Mechanical strain determines the site-specific localization of inflammation and tissue damage in arthritis.

Many pro-inflammatory pathways leading to arthritis have global effects on the immune system rather than only acting locally in joints. The reason behind the regional and patchy distribution of arthritis represents a longstanding paradox. Here we show that biomechanical loading acts as a decisive factor in the transition from systemic autoimmunity to joint inflammation. Distribution of inflammation and erosive disease is confined to mechano-sensitive regions with a unique microanatomy. Curiously, this pathway relies on stromal cells but not adaptive immunity. Mechano-stimulation of mesenchymal cells induces CXCL1 and CCL2 for the recruitment of classical monocytes, which can differentiate into bone-resorbing osteoclasts. Genetic ablation of CCL2 or pharmacologic targeting of its receptor CCR2 abates mechanically-induced exacerbation of arthritis, indicating that stress-induced chemokine release by mesenchymal cells and chemo-attraction of monocytes determines preferential homing of arthritis to certain hot spots. Thus, mechanical strain controls the site-specific localisation of inflammation and tissue damage in arthritis.

Stereological and immunogold studies on TIE1 and TIE2 localization in glomeruli indicate angiopoietin signaling in podocytes.

Angiopoietins and their TIE receptors are important regulators of vascular stability and remodeling. These molecules are involved not only in the normal development of kidney glomeruli, but also in disease, thus making them promising targets for therapies. Although TIE receptors are mainly found in endothelial cells, some reports observed TIE2 expression in glomerular podocytes as well. This suggests a role of angiopoietins in the regulation of podocytes. In the present study, we aimed to map the subcellular localization of TIE receptors in metanephric glomeruli of fetal pigs using high-resolution immunogold electron microscopy and the relative labeling index stereological approach. TIE1 and TIE2 antibody labeling was detected on the abluminal side of endothelial cell membranes. In endothelial cells, 4.5% of TIE2 was observed close to cell-cell contacts and 11.9% of TIE2 was found in closely associated pairs, which suggests the presence of homodimers. Interestingly, both receptors were also expressed in podocyte foot processes indicating that TIE1 and TIE2 may play a similar role in podocytes as in endothelial cells.

The Omental Pedicle Flap in Dogs Revised and Refined: A Cadaver Study.

OBJECTIVE: To expand current knowledge on the canine omental vasculature and refine the existing lengthening technique of the canine omentum. STUDY DESIGN: Ex vivo study. ANIMALS: Canine cadavers (n=20). METHODS: In 10 canine cadavers the omental arteries were mapped using intravascular latex injection and these results were used to create an omental pedicle flap based on the splenic artery in 10 additional cadavers. The operating range of the flap was recorded with particular attention to the main regions of interest for omental transposition in dogs (axillary and inguinal regions). RESULTS: The superficial and deep omental leaves were each predominantly supplied by a left and a right marginal omental artery that anastomosed near the caudal omental border into a superficial and a deep omental arch, respectively. Anastomoses between arteries of the superficial and the deep omental leaves were weak and inconsistent, except for 1 anastomosis that was found in 8 of 10 dogs. By transposing the intact omentum, the right axilla could be reached in 3 dogs, both axillae in 1 dog, and both groins in all cadavers. In all cases, the omental pedicle reached to and beyond the axillary and inguinal regions. By unfolding the pedicle leaves, the width of the pedicle tip could be doubled. CONCLUSION: When lengthening the omentum is necessary to reach extra-abdominal structures, the omental pedicle flap based on the splenic artery appears to preserve the omental vascular supply. These observations warrant further clinical trials to evaluate this new omtental flap technique in vivo.

Three-dimensional reconstruction of the pharyngeal tonsil innervation pattern in sheep.

The pharyngeal tonsil has recently been identified as a new participant in airborne contamination by the ovine scrapie agent. In the context of scrapie pathogenesis, we conducted a three-dimensional reconstruction of the innervation pattern in the lymphoid compartments of this tonsil. This model confirmed that very few nerve fibres penetrated the lymphoid follicles and suggested that the nerve fibre distribution in the interfollicular and subepithelial areas is more suitable with neuro-invasion through direct contact between these nerve fibres and prion-transporting cells prior to or after prion amplification in the germinal centre of the pharyngeal tonsil lymphoid follicles.

Intussusceptive angiogenesis: a biologically relevant form of angiogenesis.

Angiogenesis, i.e. the development and growth of blood vessels, is a major topic of research as it plays an important role in normal development and in various pathologies. Recent evidence revealed the existence of different mechanisms of blood vessel growth, including sprouting and intussusceptive angiogenesis, vascular mimicry, and blood vessel cooption. The latter two have only been observed in tumor growth, but sprouting and intussusceptive angiogenesis also occur in healthy, physiologically growing tissues. Despite this variety of angiogenic mechanisms, most of the current research is focused on the mechanism of sprouting angiogenesis because this mechanism was first described and because most existing experimental models are related to sprouting angiogenesis. Consequently, the mechanism of intussusceptive angiogenesis is often overlooked in angiogenesis research. Here, the mechanism of intussusceptive angiogenesis is reviewed and the current techniques and models for investigating intussusceptive angiogenesis are summarized. In addition, other mechanisms of vascular growth are briefly reviewed.

Perfusion characteristics of the human hepatic microcirculation based on three-dimensional reconstructions and computational fluid dynamic analysis.

The perfusion of the liver microcirculation is often analyzed in terms of idealized functional units (hexagonal liver lobules) based on a porous medium approach. More elaborate research is essential to assess the validity of this approach and to provide a more adequate and quantitative characterization of the liver microcirculation. To this end, we modeled the perfusion of the liver microcirculation using an image-based three-dimensional (3D) reconstruction of human liver sinusoids and computational fluid dynamics techniques. After vascular corrosion casting, a microvascular sample (±0.134 mm(3)) representing three liver lobules, was dissected from a human liver vascular replica and scanned using a high resolution (2.6 µm) micro-CT scanner. Following image processing, a cube (0.15 × 0.15 × 0.15 mm(3)) representing a sample of intertwined and interconnected sinusoids, was isolated from the 3D reconstructed dataset to define the fluid domain. Three models were studied to simulate flow along three orthogonal directions (i.e., parallel to the central vein and in the radial and circumferential directions of the lobule). Inflow and outflow guidances were added to facilitate solution convergence, and good quality volume meshes were obtained using approximately 9 × 10(6) tetrahedral cells. Subsequently, three computational fluid dynamics models were generated and solved assuming Newtonian liquid properties (viscosity 3.5 mPa s). Post-processing allowed to visualize and quantify the microvascular flow characteristics, to calculate the permeability tensor and corresponding principal permeability axes, as well as the 3D porosity. The computational fluid dynamics simulations provided data on pressure differences, preferential flow pathways and wall shear stresses. Notably, the pressure difference resulting from the flow simulation parallel to the central vein (0-100 Pa) was clearly smaller than the difference from the radial (0-170 Pa) and circumferential (0-180 Pa) flow directions. This resulted in a higher permeability along the central vein direction (k(d,33) = 3.64 × 10(-14) m(2)) in comparison with the radial (k(d,11) = 1.56 × 10(-14) m(2)) and circumferential (k(d,22) = 1.75 × 10(-14) m(2)) permeabilities which were approximately equal. The mean 3D porosity was 14.3. Our data indicate that the human hepatic microcirculation is characterized by a higher permeability along the central vein direction, and an about two times lower permeability along the radial and circumferential directions of a lobule. Since the permeability coefficients depend on the flow direction, (porous medium) liver microcirculation models should take into account sinusoidal anisotropy.

The tonsils revisited: review of the anatomical localization and histological characteristics of the tonsils of domestic and laboratory animals.

This paper gives an overview of the anatomical localization and histological characteristics of the tonsils that are present in ten conventional domestic animal species, including the sheep, goat, ox, pig, horse, dog, cat, rabbit, rat, and pigeon. Anatomical macrographs and histological images of the tonsils are shown. Six tonsils can be present in domestic animals, that is, the lingual, palatine, paraepiglottic, pharyngeal, and tubal tonsils and the tonsil of the soft palate. Only in the sheep and goat, all six tonsils are present. Proper tonsils are absent in the rat, and pigeon. In the rabbit, only the palatine tonsils can be noticed, whereas the pig does not present palatine tonsils. The paraepiglottic tonsils lack in the ox, horse, and dog. In addition, the dog and cat are devoid of the tubal tonsil and the tonsil of the soft palate.

From vascular corrosion cast to electrical analog model for the study of human liver hemodynamics and perfusion.

Hypothermic machine perfusion (HMP) is experiencing a revival in organ preservation due to the limitations of static cold storage and the need for better preservation of expanded criteria donor organs. For livers, perfusion protocols are still poorly defined, and damage of sinusoidal endothelial cells and heterogeneous perfusion are concerns. In this study, an electrical model of the human liver blood circulation is developed to enlighten internal pressure and flow distributions during HMP. Detailed vascular data on two human livers, obtained by combining vascular corrosion casting, micro-CT-imaging and image processing, were used to set up the electrical model. Anatomical data could be measured up to 5--6 vessel generations in each tree and showed exponential trend lines, used to predict data for higher generations. Simulated flow and pressure were in accordance with literature data. The model was able to simulate effects of pressure-driven HMP on liver hemodynamics and reproduced observations such as flow competition between the hepatic artery and portal vein. Our simulations further indicate that, from a pure biomechanical (shear stress) standpoint, HMP with low pressures should not result in organ damage, and that fluid viscosity has no effect on the shear stress experienced by the liver microcirculation in pressure-driven HMP.

Immunoassay of lymphocyte subsets in ovine palatine tonsils.

By means of immunohistochemistry (IHC) and triple color flow cytometry (FCM), five commercial antibodies (anti-CD2, CD4, CD8, CD21, and CD45) were evaluated to quantify and localize the B- and T-lymphocytes in the ovine palatine tonsil. The results of both techniques were compared and evaluated. For the immunohistochemical analysis, three fixation methods were evaluated for their suitability to localize the different lymphocyte populations: 3.5% formaldehyde, zinc salts-based fixative and cryopreservation. The anti-CD45 antibody showed a positive reaction after all three fixation methods. The four other antibodies tested (anti-CD2, CD4, CD8 and CD21) were compatible with zinc salts-based fixation and cryopreservation. The CD21+ B-lymphocytes were localized in the tonsillar lymphoid follicles, while the CD2+ T-lymphocytes were abundant in the interfollicular regions and rare within the lymphoid follicles. The CD8+ T-cells were concentrated adjacent to the follicles, while the CD4+ T-cells were localized in the interfollicular zones as well as in the follicles. Both by immunohistochemistry and flow cytometry, a quantification of the different lymphocyte subsets was made. When comparing the results, a reversed B/T cell ratio was noticed. Possible explanations for this observation are discussed.

Immunohistochemical detection of the angiopoietins during porcine metanephric kidney development.

Angiopoietins are growth factors involved in vascular development. They also play an important role in the development of the kidney vasculature. The localization of these growth factors was immunohistochemically investigated in developing porcine metanephric kidneys. Angiopoietin 1 was predominantly present in the maturing glomeruli, while angiopoietin 2 was observed in the early developing glomeruli as well as in maturing glomeruli. Furthermore, angiopoietin 2 was observed in the muscle layer of renal arteries, in the convoluted tubules and in the loops of Henle. In contrast to the situation in the mouse in which angiopoietin 2 has been reported to be mainly expressed in mesangial cells near the glomerular hilus, expression of angiopoietin 2 in the porcine metanephric kidney was observed in the podocytes of early developing glomeruli, but not in the cells near the glomerular hilus. The results of this study support the hypothesis that the angiopoietins play an important role during mammalian metanephric development and during glomerulogenesis in particular, but indicate that species specific characteristics must be taken into account when evaluating their involvement in glomerulogenesis.

Differences between the ovine tonsils based on an immunohistochemical quantification of the lymphocyte subpopulations.

In sheep, the pharyngeal first defence line against oral and inhaled antigens is organized in six tonsils. Since tonsils are regarded as secondary lymphoid tissue and part of the acquired immune system which is subjected to induction through contact with antigens, an evaluation of the different lymphocyte populations in tonsils is useful to determine a tendency of the specific tonsils to more inductive or more effective immunity. By means of immunohistochemistry, different lymphocyte populations were quantified and localized using a panel of eight antibodies, i.e. anti-CD45, anti-CD21, anti-CD2, anti-CD3, anti-CD4, anti-CD8, anti-WC1 and anti-Ki67. The CD21+ B lymphocytes were localized within the tonsillar lymphoid follicles. The CD2+/CD3+ T lymphocytes were numerous in the interfollicular regions and were aligned underneath and within the epithelium but were also observed at the CD21+ pole of the lymphoid follicles. Near the lingual and tubal tonsils, and the tonsil of the soft palate, the CD45+ cells around the seromucous glands and in the lamina propria were mainly CD3+ T cells. In all tonsils, the WC1+ gamma delta T cells formed a small lymphocyte population which harboured the lamina propria and the interfollicular region. The relative percentages of the different lymphocyte populations of the large palatine and pharyngeal tonsils, which are macroscopically the most developed, were comparable. In contrast, the lingual tonsil was significantly different from the other tonsils not only by its small size and lack of lymphoid follicles, but also by the lymphocyte populations. Based on the lymphocyte populations, the ovine tonsils can be divided in three groups with the tonsil of the soft palate, the tubal and paraepiglottic tonsil forming an intermediate between the palatine and pharyngeal tonsils as true tonsils on the one side, and the lingual tonsil as a scattered lymphocyte aggregation on the other side.

Topography of the rabbit paranasal sinuses as a prerequisite to model human sinusitis.

BACKGROUND: Anatomical studies of the rabbit paranasal cavities are impelled by the increasing interest in the rabbit model to investigate human sinusitis. Although several such studies have already been performed, the topography of the rabbit dorsal conchal and maxillary sinuses is described ambiguously and the existence of the ethmoidal, frontal and sphenoidal sinuses is controversial. METHODOLOGY: The paranasal cavities were investigated using corrosion casting, gross and histological cross-sectioning, and micro-CT scanning of rabbit noses followed by computerized three-dimensional reconstruction. RESULTS: Micro-CT scanning was most useful to illustrate the dorsal conchal sinus, the large maxillary sinus consisting of a dorsal and a ventral recess, and the sphenoidal sinus. All these sinuses are paired and symmetrical. A large connection is present between the dorsal conchal sinus and the maxillary sinus resulting in one large conchomaxillary cavity. The sphenoidal sinus lies most caudal and is surrounded by the presphenoid bone. The openings from the nasal cavity into the conchomaxillary cavity and the sphenoidal sinus are very small. CONCLUSIONS: The absence of frontal and ethmoidal sinuses in any of the rabbits examined is a major difference between the rabbit and human sinuses. The rabbit maxillary sinus seems most appropriate for experimental work.

Validation of the murine aortic arch as a model to study human vascular diseases.

Although the murine thoracic aorta and its main branches are widely studied to gain more insight into the pathogenesis of human vascular diseases, detailed anatomical data on the murine aorta are sparse. Moreover, comparative studies between mice and men focusing on the topography and geometry of the heart and aorta are lacking. As this hampers the validation of murine vascular models, the branching pattern of the murine thoracic aorta was examined in 30 vascular corrosion casts. On six casts the intrathoracic position of the heart was compared with that of six younger and six older men of whom contrast-enhanced computer tomography images of the thorax were three-dimensionally reconstructed. In addition, the geometry of the human thoracic aorta was compared with that of the mouse by reconstructing micro-computer tomography images of six murine casts. It was found that the right brachiocephalic trunk, left common carotid artery and left subclavian artery branched subsequently from the aortic arch in both mice and men. The geometry of the branches of the murine aortic arch was quite similar to that of men. In both species the initial segment of the aorta, comprising the ascending aorta, aortic arch and cranial/superior part of the descending aorta, was sigmoidally curved on a cranial/superior view. Although some analogy between the intrathoracic position of the murine and human heart was observed, the murine heart manifestly deviated more ventrally. The major conclusion of this study is that, in both mice and men, the ascending and descending aorta do not lie in a single vertical plane (non-planar aortic geometry). This contrasts clearly with most domestic mammals in which a planar aortic pattern is present. As the vascular branching pattern of the aortic arch is also similar in mice and men, the murine model seems valuable to study human vascular diseases.

Ultramicroscopic examination of the ovine tonsillar epithelia.

As solid morphological knowledge of ovine tonsillar epithelia might contribute to a better understanding of the pathogenesis of several diseases including prion diseases, the epithelia of all tonsils of 7 one-year-old Texel sheep were examined using scanning and transmission electron microscopy. Major parts of the pharyngeal and tubal tonsils were covered by pseudostratified columnar ciliated epithelia that were interrupted by patches of epithelium containing cells with densely packed microfolds or microvilli, and cells with both microvilli and cilia. Smaller parts were covered by either flattened polygonal cells with densely packed microvilli or microfolds, squamous epithelial cells, or patches of reticular epithelium. The palatine and paraepiglottic tonsils were mainly lined by squamous epithelial cells with apical microplicae or short knobs. Additionally, regions of reticular epithelium containing epithelial cells with apical microvilli were seen. The lingual tonsil was uniformly covered by a keratinized squamous epithelium and devoid of microvillous cells and patches of reticular epithelium. The rostral half of the tonsil of the soft palate was lined by a pseudostratified columnar ciliated epithelium with characteristics of the pharyngeal and tubal tonsils. The epithelium of the caudal part resembled the epithelia of the palatine and paraepiglottic tonsils. Putative M cells, mainly characterized by apical microvilli or microfolds and a close association with lymphoid cells, seem manifestly present on the nasopharyngeal tonsils. The reticular epithelium of the palatine and paraepiglottic tonsils also harbor cells with small apical microvilli. The exact nature of these presumptive M cells should, however, be elucidated in functional studies.

Morphometric data on the accessory ligament of the deep digital flexor tendon in the equine hindlimb.

Desmopathy of the accessory ligament of the deep digital flexor tendon (ALDDFT) in the hindlimb has recently been described as a cause of lameness in horses. However, there is limited morphometric data on this ligament. In the present study, the ALDDFT was carefully dissected in 165 hindlimbs that were collected from an abattoir. Length, width (lateral-to-medial) and thickness (plantar-to-dorsal) of the ligament were measured. It was found that the ALDDFT may be absent in a minority of horses, but when it was present the general morphology was variable. The ALDDFT can be a single rectilinear structure ranging from slender or poorly developed to quite large and can be divided, in part or in its entirety, into two or three bundles.

Morphological and immunological characteristics of the bovine temporal lymph node and hemal node.

Anatomical dissection of the temporal regions of 62 cattle demonstrated that lymph nodes and hemal nodes are present in 89% of the animals (bilaterally in 65% and unilaterally in 24% of the cases). Lymph nodes accounted for 60% and hemal nodes for 40% of all examined nodes. They are nearly always round with diameters ranging from 1 to 9mm. Injections of India ink showed that their drainage area consists of the forehead, the upper eyelid, the base of the horn and the temporal muscle. Immunohistochemistry and flow cytometry revealed that the distribution and percentages of the different cell populations in the lymph nodes and hemal nodes are similar to other cranial lymph nodes. Based on its anatomical location the name temporal lymph node (lymphonodus temporalis) is proposed.

Larynx-associated lymphoid tissue (LALT) in young cattle.

This paper describes the presence of lymphoid tissue in the mucosa of the bovine larynx. A total of 15 bovine larynges were examined both macroscopically after tissue fixation in acetic acid and microscopically using histology. It was found that no paraepiglottic tonsil was present in cattle, although a few lymphoid follicles were present in the mucosa at the base of the epiglottis. This result is in accordance with previous reports. In contrast, numerous lymphoid follicles were seen in the mucosa of the epiglottis and the corniculate processes of the arytenoid cartilages. This suggests that larynx-associated lymphoid tissue is present in cattle. Our observation could be of clinical importance, e.g. in the framework of the development of aerosolized vaccines.

Three-dimensional reconstruction of the remodeling of the systemic vasculature in early pig embryos.

Current research on angiogenesis and vascular regression is mainly focused on pathological conditions such as tumor growth and diabetic retinopathy, while a suitable physiological model to study the controlling factors in these processes is still lacking. The remodeling pattern of the embryonic vasculature into the adult configuration, such as the branchial arch arterial system developing into the aorta or the early embryonic veins building the caudal vena cava can potentially serve as a model. However, practical applications of the embryonic vascular patterning are impeded by the current controversy over the exact development of the caudal vena cava in mammals. To elucidate these ambiguities, specific developmental stages of vascular development in pig embryos were mapped by means of computer-assisted 3D reconstructions starting from histological serial sections of Bouin's fixed embryos. Special attention was given to venous segments in the lumbar region, as their origin and fate are equivocally described in literature. Here we demonstrate that these venous segments originate from the caudal cardinal veins which are forced to migrate during development into a more dorsal position due to the expansion of the developing metanephroi and the more dorsal relocation of the umbilical arteries. These findings are in contrast with the generally accepted theory that the venous segments in the lumbar region arise from newly formed veins that are located dorsal to the early caudal cardinal system.

Anatomical and histological aspects of the bovine lingual tonsil.

The localization of the bovine lingual tonsil is described as a prerequisite for the removal of specified risk material from the tongue meat in order to restrict the risk arising from bovine spongiform encephalopathy (BSE) to public health. The major part of this tonsil can be located macroscopically by the openings of its follicular crypts at the root of tongue. This part consists of organized aggregations of lymph nodules. Additional solitary primary lymph nodules and diffuse accumulations of lymphocytes are macroscopically invisible but are bilaterally present in the area extending 2 cm caudal to 3 cm rostral to the last vallate papillae. By sectioning the tongue 3 cm rostral to the last vallate papillae, undermining the lingual mucosa to the level of these papillae and making a transverse cut towards the lingual process of the basihyoid bone, the greater part of the lingual tonsil can efficiently be removed. Finally, immunohistochemical staining demonstrated the presence of T and B lymphocytes, suggesting that the bovine lingual tonsil can be considered as a site where an immune response can be induced.

The buccal lymph node (lymphonodus buccalis) in dogs: occurrence, anatomical location, histological characteristics and clinical implications.

Three dogs were presented for clinical examination with bilateral buccal nodules which were identified as enlarged buccal lymph nodes. As little is known about this pathology, 150 dogs were examined by anatomical dissection for the presence of buccal lymph nodes. They were found in 13 dogs, occurring bilaterally in six dogs and unilaterally in seven dogs. Two buccal lymph nodes were bilobulated and one was double. The lymph nodes were always located dorsal to the zygomatic muscle and rostral to the masseter muscle in the region where the superior labial vein drains into the facial vein. Histology demonstrated a large amount of intranodal adipose tissue scattered throughout the lymphoid tissue. The canine buccal lymph node should not be confused with the accessory parotid or ventral buccal salivary gland and is clinically important as it can enlarge due to tumour metastasis or inflammation of the buccal region.