Ident system (Analytab Products, Inc., Plainview , N.Y.). Of 195 isolates from dogs, 179 (91.8%) were Staphylococcus intermedius, as were 9 of 25 (36%) isolates from horses, 7 of 15 (46.6%) isolates from cats, and 4 of 6 (66.6%) isolates from goats. Only 1 of 10 isolates from monkeys and none of 7 isolates from cows were S. intermedius. Of the remaining 68 cultures, 63 were identified as Staphylococcus aureus and 5 as Staphylococcus hyicus. The latter identifications were rendered doubtful on the basis of conventional tests. Identification appeared to be more certain in the S. aureus sample than in the S. intermedius sample. Distribution of biotypes within the two bacterial species as represented by different API profile numbers and reactivity on test substrates showed no significant variations among the host species, except for the S. aureus biotypes in dogs. Both Staphylococcus species were represented about equally among samples from different tissues and lesions, apart from skin-related infections in dogs, which were associated exclusively with S. intermedius (P less than 0.01). Differences between S. aureus and S. intermedius in antimicrobial susceptibility patterns, prevalence of clumping factor, and occurrence of beta-toxin were found to be not significant.     

The microtexture of amnion and chorion connective tissue

On microscopical, stereo-scan and TEM observations the microtexture of the connective tissue of amnion and chorion have been analysed. The "compact layer" of the amnion-connective tissue consists of single collagen fibrils, which are arranged felt-like in parallel layers. In the "fibroblast layer" the fibrils are mostly arranged in bundles, forming a network. Single fibrils and bundles of fibrils in both layers are arranged surface parallel. The superficial layer of the chorion's connective tissue is predominated by wave-like bundles of fibrils in parallel and latticed arrangements, which are consolidated by transversely and diagonally running fibres. In the inferior layer the fibres leave their formation, run diagonally in the direction of the trophoblast and interweave with each other to a mat of fibrils at the border to the trophoblast. The tensile strength of the amnion connective tissue is due to the felt-like fibril texture of the "compact-layer" and the special arrangement of surface-parallel collagen fibres and fibrils.     

The potential aggressiveness of synovial tissue in rheumatoid arthritis

Operation specimens of articular tissue from about 8000 patients with clinically evident rheumatoid arthritis have been studied by light microscopy and in 583 cases by electron microscopy over a period of several years. We examined the tissue in closest contact with eroded cartilage. In approximately 70 per cent. of cases this was a fibrous pannus of compact collagenous connective tissue with few fibrocytes; in approximately 25 per cent. of cases a pannus, consisting of loose granulation tissue, moderately rich in collagen fibres, fibroblasts, and small blood vessels; in approximately 5 per cent. of cases, however, we found, that the zone in close contact consisted of uniform layers of immature-looking synovial cells with large nuclei. These immature-looking cells encroach on the cartilage from the junction of synovium with the articular cartilage. The development of these aggressive cell structures seemed to be preceded by a fibrinous inflammatory exudate. The cells contain lysosomal enzymes which are able to destroy prcteoglycans and collagen fibres and could therefore be the basis of erosion of cartilage. The cell structures seem to be short-lived and they are avascular at least initially. Most of the cells seem to die within a few days. The remaining cells assume the appearance of fibroblasts and form the later pannus. The cell content decreases as the pannus gets older. It is suggested that the destruction of articular cartilage takes place only during the short time in which these aggressive cell structures are present. The erosive synovial cells may however reappear during the intermittent course of the disease, after episodes of quiescence and acute inflammation.