Loss of connexin43 in murine Sertoli cells and its effect on blood-testis barrier formation and dynamics.

Connexin43 (Cx43) is the predominant testicular gap junction protein and in cases of impaired spermatogenesis, Cx43 expression has been shown to be altered in several mammals. Amongst other functions, Cx43 is supposed to regulate junction formation of the blood-testis barrier (BTB). The aim of the present study was to investigate the expression pattern of different tight junction (TJ) proteins of the murine BTB using SC-specific Cx43 knockout mice (SCCx43KO). Adult homozygous male SCCx43KO mice (SCCx43KO-/-) predominantly show an arrest of spermatogenesis and SC-only tubules that might have been caused by an altered BTB assembly, composition or regulation. TJ molecules claudin-3, -5 and -11 were examined in adult wild type (WT) and SCCx43KO-/- mice using immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR). In this context, investigation of single tubules with residual spermatogenesis in SCCx43KO-/- mice was particularly interesting to identify a potential Cx43-independent influence of germ cells (GC) on BTB composition and dynamics. In tubules without residual spermatogenesis, a diffuse cytoplasmic distribution pattern for claudin-11 protein could be demonstrated in mutant mice. Nevertheless, claudin-11 seems to form functional TJ. Claudin-3 and -5 could not be detected immunohistochemically in the seminiferous epithelium of those tubules. Correspondingly, claudin-3 and -5 mRNA expression was decreased, providing evidence of generally impaired BTB dynamics in adult KO mice. Observations of tubules with residual spermatogenesis suggested a Cx43-independent regulation of TJ proteins by GC populations. To determine initial BTB formation in peripubertal SCCx43KO-/- mice, immunohistochemical staining and qRT-PCR of claudin-11 were carried out in adolescent SCCx43KO-/- and WT mice. Additionally, BTB integrity was functionally analysed using a hypertonic glucose fixative. These analyses revealed that SCCx43KO-/- mice formed an intact BTB during puberty in the same time period as WT mice, which however seemed to be accelerated.

Comparison of clinical and immunological findings in gnotobiotic piglets infected with Escherichia coli O104:H4 outbreak strain and EHEC O157:H7.

BACKGROUND: Shiga toxin (Stx) producing Escherichia coli (E. coli) (STEC) is the most frequent cause of diarrhoea-positive haemolytic uraemic syndrome (D + HUS) in humans. In 2011, a huge outbreak with an STEC O104:H4 strain in Germany highlighted the limited possibilities for causative treatment of this syndrome. The responsible STEC strain was found to combine Stx production with adherence mechanisms normally found in enteroaggregative E. coli (EAEC). Pathotypes of E. coli evolve and can exhibit different adhesion mechanisms. It has been shown previously that neonatal gnotobiotic piglets are susceptible for infection with STEC, such as STEC O157:H7 as well as for EAEC, which are considered to be the phylogenetic origin of E. coli O104:H4. This study was designed to characterise the host response to infection with the STEC O104:H4 outbreak strain in comparison to an STEC O157:H7 isolate by evaluating clinical parameters (scoring) and markers of organ dysfunction (biochemistry), as well as immunological (flow cytometry, assessment of cytokines/chemokines and acute phase proteins) and histological alterations (light- and electron microscopy) in a gnotobiotic piglet model of haemolytic uraemic syndrome. RESULTS: We observed severe clinical symptoms, such as diarrhoea, dehydration and neurological disorders as well as attaching-and-effacing lesions (A/E) in the colon in STEC O157:H7 infected piglets. In contrast, STEC O104:H4 challenged animals exhibited only mild clinical symptoms including diarrhoea and dehydration and HUS-specific/severe histopathological, haematological and biochemical alterations were only inconsistently presented by individual piglets. A specific adherence phenotype of STEC O104:H4 could not be observed. Flow cytometric analyses of lymphocytes derived from infected animals revealed an increase of natural killer cells (NK cells) during the course of infection revealing a potential role of this subset in the anti-bacterial activity in STEC disease. CONCLUSIONS: Unexpectedly, E. coli O104:H4 infection caused only mild symptoms and minor changes in histology and blood parameters in piglets. Outcome of the infection trial does not reflect E. coli O104:H4 associated human disease as observed during the outbreak in 2011. The potential role of cells of the innate immune system for STEC related disease pathogenesis should be further elucidated.

The histological components of the phoniatrical body-cover model in minipigs of different ages.

Pigs are models in human phoniatry. However, features of maturation and ageing have not been considered with regard to the so-called body-cover model in this species. Therefore, the glottis of "young" (2-3 months; n = 6) and "old" (4-7 years; n = 6) minipigs was investigated. Their cranial (CraF) and caudal (CauF) vocal folds were histomorphometrically and stratigraphically analysed with emphasis on their amounts of collagen structures and elastic fibres. A dense subepithelial layer (SEL) was a distinct feature of CraF and CauF of both age groups; it was spread upon the underlying loose, flexible "cover" like a fibro-elastic membrane. The "cover" was characterised by the so-called superficial layer (SL), which was distinctly loose in the "young" minipigs, but had a much denser texture in the "old" minipigs. Here, the SL was dominated by elastic fibres in the CraF, but was of mixed qualities (collagenous and elastic) in the CauF. The structural requirements for the SL's function as a loose "cover" were thus met only in the "young" animals. A clearly demarcated intermediate layer (IL)--characterised by high amounts of elastic fibres (as in humans)--was only found in the CraF of the "young" animals. In the "old" animals, it had lost its demarcation. In the depth of the CraF of the "old" animals, many thick collagen fibre bundles were detected in a location equivalent to that of the vocal muscle in the CauF. The development of their large diameters was interpreted as part of the maturation process, thereby supporting the hypothesis of their functional importance as a component of the "body." In the CauF, the amounts of collagen structures increased throughout the entire lamina propria, resulting in a loss of demarcated stratigraphical subdivisions in the "old" minipigs. This situation resembled that described in the vocal fold of geriatric humans.

PR-39, a porcine host defence peptide, is prominent in mucosa and lymphatic tissue of the respiratory tract in healthy pigs and pigs infected with Actinobacillus pleuropneumoniae.

BACKGROUND: Host defence peptides are important components of mammalian innate immunity. We have previously shown that PR-39, a cathelicidin host defence peptide, is an important factor in porcine innate immune mechanisms as a first line of defence after infection with Actinobacillus pleuropneumoniae. PR-39 interacts with bacterial and mammalian cells and is involved in a variety of processes such as killing of bacteria and promotion of wound repair. In bronchoalveolar lavage fluid of infected pigs PR-39 concentrations are elevated during the chronic but not during the acute stage of infection when polymorphonuclear neutrophils (known as the major source of PR-39) are highly increased. Thus it was assumed, that the real impact of PR-39 during infection might not be reflected by its concentration in bronchoalveolar lavage fluid. RESULTS: Using immunohistochemistry this study demonstrates the actual distribution of PR-39 in tissue of the upper and lower respiratory tract of healthy pigs, and of pigs during the acute and chronic stage of experimental infection with Actinobacillus pleuropneumoniae.During the acute stage of infection PR-39 accumulated adjacent to blood vessels and within bronchi. Immune reactions were mainly localized in the cytoplasm of cells with morphological characteristics of polymorphonuclear neutrophils as well as in extracellular fluids. During the chronic stage of infection pigs lacked clinical signs and lung alterations were characterized by reparation and remodelling processes such as tissue sequestration and fibroblastic pleuritis with a high-grade accumulation of small PR-39-positive cells resembling polymorphonuclear neutrophils. In healthy pigs, PR-39 was homogenously expressed in large single cells within the alveoli resembling alveolar macrophages or type 2 pneumocytes. PR-39 was found in all tissue samples of the upper respiratory tract in healthy and diseased pigs. Within the tracheobronchial lymph nodes, PR-39 dominated in the cytoplasm and nuclei of large cells resembling antigen-presenting cells located in the periphery of secondary follicles. CONCLUSIONS: These immunohistochemical findings indicate that, in addition to polymorphonuclear neutrophils, other cells are involved in the expression, storage, or uptake of PR-39. The presence of PR-39 in healthy lung tissue showed that this antibacterial peptide might be important for the maintenance of health.

Experimental Actinobacillus pleuropneumoniae challenge in swine: comparison of computed tomographic and radiographic findings during disease.

BACKGROUND: In pigs, diseases of the respiratory tract like pleuropneumonia due to Actinobacillus pleuropneumoniae (App) infection have led to high economic losses for decades. Further research on disease pathogenesis, pathogen-host-interactions and new prophylactic and therapeutic approaches are needed. In most studies, a large number of experimental animals are required to assess lung alterations at different stages of the disease. In order to reduce the required number of animals but nevertheless gather information on the nature and extent of lung alterations in living pigs, a computed tomographic scoring system for quantifying gross pathological findings was developed. In this study, five healthy pigs served as control animals while 24 pigs were infected with App, the causative agent of pleuropneumonia in pigs, in an established model for respiratory tract disease. RESULTS: Computed tomographic (CT) findings during the course of App challenge were verified by radiological imaging, clinical, serological, gross pathology and histological examinations. Findings from clinical examinations and both CT and radiological imaging, were recorded on day 7 and day 21 after challenge. Clinical signs after experimental App challenge were indicative of acute to chronic disease. Lung CT findings of infected pigs comprised ground-glass opacities and consolidation. On day 7 and 21 the clinical scores significantly correlated with the scores of both imaging techniques. At day 21, significant correlations were found between clinical scores, CT scores and lung lesion scores. In 19 out of 22 challenged pigs the determined disease grades (not affected, slightly affected, moderately affected, severely affected) from CT and gross pathological examination were in accordance. Disease classification by radiography and gross pathology agreed in 11 out of 24 pigs. CONCLUSIONS: High-resolution, high-contrast CT examination with no overlapping of organs is superior to radiography in the assessment of pneumonic lung lesions after App challenge. The new CT scoring system allows for quantification of gross pathological lung alterations in living pigs. However, computed tomographic findings are not informative of the etiology of respiratory disease.

Isolation and characterization of multipotent mesenchymal stromal cells from the gingiva and the periodontal ligament of the horse.

BACKGROUND: The equine periodontium provides tooth support and lifelong tooth eruption on a remarkable scale. These functions require continuous tissue remodeling. It is assumed that multipotent mesenchymal stromal cells (MSC) reside in the periodontal ligament (PDL) and play a crucial role in regulating physiological periodontal tissue regeneration. The aim of this study was to isolate and characterize equine periodontal MSC. Tissue samples were obtained from four healthy horses. Primary cell populations were harvested and cultured from the gingiva, from three horizontal levels of the PDL (apical, midtooth and subgingival) and for comparison purposes from the subcutis (masseteric region). Colony-forming cells were grown on uncoated culture dishes and typical in vitro characteristics of non-human MSC, i.e. self-renewal capacity, population doubling time, expression of stemness markers and trilineage differentiation were analyzed. RESULTS: Colony-forming cell populations from all locations showed expression of the stemness markers CD90 and CD105. In vitro self-renewal capacity was demonstrated by colony-forming unit fibroblast (CFU-F) assays. CFU-efficiency was highest in cell populations from the apical and from the mid-tooth PDL. Population doubling time was highest in subcutaneous cells. All investigated cell populations possessed trilineage differentiation potential into osteogenic, adipogenic and chondrogenic lineages. CONCLUSIONS: Due to the demonstrated in vitro characteristics cells were referred to as equine subcutaneous MSC (eSc-MSC), equine gingival MSC (eG-MSC) and equine periodontal MSC (eP-MSC). According to different PDL levels, eP-MSC were further specified as eP-MSC from the apical PDL (eP-MSCap), eP-MSC from the mid-tooth PDL (eP-MSCm) and eP-MSC from the subgingival PDL (eP-MSCsg). Considering current concepts of cell-based regenerative therapies in horses, eP-MSC might be promising candidates for future clinical applications in equine orthopedic and periodontal diseases.

Absence of lymphatic vessels in the dog dental pulp: an immunohistochemical study.

In spite of numerous investigations it has not been precisely determined whether lymphatic vessels are present in the dental pulp of dogs. Therefore, this study attempted a specific immunohistochemical detection of lymphatic endothelium. The canine teeth of 19 healthy beagle dogs were dissected into three segments (apical, intermediate and occlusal). After decalcification, specimens were embedded in paraffin wax and histologic cross-sections were stained immunohistochemically using a reliable antibody (anti-Prox-1) against the homeobox transcription factor Prox-1, which is located within the nucleus of lymphatic endothelium. Anti-Prox-1 reacted positively with canine control tissues (lymph nodes, gingiva, nasal mucosa), but showed no staining in tissue sections of the dental pulp. The dog dental pulp contained no vascular structures lined with lymphatic endothelium. This suggests that drainage of interstitial fluid makes use of other routes, i.e. extravascular pathways.

Biomechanical evaluation of the equine masticatory action: calculation of the masticatory forces occurring on the cheek tooth battery.

The forces generated during equine mastication influence the teeth, as well as the periodontium, the jaw bones, the temporomandibular joints and materials used in dental therapy. Due to the limited accessibility of the cheek teeth located farther caudal in the equine oral cavity the measurement of the masticatory forces on these teeth is virtually impossible. The calculation of such forces is an appropriate, indirect alternative to direct measuring. Hence, a mathematical model, which respects several morphological features of the equine dentition, e.g. the Curve of Spee, is presented. The relevant parameters were measured on laterolateral radiographs of 24 cadaver heads and 6 skulls. Our calculations yielded two major results. (1) The masticatory forces increased from rostral to caudal. This increase was due to distinct anatomical features of the equine dentition, i.e. the Curve of Spee, the position of the teeth, and the length of the mandible. (2) The masticatory forces decreased with age. This decrease was due to the reduced height of the Curve of Spee in older horses. These results are discussed in view of veterinary dental treatment, with special reference to the geriatric horse.

Measurement of the Curve of Spee in horses.

The Curve of Spee is well known, yet hardly understood in the horse, as there is a striking lack of biometrically related data which could help to explain its biomechanical significance. Older horses display drastic alterations of the Curve of Spee in routine clinical examinations. Since the Curve of Spee is known to be biomechanically related to the masticatory forces in humans, the same may apply to the equine, and masticatory deficits can be expected in the geriatric horse. Quantitative data of the Curve of Spee cannot be obtained directly by means of measurements in the horse's long and narrow oral cavity. Instead, the radiographs of 24 heads and 6 skulls of horses of different age were used to develop a geometrical procedure facilitating a quantitative analysis of the height of the Curve of Spee. The geometrical values gave clear evidence that the height of the Curve of Spee was smaller in groups of older horses compared with those of younger horses. This finding was in accordance with biomechanical studies which reported decreased masticatory forces in older horses.

Biomechanical evaluation of equine masticatory action: position and curvature of equine cheek teeth and age-related changes.

The equine cheek tooth battery is part of a very dynamic system. The aim of this study was to investigate whether the curvature and position of the teeth are also involved in such dynamical processes. The alveolar crest was labelled with a radiodense marker (48 cadaver heads, 15 skulls) and laterolateral radiographs were taken. Then a geometrical method was elaborated to determine a cheek tooth's curvature and its position by means of specific angles. This method respects the remarkable changes of the equine dentition throughout life by considering two items: (1) the alveolar crest was taken as a constant landmark, (2) the central axis of the curved dental crown was determined by calculation of a linear regression equation. This equation considered several geometrically determined points on the curved dental crown which had been marked in the radiographs. Our study yielded the following results: Mandibular cheek teeth became more curved with age, but their positions (represented by the so-called mesio-occlusal angle between tooth and alveolar crest) did not change significantly. In maxillary cheek teeth, however, the mesio-occlusal angle became larger with age (indication of change of dental position), while their curvature did not change. Even though changes of the dental position were not always statistically significant, they are discussed as being biologically/functionally relevant. The mandibular anticlinal tooth, i.e. the tooth positioned at a mesio-occlusal angle of about 90 degrees , was not in contact with the maxillary anticlinal tooth. Interestingly, the maxillary anticlinal tooth is known to cause most clinical dental problems.

Simulation of local anaesthetic nerve block of the infraorbital nerve within the pterygopalatine fossa: anatomical landmarks defined by computed tomography.

To provide anaesthesia of the maxillary cheek teeth, a local block of the infraorbital nerve in the pterygopalatine fossa has been suggested. The aim of this study was to re-examine the anatomy of the pterygopalatine fossa, giving special attention to relevant arteries, veins and nerves; simulate the infiltration of an anaesthetic by injecting a contrast medium; improve the injection technique to avoid puncturing of relevant anatomical structures. Five heads and two living horses were investigated using contrast medium injections and computed tomography (CT). Needles were inserted using two insertion techniques: "Palatine Bone Insertion" (PBI) and "Extraperiorbital Fat Body Insertion" (EFBI). Both techniques are suitable for achieving a consistent distribution of contrast medium around the infraorbital nerve. The periorbita prevents the contrast medium from penetrating into the intraperiorbital compartment. The EFBI-technique is most appropriate for providing a sufficient infraorbital nerve block with a minimised risk of complications.

The blood vessel system in the periodontal ligament of the equine cheek teeth--part I: The spatial arrangement in layers.

Corrosion casts of blood vessels in the periodontium of cheek teeth from eight horses were observed three-dimensionally with a dissection microscope. Selected specimens were examined in a scanning electron microscope. Periodontal blood vessels communicated with those from the gingiva, the alveolar bone, and the apical region. In the upper jaw, there were anastomoses with the blood vessels of the mucosa of the maxillary sinus. The periodontal vascular system was organized in two or three layers. The peripheral layer was mainly composed of large venules, the inner one consisted of capillaries. In the intermediate layer, blood vessels were post-capillary venules. This layer was developed only in horses under 10 years of age. In all layers the vascular orientation was mainly occluso-apical, this was defined as the standard pattern. There were many variations displayed in different courses of certain blood vessels. The vascular organization is discussed with regard to the specialized functions of the periodontal ligament (PDL). The wide vessels of the outer layer are thought to play a mechanical role as part of a shock absorbing system. The capillaries of the inner layer meet nutritional requirements. The disappearance of the intermediate layer in horses older than 10 years is taken as an adaptation to the remodelling of the PDL. Modifications in the standard pattern of vascular arrangements are also interpreted as adaptations to life-long changes in the periodontal space. Anastomoses between the periodontal vasculature and the blood vessels of the maxillary sinus indicate that periodontal disease may be transferred into the sinus.

The blood vessel system in the periodontal ligament of the equine cheek teeth--part II: The micro-architecture and its functional implications in a constantly remodelling system.

The micro-vasculature of the equine periodontal ligament (PDL) was investigated using corrosion casts for scanning electron microscopy. Specimens from eight healthy warm-blooded horses were examined. Specific vascular features such as large ampullae with a diameter of up to 300 microm and blind vascular casts were found in the specimens of the equine PDL. The ampullae-shaped venules occurred only in the peripheral layer, where they were formed at the confluence of several vessels. Two types of blind stumps were identified. The first was developed in all three layers, while the second was found only in the peripheral layer. Blood-flow-regulating structures were not present in the equine PDL. As vascular valves are absent, the blood can flow in all directions. This polydirectional shift exists in all vascular layers. The vascular ampullae, present exclusively in the peripheral layer, are discussed here as specific features of a mechanism that can receive masticatory forces. Apart from its mechanical role, the vasculature must constantly adapt to the spatial changes resulting from the life-long eruption of the equine cheek tooth. This is facilitated by vascular sprouting and intussusceptive vascular remodelling. The blind ending stumps of the corrosion casts are typical equivalents of vascular sprouting. The large ampullae are interpreted as the first step in an intussusceptive process.

Collagen fiber architecture of the periodontal ligament in equine cheek teeth.

The objective of this study was to examine the spatial arrangement of the fiber apparatus of the equine periodontium which is supposed to meet two contrary requirements: (1) to attach the tooth firmly and elastically to the alveolar bone; and, to be appropriately remodeled and reconstructed in order to facilitate the prolonged eruption of the tooth. Specimens of periodontal ligament were obtained from the buccal and lingual/palatal aspects of the first molars from the maxilla and mandible of 12 horses. The animals were assigned to three age groups. Histological sections were prepared from three specific horizontal levels of the periodontal ligament and examined with conventional and polarized-light microscopy. At the gingival level, collagen fascicles (diameter > 200 microm) were densely packed. Their spatial alignment was the same in all age groups. The architecture of the collagen fiber apparatus differed at the middle and apical levels in the three age groups. There was a clear distinction between fibers, bundles, and fascicles. Bundling of collagen fibers, density of the fiber arrangement, and collagen fascicles with an alveolo-cemental orientation increased with age. The collagen fiber apparatus of the equine periodontal ligament is highly adaptive, responding continuously to the dynamic changes in the periodontal environment. Site-specific arrangements and age-dependent structural variations are assumed to maintain tooth support as the reserve crown gradually decreases in length with progressive dental attrition. Most of the age-dependent changes to the periodontal ligament in teeth examined in this study occurred at the apical level. The apical region of young teeth had no periodontal attachment, while the roots of older teeth were firmly attached to the alveolus. When evaluating periodontal ligament development, the individual tooth's 'dental age' should be considered rather than the animal's age to account for individual tooth eruption times.

The equine periodontium as a continuously remodeling system: morphometrical analysis of cell proliferation.

OBJECTIVE: The hypsodont equine cheek tooth erupts continuously throughout life. Tooth eruption is inevitably associated with a remodeling of the periodontium. One major process of remodeling in the PDL is cell proliferation. The aim of this study was to detect cell proliferation at different sites of the equine PDL in order to examine the dynamics of the periodontal cell population. DESIGN: Specimens from nine warm-blood horses were taken-- containing the PDL interposed between the cementum and the alveolar bone--at three designated levels: subgingival, middle, and apical. Cell proliferation was detected immunohistochemically by use of anti-Ki-67 in combination with an elaborate morphometrical procedure using an image-analysis program. Three zones of the PDL were distinguished in each specimen: a zone next to the dental cementum, a central zone, and a zone next to the alveolar bone. The PDL was divided into three levels and three zones, resulting in nine defined regions of interest. RESULTS: The proliferation index was high at the apical level and in the zone next to the alveolar bone. The opposite was true of cell density, which was high at the subgingival level and in a zone next to the dental cementum. CONCLUSIONS: Evaluation and statistical analysis of the parameters proliferation index and cell density in distinct, biologically appropriate sites of the equine PDL suggest the presence of dynamic processes like cell migration by which the cells of the PDL move from an area next to the alveolar bone towards the dental cementum, and in an apico-occlusal direction. Furthermore, our study confirms the close relationship between cell proliferation, cell migration, and eruption.

Distinct fibro-vascular arrangements in the periodontal ligament of the horse.

OBJECTIVE: The periodontal ligament (PDL) of the hypsodont cheek teeth of the horse meets specific requirements of tooth support: (1) to fix the tooth; (2) to withstand pressure and tension during the laterolateral mastication movement; (3) to restore the original tooth position. These requirements are assumed to be met by the periodontal collagen fiber apparatus and by the vascular system. Distinct fibro-vascular arrangements are described here in order to reveal the structural prerequisites for these distinct functions. DESIGN: Specimens from nine horses were processed for (1) collagen labeling with fluorescein, (2) oxytalan fiber staining, (3) immunostaining of smooth muscle actin, and (4) TEM examinations. RESULTS: Blood vessels were found to be integrated in the PDL in three distinct ways: CONCLUSIONS: The sheaths formed by veil cells (type-I arrangement) isolate distinct groups of blood vessels from deformations of the surrounding tissue. In contrast, the distinct fibro-vascular arrangements of anchored blood vessels (type-II arrangement) and ballooned venules (type-III arrangement) are assumed to accept the forces generated by masticatory tooth movements. The anchored blood vessels sustain tractive forces by means of what is called a lateral compression mechanism, whereas the ballooned venules act as a hemodynamic cushion.

Immunohistochemical identification of lymphatic vessels in the periodontium of equine cheek teeth.

Immunohistochemical detection of lymphatic capillaries was performed in the periodontium of maxillary and mandibular cheek teeth from 6 horses (aged 3-23 years). Tissue sections of the periodontium were taken at 4 different horizontal levels along the long axis of the tooth. The specimens were processed for immunoreaction with anti-Prox1, in order to distinguish lymphatic endothelium from blood vascular endothelium. Lymphatic vessels were detected in all periodontal tissues except for the dental cementum. Lymphatic capillaries were most densely distributed in the gingiva compared to other tissues of the periodontium. Lymphatic capillaries were found most consistently in samples taken from the gingival and subgingival regions in all horses examined. Within these levels, the gingiva as well as the spongiosa of the maxillary and mandibular bone had the greatest incidence of lymphatic vessels. Considering the distinct distribution of the lymphatic capillaries in the periodontium of the maxillary and mandibular cheek teeth, two complementary lymphatic drainage pathways are proposed: (1) superficial lymph drainage via the gingiva, emptying into the mandibular lymph nodes; (2) deep lymph drainage via the mandibular and maxillary spongiosa, emptying into the mandibular and retropharyngeal lymph nodes, respectively.

A simple fluorescence labeling method to visualize the three-dimensional arrangement of collagen fibers in the equine periodontal ligament.

In order to display the collagen-fiber arrangement in the equine periodontal ligament an inexpensive and easy staining procedure with fluorescein was applied to paraffin sections. After fluorescein labeling a section was suitable for successful examination with three special microscopical systems: a) fluorescence microscopy b) phase contrast microscopy and c) polarized light microscopy. Collagen fibers were clearly displayed as compact structures in the fluorescence microscope. This distinct feature of the fluorescent image generated an almost three-dimensional impression of the fiber arrangement. Phase contrast microscopy and polarized light microscopical investigations of the same section supplemented the findings with further structural details. This contributed to demonstration of the complex architecture of the PDL, i. e. the varying sizes of the fiber bundles, their specific spatial alignment, and the entheses to the dental cementum.

Blood vessels of the rat tail: a histological re-examination with respect to blood vessel puncture methods.

The rat tail vascularization is histologically re-examined especially with respect to blood sampling and vascular-injection methods. The terminal third of the tail is recommended for blood vessel puncturing. In this segment, the arteries and veins are most prominent, since the structures of the musculo-skeletal system diminish towards the tip of the tail. In addition to the commonly-known blood vessels (one ventral artery, two lateral veins), there is a dorsal vein in the rat tail that is well suited for puncture and cannulation.