bluuebunny/crossref_metadata_2025 huggingface dataset:
The Ovine Tonsilla Lingualis: Scattered Lymphocyte Aggregations Rather Than a Well‐defined Tonsillar Structure
Authors: Van den Broeck W. , Cocquyt G. , Simoens P.
Published: November 2005

Recently, it has been hypothesized that the BSE‐causing agent PrPSc can infect sheep, making these animals a possible source of infection for man. Furthermore, experimental studies in inoculated sheep have demonstrated that PrPSc is not only present in neuronal tissue, but also in lymphoid tissue. Therefore, ovine tonsils are considered as high‐risk material and consequently have to be removed after slaughter. The present study is part of an elaborate project in which anatomical, histological and immunological aspects of ovine tonsils are studied within the framework of BSE/TSE research. The tongues of two sheep of 9‐months‐old were collected immediately after slaughter. The lingual root between the base of the epiglottis and the torus linguae was dissected and the 4.5 cm wide and 8 cm long sample was cut into 24 pieces which were subsequently fixed in 3.5% formaldehyde and embedded in paraffin. From each tissue block, ten 8‐μm‐thick sections with 1000 μm interval were cut and stained with eosin–haematoxylin. Microscopic analysis revealed that lymphocytes are mainly present at the lateral side of the tongue forming numerous small aggregates, while in the more central part the amount of aggregates was lower. In both lateral and central parts, the aggregations were located in an area ranging from 1–2 cm till 6 cm rostral to the epiglottis. These lymphatic clusters were in general smaller than the lymphatic nodules seen in the other pharyngeal lymphoid tissue, and a differentiation into secondary nodules was never observed. The epithelium covering these lymphocyte aggregations was a stratified squamous epithelium without infiltration of lymphocytes. It was concluded that the tonsilla lingualis in sheep consists of scattered lymphocyte aggregations without forming a well‐defined tonsillar organization. Further studies are planned to examine the tongues of more sheep and to determine the presence of dendritic cells, follicular dendritic cells and different B‐ and T‐cell populations.

This study was supported by the BOF project grant #011B4101 of Ghent University.
Structure of the Seminal Pathway in the European Chub (Leuciscus cephalus)
Authors: Walter I. , Tschulenk W. , Schabuss M. , Miller I. , Grillitsch B.
Published: November 2005

The European chub, Leuciscus cephalus (Cyprinidae), is a fish species employed in bioindication for the effects of endocrine disruption chemicals (EDC) upon the reproductive system. In applied histopathology, a useful tool in aquatic ecotoxicology, detailed knowledge on the intact structural integration of an organism is an indispensable prerequisite for the diagnosis of pathological alterations. This is particularly evident for gonadal structures in bioindication for the effects of EDCs interacting with the hypothalamus‐pituitary‐gonadal‐liver axis. However, little is known about the histology of L. cephalus until now, especially the extratesticular duct system is widely disregarded. Therefore, in the present study the testicular efferent duct system was described for three phases of testicular development (pre‐spawning, spawning, post‐spawning). Testicular main ducts were analyzed by means of histology and transmission electron microscopy. Additional techniques applied were lectin histochemistry to determine the secretory activity of epithelial cells, as well as immunohistochemistry to demonstrate cell proliferation activity and muscle actin to show distribution and amount of contractile cells. Remarkable variations of the extratesticular ducts mainly concerning the epithelium, were observed during the reproductive phases. Contribution of the main ducts’ epithelia (secretion and cellular detachment) and of degenerating spermatocytes, as sources of carbohydrates and phospholipids, to the seminal fluid were confirmed. Epithelial cell proliferation and secretory activity were dependent on the spawning phase of the specimen. Lectin binding experiments in the present study demonstrated sugar residues (mannose, glucose, galactose, N‐acetyl‐glucosamine) in the epithelial cell cytoplasm and apical glycocalyx, which is important in cell recognition. In the stroma of the testicular efferent ducts, a high number of cells expressed muscle actin and tropomyosin indicating the ducts’ contractile potential for the transportation of seminal fluid towards release. Adjacent to these contractile cells, numerous nerve fibres were found indicating neuronal control of sperm fluid flow.
Immunhistochemical Demonstration of Lumbar Intervertebral Disc Innervation in the Dog
Authors: Willenegger S. , Friess A. E. , Stoffel M. H.
Published: November 2005

Low back pain is a common ailment in dogs, particularly in specific breeds as for instance the German shepherd dog. A number of structures such as facet joint capsules, ligaments, dorsal root ganglia, periosteum, vertebral endplate, and meninges have been associated with this condition. However, in a substantial proportion of all cases, the origin of pain remains obscure. A further structure often being involved in vertebral column disorders is the intervertebral disc. However, the presence of nerves is a precondition for pain sensation and, consequently, structures lacking sensory innervation can be left out of consideration as a cause for low back pain. Nerve fibres have been demonstrated at the periphery of intervertebral discs in man, rabbit and rat. With regard to the dog, however, extent of intervertebral disc innervation still is disputed. The goal of the present study, therefore, was to assess previous findings based on silver impregnation. Protein gene product (PGP) 9.5 was used as a marker for immunohistochemical examination of serial transversal and sagittal paraffin sections of lumbar discs from adult dogs. This technique revealed nerve fibres to be confined to the periphery of the intervertebral discs. Thus, even limited pathological processes affecting the outer layers of the intervertebral disc are prone to cause low back pain.
A New Look on the Origin of the Gonad and the Müllerian Duct: the Sturgeon (Acipencer) as a Model for Vertebrate Urogenital Development
Authors: Wrobel K. ‐H.
Published: November 2005

The origin of the vertebrate gonad and the Müllerian duct are still a matter of debate. According to the majority of recent textbooks, the gonad is a product of the proliferating coelothelium and therefore derived from the mesoderm of the lateral plate region. The Müllerian duct grows parallel to the Wolffian duct, but it is not clear to what extent the latter contributes actively to the development of the former. In the last decade, we reinvestigated early gonadogenesis and Müllerian duct development in a number of vertebrate model species using various morphological techniques (TEM, SEM, immunohistochemistry). The conclusion of our studies is that rudimentary or regressing nephrostomial tubules, particularly cells of their nephrostomes, must be regarded as the immediate precursors of the somatic cells of the gonadal crest and the Müllerian infundibular field. According to this concept, both structures are derivatives of the intermediate mesoderm. Nephrostomial tubules are regular components of the primitive pro‐ and mesonephros. They connect the nephric tubule or the nephric corpuscle to the coelomic cavity and open into the latter by means of a funnel‐like mouth, the nephrostome (coelomostome). In the larval sterlet, Acipenser ruthenus, short, segmentally arranged nephrostomial tubules with well‐developed nephrostomes are present in the region of the cranial opisthonephros. Cells of the medial nephrostomial lips proliferate, surround the germ cells that have accumulated in this location and form a continuous gonadal crest. Cells of the lateral nephrostomial lips proliferate also, spread out on the coelomic surface, replace the original flat mesothelial cells over the Wolffian duct and the cranial opisthonephros and form the Müllerian infundibular field. At about 28 days, a flat pocket begins to invaginate the infundibular field. This pocket is the primordium of the Müllerian ostium abdominale. The findings in Acipenser can be generalized and transferred to other vertebrates.
The Isolated Haemoperfused Distal Cow Limb as an Experimental Model for Studies on the Pathomechanisms of Bovine laminitis
Authors: Wüstenberg R. Y. , Mülling Ch. K. W. , Budras K. ‐D.
Published: November 2005

Introduction:  The pathomechanisms of bovine laminitis are still poorly understood. Isolated perfused organ models are promising tools to study the biological mechanisms that lie behind diseases. They are preferable for ethical and financial reasons but also for experimental necessities.

Aims:  To develop an isolated haemoperfused distal cow limb model, basing on an already existing porcine limb model used in experiments studying the effects of bioactive molecules.

Methods:  Isolated limbs and blood were obtained from routinely slaughtered cows and subsequently perfused in the laboratory of a biotech company (Vitro‐Tec, Berlin) with expertise in organ perfusion. A standard perfusion apparatus enables perfusion for up to six hours under close to physiological conditions. Perfusion pressure and flow were calculated based on values available for horses and pigs. The oxygen saturation was kept at 100% and the pH between 7.35 and 7.45. Glucose was added as nutrient. The lactate concentration was measured as a parameter for oxygen supply and free haemoglobin as an indicator for haemolysis. Potassium content and pressure served as vitality indicators. Additionally, morphological criteria of tissue integrity were assessed using light and electron microscopy.

Results:  A constant flow of 150–200 ml/min was optimal. The resulting pressure ranged from 30 to 117 mmHg. Light microscopy revealed, with exception of a few thromboses, no cellular damage after 4 h. The perfusion was successfully extended to 5 h. Then first pressure related alterations including vascular dilatations and cellular degeneration occurred in the dermal lamellae but not in the other regions. Potassium never exceeded physiological values.

Conclusion:  The model is now available for experiments. Equipment and technical implementation have been standardized and described in a standard operating procedure. The model will be challenged by bioactive molecules such as lactate, neuropeptides and chemokines. Ischaemic conditions will be simulated and studied.

This work was supported by the European Communities under the Lame Cow Project QLK5‐CT‐2002‐00969.The authors are solely responsible and the work does not necessarily represent the opinion of the European Communities.
Double Labelling Immunohistochemistry on the Paravertebral Ganglion Related to the Smooth Vasal Musculature of the Swine Cremaster Muscle
Authors: Botti M. , Gazza F. , Ragionieri L. , Acone F. , Bo Minelli L. , Panu R. , Palmieri G.
Published: November 2005

Retrograde neuronal tracing and double labelling immunohistochemistry methods were used to study the co‐existence of tyrosine hydroxylase (TH) with vesicular acetylcholine transporter (VAChT), neuronal nitric oxyde synthase (nNOS), calcitonine gene related peptide (CGRP), leucine‐enkephaline (LENK), neuropeptide Y (NPY), substance P (SP) and vasoactive intestinal polypeptide (VIP) in the peripheral paravertebral neurons projecting to the smooth vasal musculature of the swine cremaster muscle (CM). The fluorescent tracer Fast Blue (FB) was injected into the left CM of 4 50 Kg non‐emasculated pigs. After 7‐day survival time, the ipsilateral paravertebral L4 (PaGL4) ganglion, the most positive among the autonomic ganglia, was collected. 12 μm‐thick cryostat sections were observed under a fluorescent microscope. In the PaGL4, there were 575.5 ± 152.93 FB+ neurons. On 206 ± 51.07 FB+ cells tested for TH/ VAChT, 24.52% ± 5.6% were TH+/VAChT+, 33.65% ± 7.33% TH+/VAChT‐, 7.46% ± 2.77% TH‐/VAChT+, 34.38 ± 2.66% TH‐/VAChT‐. 211.5 ± 47 FB+ neurons were tested for TH/nNOS: 24.72% ± 5.43% were TH+/nNOS+; 29.35% ± 7.71% were TH+/nNOS‐; 7.23% ± 3.22% were TH‐/nNOS+; 38.7% ± 2.88% were TH‐/nNOS‐. On 213.25 ± 71.82 positive neurons tested for TH/CGRP, 29.4% ± 4.36% were TH+/CGRP+, 29.09% ± 7.61% TH+/CGRP‐, 11.45% ± 4.43% TH‐/CGRP+, 30.06% ± 3.7% TH‐/CGRP‐. On 248.5 ± 73.82 FB+ neurons tested for TH/LENK, 37% ± 4.25% were TH+/LENK+, 40.52% ± 5.96% TH+/LENK‐, 3.54%± 1.37% TH‐/LENK+, 18.94% ± 2.09% TH‐/LENK‐. 210 ± 53.22 FB+ cells were tested for TH/NPY: 50.72 ± 2.8% were TH+/NPY+; 15.38% ± 2.55% TH+/NPY‐; 11.12%±4.32 TH‐/NPY+; 22.79 ± 2.78% TH‐/NPY‐. 218.75 ± 65.02 of positive cells were tested for TH/SP: 26.76 ± 4.73 were TH+/SP+; 36.82% ± 7.58 TH+/SP‐; 3.78% ± 2.43% TH‐/SP+; 32.64 ± 3.75% TH‐/SP‐. On 202.5 ± 47.65 FB+ neurons tested for TH/VIP, 30.95% ± 8.2% were TH+/VIP+, 29.51% ± 3.87 TH+/VIP‐, 8.38%± 2.33% TH‐/VIP+, 31.16%± 6.04% TH‐/VIP‐. The obtained results allow us to document that the neurochemical content of the paravertebral neurons projecting to the CM differ considerably. The contemporaneous presence of adrenergic, peptidergic, nitrergic, and cholinergic neurons shows the complex role that these autonomic ganglia have in the control of smooth vasal musculature.
A Potential Role for Platelet‐activating Factor (PAF) During Bovine Placentation
Authors: Bücher K. , Pfarrer C. , Leiser R.
Published: November 2005

Platelet‐activating factor (PAF) is an important phospholipid messenger involved in diverse physiological and pathological conditions. Actions of PAF are mediated by a specific PAF receptor (PAF‐R) and the inhibition of biological activity by PAF acetylhydrolase (PAF‐AH). To elucidate the potential role of PAF in stimulating angiogenesis and trophoblast giant cell migration during gestation, we examined the expression and localization of PAF‐R mRNA and protein as well as PAF‐AH mRNA in the bovine placenta. RT‐PCR, in situ hybridization and immunohistochemistry were applied to placentomes from days 20, 80, 150, 200, 240, 270 of gestation, and immediately pre‐partum (around day 280). PAF‐R mRNA and protein were colocalized in immature trophoblast giant cells (TGC), uterine epithelial cells, and endothelial cells of fetal and maternal vessels. There was no apparent difference in placental PAF‐R mRNA or protein levels until day 270. In contrast, in placentomes taken immediately pre‐partum the localization of the immunohistochemical and hybridization signals switched from immature TGC and maternal epithelium to cells of the maternal stroma, particularly endothelial cells of maternal vessels. mRNAs for PAF‐AH isoforms Ib (β‐ and γ‐subunit), II, and plasma‐isoform were detected by RT‐PCR in placentomes throughout gestation. In tissue sections mRNA of the PAF specific PAF‐AH‐Ib β‐subunit was predominantly found in TGC, while the intensity of the signal was weaker in maternal epithelium and endothelium of fetal and maternal vessels. Corresponding to the PAF‐R expression, mRNA of PAF‐AH Ib was detected mainly in maternal endothelial cells prior to parturition. The constitutive expression and localization of PAF‐R and PAF‐AH in epithelial and endothelial cells during gestation, together with the parturition‐related switch to the maternal stroma suggests a functional role of PAF for the interaction of TGC with the maternal epithelium, and thus may be associated with TGC migration, placental angiogenesis and pre‐partum tissue programming.

Acknowledgement:  This study was supported by the German Research Foundation.
Distribution and Activity of Fatty Acid Amide Hydralase (FAAH) in the Gastrointestinal Neurons of Mouse
Authors: Costagliola A. , Russo D. , Izzo A. A. , Urbani P. , di Marzo V. , Cecio A.
Published: November 2005

The endocannabinoid anandamide may regulate intestinal motility through activation of CB1 receptors. Anandamide is then inactivated by fatty acid amide hydrolase (FAAH), a membrane bound enzyme. Under pathological conditions, inactivation of such enzymatic activity may lead to inhibition of the intestinal motility. Here, preliminary reports on the distribution of Fatty Acid Amide Hydrolase (FAAH) immunoreactivity in the mouse gastrointestinal neurons, and the pharmacological effects of N‐arachidonoylserotonin (AA‐5HT), a selective inhibitor of FAAH, are reported. FAAH was revealed by an indirect immunofluorescence. Laminar preparations containing the myenteric or the submucous plexus adhered, were peeled off after the whole gut wall had been stretched out and fixed in 4% paraformaldehyde. They were subsequently incubated with a polyclonal anti‐serum directed against a region near the N‐terminus of the human FAAH and revealed by a FITC‐conjugated goat anti‐rabbit secondary anti‐serum. FAAH‐immunoreactive neurons were observed within the myenteric ganglia throughout the GIT. The positive nerve cells varied in size and density of immunoreactivity. Stomach and large intestine showed the highest neuronal density. AA‐5HT significantly reduced both gastric emptying and gastrointestinal tract transit. Such inhibitory effect was reduced by the C1 receptor antagonist SR141716A. Both morphological and pharmacological results suggest that FAAH may play a critical role in controlling gut anandamide levels.
Localization of Estrogen Receptor Beta (ERß) mRNA within Different Bovine Ovarian Follicles
Authors: D’Haeseleer M. M. , Van den Broeck W. L. M. , Simoens P.
Published: November 2005

The oestrogen receptor beta (ERß) is largely distributed in the ovary of many species but data for the bovine ovary are scare. Therefore, the expression of ERß mRNA in the different follicles of the bovine ovary was studied using in situ hybridization. Ovarian tissue sections of three cows with different plasma progesterone concentrations were used (cow 1: 3.50 ng/ml; cow 2: 1.00 ng/ml, cow 3: 0.35 ng/ml). A 602 bp fragment of ERß mRNA was cloned, sequenced and digoxigenin (DIG)‐labelled. Subsequently, in situ hybridization was performed by incubating the sections with the DIG‐labelled RNA anti‐sense probe. For the semi‐quantitative evaluation of ERß mRNA expression the ERß mRNA score (SER) was determined for the different follicular cell types using the formula: SER = 0.n0 + 1.n1+ 2.n2 + 3.n3 with n0, n1, n2, n3 indicating the percentage of cells exhibiting a staining intensity 0 (absent), 1 (weak), 2 (moderate) or 3 (strong), respectively. High ER mRNA levels were noticed in primordial and primary follicle cells, and suggest a role of ER mRNA in early folliculogenesis. A lower SER was observed in the granulosa cells of secondary and tertiary follicles. This significant difference in the SER of follicle cells during follicular growth may be associated with cell proliferation. In obliterative and cystic atretic follicles high SER were observed, although ERß mRNA levels in obliterative follicles showed much inter‐individual variation. This is suggestive for ERß mediated oestrogen action in atretic follicles. In the corpora lutea moderate ERß mRNA levels were noticed. Our findings are in accordance with studies in the ewe in which corpora lutea cells synthesize estrogen. These preliminary findings will be further elaborated in a higher number of cows to examine the role of ERß in the ovary throughout the oestrus cycle.
The ligamentum olecrani of the Elbow Joint in Dogs and Cats
Authors: Engelke E. , Koch R. , Waibl H.
Published: November 2005

The olecranon ligament (ligamentum olecrani) is described as an elastic ligament of the elbow joint in carnivores that tenses the caudomedial part of the joint capsule. The aim of the study was to compare the course and the microscopic structure of the ligament in dogs and cats. The elbow regions of 25 dogs and 15 cats were dissected to examine the topography of the ligament in extension and flexion. Furthermore, the olecranon ligaments of five dogs and five cats were studied using routine histological methods. Additional sections were stained with Resorcin–Fuchsin and Orcein to detect elastic fibres. In both species the olecranon ligament originates at the lateral surface of the epicondylus medialis humeri and inserts at the cranial crest of the olecranon extending distally to the roof of the processus anconeus. Tension of the ligament only occurs when the elbow joint is flexed maximally. This tension is increased by a slight supination of the forearm, which takes place automatically in this joint position. In dogs the ligament is long (30–40 mm in medium sized breeds) and relatively slim (approx. 2–4 mm). In cats the ligament is short (10–12 mm) and relatively strong (5–8 mm). The histological examination of the olecranon ligament shows all signs of a tight collagenous ligament with a negligible amount of elastic fibres. The olecranon ligament helps to limit the maximal flexion of the elbow joint. In addition, it controls a slight lateral movement of the processus anconeus during the automatic supination of the antebrachial bones in extreme flexion of the elbow joint.
Expression and Role of Connexin Proteins in Bovine Claw Epidermis
Authors: Frohberg‐Wang D. , Mülling Ch. K. W. , Budras K. D.
Published: November 2005

Gap junction mediated inter‐cellular communication plays a crucial role in maintaining tissue homeostasis in skin. Gap junctions consist of hemi‐channels (connexons), which in turn are established by transmembrane proteins (connexins, Cx). The pattern of Cx‐ protein expression is characteristic for tissues and depends on the differentiational and functional state.

Aim:  As part of our investigations of cell biological causes for lameness in dairy cows we wanted to assess the expression of Cx‐proteins in relation to differentiation in bovine claw epidermis.

Methods:  We examined hoof tissue from five defined regions of the claws of 10 cows from the abattoir by immunohistochemistry, Western blot and by transmission electron microscopy. The following monoclonal antibodies were used: anti‐ Cx26, Cx31, Cx37 (ADI, USA) and anti‐Cx43 (Chemicon, USA). Immunohistochemistry was carried out on frozen and paraffin sections.

Results:  Cx43 was expressed in the bovine claw epidermis in a pattern described for skin. Cx43 expression was strongest in the stratum basale in all regions. Cells of the stratum spinosum expressed lower levels, and in terminal differentiating cells Cx43 was disappearing. Expression of Cx26, 31 and 37 showed a high variation between individuals and regions. Protein expression was verified by Western blotting. The formation of gap junctions in the respective layers was confirmed by electron microscopy.

Conclusion:  Our results provide evidence that Cx 43 is expressed in bovine claw epidermis in a distribution similar to skin. Cx 43 is progressively downregulated with the progress of differentiation. This suggests a role of Cx43 in cellular coordination particularly in the early stages of differentiation. Results for Cx 26, 31 and 37 will be confirmed in further studies also on the mRNA level to get deeper insight in the regulation of Cx expression in claw epidermis. We hypothesize that Cx 43 plays a similar role in controlling differentiation as in skin and may be involved in disruption of claw horn formation in claw disorders.

Acknowledgement:  This work was supported by the European Communities under the Lamecow project QLK5‐CT‐2002‐00969. The authors are solely responsible and the work does not necessarily represent the opinion of the European Communities.
S100 Expression in the Lateral Line System of Zebrafish from Juvenile to Adult Stages
Authors: Germanà A. , Abbate F. , Guerrera M. , Laurà R. , Levanti M. B. , Germanà G.
Published: November 2005

The lateral line system (LLS) is a sensory organ present in fishes and in the larval stage of aquatic amphibians, which provides the animal with information about its surrounding environment. Mechanoreceptive sensory cells in the LLS of fishes are grouped into neuromasts that are considered the morphological unit of this system. Structurally the neuromast consists of a central cluster of hair cells with a ring of support cells with some lateral mantle cells. The LLS is localized in both the body surface (superficial neuromast or pit organs) and in a system of canals distributed along the head, trunk and tail (deep neuromast) (Cernuda‐Cernuda et al., 1996). The study of hair cells in the neuromast of fishes is becoming of a great interest, since they are similar in structure and function to the sensory cells of the inner ear and show some identical immunohistochemical properties (Foster et al., 1993). Recently, we have demonstrated the occurrence and the presence of S100 protein in the neuromast hair cells of some teleostean species (Abbate et al., 2002; Germanà et al., 2004). The zebrafish has become an ideal model for the study of developmental genetics, and the neuromast has been used as a model for the study of vertebrate auditory system (Withfield, 2002), so the need for cell selective markers has increased dramatically. Therefore, in this study we have investigated by tissue and whole‐mount immunohistochemistry the presence of S100 protein in both deep and superficial neuromasts in the zebrafish larval, juvenile and adult stages. The results of this work demonstrated that the localization of S100 protein was restricted only to the hair cells of the neuromast at all the examined ages. The mantle cells and supporting cells within the neuromast were unreactive. Nevertheless, heterogeneity has been detected in the S100 protein expression by hair cells, independent of their localization within the neuromast. Our data suggest that the S100 protein is a specific marker for sensory cells in the neuromast of zebrafish at post‐embryonic different stages of development, and it could be important for the analysis of development, regeneration and turnover of hair cells.
Morphological and Ultrastructural Study of Vaccine‐induced Granuloma in Atlantic Salmon (Salmo salar)
Authors: Haugarvoll E. , Bjerkås I. , Poppe T. T. , Koppang E. O.
Published: November 2005

Aim:  The aims of this study were immunhistochemical (IHC) and ultrastruktural characterization of cells involved in vaccine granuloma formation in Atlantic salmon.

Methods:  Fish suffering from severe reaction 6 to 9 months after vaccination were selected. We investigated granuloma from peritoneum, at the injection site. Morphological staining techniques, IHC to visualise MHC+ cells and ultrastructural analysis were performed.

Results:  Microbial investigations revealed no infective agents. Histological and ultrastructural studies revealed Russel bodies, macrophages, multinucleated giant cells, melanomacrophages (MMP), mast cells (MC) and Splendore–Hoepply material in the lesions.

Conclusion:  Signet ring cells identified after vaccination have been hitherto described as svollen mast cells. This study shows that these cells fit the Russel body characteristic, consistent with humoral stimulation following vaccination and foreign material precipitate, Splendore‐Hoepply material. MHC+ cells were located circumferal to granuloma centers. MMP were present containing melanosomes in different developmental stages.
Biometrical Studies on the Cornea in Some Domestic Animals
Authors: Hifny A. , Abdalla K. E. H. , Aly K. H. , Kamel G.
Published: November 2005

This work was carried out on the cornea of the eyeball of the donkey, cow, buffalo, camel, sheep, goat, pig and dog. The following dimensions were measured: the outer and inner dorsoventral and mediolateral dimension, also the plain, weight and the volume of the cornea and fibrous coat were recorded. The morphometric studies include also thickness of the cornea and its different layers centrally and peripherally. Generally, the thickness of the cornea is about 0.5–0.8 mm, the thickest cornea was observed in cow followed by pig and donkey, the thinnest cornea was observed in sheep followed by camel, goat and buffalo, while the cornea of the goat and buffalo has nearly the same thickness. The biometry of the cornea has been useful for assessment of certain pathologic and congenital abnormalities such as microcornea, megalocornea moreover this measurement are of utmost important when superficial keractectomy and keratoplasty are indicated.
Vascular Mimicry of Granulosa Cells: a New Concept of Corpeus Luteum Development?
Authors: Hirschberg R. M. , Plendl J.
Published: November 2005

So far, it was generally accepted that newly formed blood vessels are exclusively comprised of endothelial cells, and complemented by pericyte and myocyte recruitment during vessel maturation. Accordingly, participation of non‐endothelial cells in the formation of blood vessels has rarely been suggested. Recently, evidence supporting the existence of tumour vessels lined by non‐endothelial cells has emerged. Consequently, the concept of the inherent capacity of non‐endothelial cells to behave like endothelial cells has been discussed for tumours, and this pathomechanism has been termed vascular mimicry. The corpus luteum is one of the most intensely vascularized tissues, and angiogenesis in the corpus luteum is more effective than in highly malignant tumours. Our results indicate active involvement of granulosa cells in luteal angiogenesis, and the aim of this study was to shed more light on this exciting prospect. The study was based on cultured granulosa cells isolated from the bovine ovary in different stages of follicle maturation. Morphology of angiogenic granulosa cells was studied by phase contrast, transmission electron and scanning electron microscopy. Expression of angiogenesis‐regulating factors and their receptors was demonstrated by polymerase chain reaction (RT‐PCR). Cultured granulosa cells underwent changes reminiscent of endothelial angiogenesis, i.e., migration, proliferation, differentiation and three‐dimensional organization, and expressed angiogenesis‐regulating factors and their receptors. Our results suggest a tight regulatory and structural association of endothelial and granulosa cells in luteal angiogenesis, suggesting physiological vascular mimicry in the ovary.
How to Open the Mouth: the Kinetic Skull, and the Jaw and Branchial Apparatus in Sharks
Authors: Homberger D. G. , Westphal K. A. , Gudo M. , Bragulla H. H.
Published: November 2005

Aims:  Mammals cannot swallow if their necks and, hence, their mm. sternohyoidei et sternothyroidei are stretched. In order to understand the functional morphology of mastication and swallowing in mammals, it is useful to understand its evolutionary origin.

Material and Methods:  Five formalin‐preserved specimens of the Spiny Dogfish (Squalus ancanthias) were used for dissection or to prepare sagittal or transverse serial sections.

Results and Conclusions:  The feeding apparatus of the shark Squalus acanthias as a representative of the cartilaginous fishes serves as an excellent model of that of an ancestral vertebrate with a kinetic skull, in which the chondrocranium is movable relative to the jaw apparatus. To open the mouth, the cranium is raised by the epibranchial musculature, the palatoquadrate cartilage (i.e., upper jaw) is entrained, and the mandibular cartilage is held in place and prevented from following the upper jaw by the coracomandibular muscle. At the same time, the branchial basket and oropharyngeal cavity are expanded and held in place by the coracohyoid and coracoarcual muscles, which prevent the hyoid and branchial arches from being entrained cranially by the raised chondrocranium. The mandible is not retracted during the opening of the mouth in sharks because the branchial region would have to be compressed instead of expanded. In the course of the evolutionary modifications leading to mammals, the heart and respiratory apparatus became decoupled from the feeding apparatus through the formation of a neck. The skull became akinetic through the integration of the upper jaw into the base of the cranium. The mouth is not opened any longer by lifting the cranium, but by depressing the mandible through a special muscle, the m. depressor mandibulae. The coracomandibular muscle lost its function as a stabilizer of the mandible during mouth opening and became modified into the lingual and geniohyoid muscles.
Morphology as a Tool in Embryo Technology
Authors: Maddox‐Hyttel P.
Published: November 2005

The increased use of embryo technologies, in particular in cattle, has emphasized the need for assessing embryo quality by morphological techniques, such as transmission electron microscopy, immunohistochemistry, immunocytochemistry for confocal laser scanning microscopy, and fluorescence in situ hybridization. By a combination of these techniques, we have demonstrated that (1) the rRNA gene activation in the 8‐cell bovine embryo as monitored by nucleolar development is comparable in embryos developed in vivo and produced in vitro, whereas embryos reconstructed by cloning by nuclear transfer display deviations in nucleolar development, (2) generating bovine embryos by in vitro production and reconstruction by nuclear transfer is associated with increased occurrence of apoptosis, in particular in the inner cell mass of the blastocyst, (3) the same two embryo production techniques are associated with increased occurrence of mixoplody, i.e. embryos presenting a major population of normal diploid cells and a minor population of abnormal haploid or polyploid cells, and (4) upon hatching from the zona pellucida, the inner cell mass of the bovine blastocyst differentiate into hypoblasts and epiblasts, the latter of which forms a laminin enclosed pluripotent cell niche staining positive for the pluripotent transcription factor Oct‐4; a morphology that resembles that maintained by bovine ES‐like cells cultured through several passages in vitro.
Fish immunology. Fish Erythrocytes Release Cytokine‐like Factors that Modulate Autologous Macrophage Function
Authors: Jirillo E. , Passantino L. , Ribaud M. R. , Passantino G. F.
Published: November 2005

According to our previous findings (Passantino et al. 2002a,b; 2004) we discovered that fish erythrocytes (FE) act as immune cells in that they bind and engulf Candida albicans (CA). Then, these activated FE interact with macrophages (MØ) so that a so‐called rosette results from such an interplay. A rosette consists of one or more MØ containing ingested CA, centrally located and surrounded by CA activated FE. Quite interestingly, we found that supernatants of CA‐activated FE contain cytokine‐like factors able to modulate MØ function. In particular, active supernatants are able to inhibit MØ migration under agarose and this activity was identified as macrophage inhibiting factor (MIF) – like function, by analogy to human MIF. On the other hand, same supernatants could enhance MØ phagocytosis of CA, thus contributing to fungus killing. Taken together, these results suggest that FE act as immune cells by secreting cytokine‐like factors, which contribute to the rosette formation through modulation of MØ.

References  Passantino, L., M. Altamura, A. Cianciotta, R. Patruno, A. Tafaro, E. Jirillo, and Passantino G.F., 2002a: Fish immunology. I. Binding and engulfment of Candida albicans by erythrocytes of rainbow trout (Salmo gairdneri Richardson). Immunopharmacol. Immunotoxicol. 24: 665–678

Passantino, L., A. Tafaro, M. Altamura, R. Arena, G.F. Passantino, E. Jirillo, 2002b: Fish immunology. II. Morphological and cytochemical characterization and phagocytic activities of head kidney macrophages from rainbow trout (Salmo gairdneri Richardson). Immunopharmacol. Immunotoxicol.24: 679–691

Passantino, L., M. Altamura, A. Cianciotta, F. Jirillo, M.R. Ribaud, E. Jirillo, and G.F. Passantino, 2004: Maturation of fish erythrocytes coincides with changes in their morphology, enhanced ability to interact with Candida albicans and release of cytokine‐like factors active upon autologous macrophages. Immunopharmacol. Immunotoxicol. 26 (in press).
On the Development of the Feline Skeleton
Authors: Knospe C. , Roos H. , Vollmerhaus B.
Published: November 2005

To find precise data about the development of the skeleton of the domestic cat (Felis catus) embryological sections cleared specimens stained for bone and cartilage and radiographs were used (for details see our upcoming publication). The pre‐natal development is characterized by three main phases: the mesenchymal skeleton, already formed around stage 5 (Knospe, 2002) of the cat's development, with the notochord, somites, pharyngeal arches and limb buds, the cartilage skeleton arising during stage 14, and the bony skeleton formed by intramembranous and endochondral ossification from stage 16/17 on. The mesenchymal skeleton survives as periosteum in different ligaments, joint capsules, syndesmoses and inter‐vertebral disks. The cartilage skeleton is reduced up to birth to the epiphyseal and tympanic cartilages, and survives as laryngeal, tracheal, joint, rib, sternal, nasal cartilages and synchondroses. In the newborn cat all primary centres are present, except the carpal and distal tarsal bones, secondary centres aren not established radiographically yet. The secondary and tertiary centres are formed post‐natal from the first week on up to the second year. The results are presented in nine slides showing histological pictures with the main pre‐natal events and schemes of the post‐natal development with the skull, trunk and limb centres and their first appearance.
A Novel In Vitro Model of Canine Malignant Hemangioendothelioma
Authors: Kühn D. , Kohn B. , Brunnberg L. , Plendl J.
Published: November 2005

Introduction and Aim:  Canine malignant haemangioendothelioma is an aggressive neoplasia that affects mostly older dogs of large breeds with a strong predilection for the spleen, liver, heart and skin. The tumour originates in the vascular endothelium and consists of transformed cells forming large and leaky vessel‐like structures. Prognosis is poor because surgery and chemotherapy have limited success in prolonging survival times and increasing quality of patients. A new strategy to treat this malignancy could be anti‐angiogenic therapy based on the inhibition of proliferation, migration and three‐dimensional organization of transformed cells. In order to reduce animal experiments, in vitro‐models are required to test the safety and efficacy of anti‐angiogenic drugs. So far only few models of angiogenesis are available using mostly human, rodent and bovine cells. Therefore, the aim of our study was to establish an in vitro model of canine haemangioendothelioma.

Materials and Methods:  Tumours were collected from dogs during surgery or immediately after euthanasia. Isolation of cells was done from different areas of the tumours and by enzymatic digestion of the tissue. Cells were incubated in culture media with and without endothelial growth factors. Cells were characterized by lectin histochemistry using Dolichos biflorus agglutinin, Ulex europaeus agglutinin and Bandeiraea simplicifolia agglutinin I. Moreover, RT‐PCR (polymerase chain reaction) was employed to investigate the expression of vascular endothelial growth factor (VEGF) and its endothelium‐specific receptors VEGF‐R1 and ‐R2.

Results and Conclusions:  Different populations of cells were isolated and cultured successfully from canine malignant haemangioendothelioma. Cells show characteristics of microvascular endothelial cells of an angiogenic phenotype, i.e. the formation of spheroids and tube‐like structures as well as strong labelling for Bandeiraea simplicifolia agglutinin I. Thus, morphological and glycohistochemical results confirm the vascular character of the cells isolated. RT‐PCR showed expression of VEGF. However, endothelium‐specific VEGF receptors were not expressed. Loss of typical receptors is common in cancer and may correlate with increased tumour dedifferentiation.
The Development of Parabronchi in the Duck
Authors: Mansour A. A. , Abdelmohdy F. E. , Kasab M. A.
Published: November 2005

This work was carried out on the parabronchi of ducks during the pre‐ and post‐hatching periods. For this purposes, the samples were taken from the embryo of incubated eggs from the 10th to 28th day of incubation, at regular interval, and from adult ducks under anaesthesia. The parabronchi first appeared at 14th day of incubation as out‐pouches, from the secondary bronchi, with solid free end invading the surrounding mesenchym. These pouches was lined by high cuboidal cells rested on PAS positive basal lamina, the cells showed free luminal quarter, which indicated an apocrine secretion. At 17th to 22nd day of incubation, irregularities invading the underlying tissue were observed. This invading part was club‐shape forming the future atrium and air capillaries. At 24th to 28th day of incubation the parabronchi were observed at the center of a polygonal shape lobule (lung lobule), it was lined with squamous cells at the head of the clubs, while atrium was lined with cuboidal cells at both side of the stalk (interatrial septum), and the air capillaries was lined with attenuated squamous cells. Because of the breathing of the duck started before hatching the structure of the parabronchi was similar in post‐hatching period to that of late stage of pre‐hatching period. The wall of parabronchi was pierced by a number of openings leading to the atrium. The lining epithelium was followed by a layer of smooth muscles, which formed a sphincter around the opening of the atrium regulating the amount of air passed to the atrium and air capillaries to avoid hyperventilation. The E.M. picture revealed that, the epithelium of parabronchi was squamous and displayed a scarce number of microvilli, while that of the atrium was cuboidal with a large number of microvilli, the cells of both contained osmiophilic multilamiller bodies that extruded to the surface. Under the epithelium of parabronchi, there were: smooth muscle cells, macrophages, mast cells, collagen fibers, and fibroblasts, while in the interatrial septum between the epithelium on both side there were collagen fibers and fibroblasts only.
Anatomy of the Thorax Structures in a Newborn Dromedary Camel by Computed Tomography
Authors: Arencibia A. , Rivero M. A. , Gil F. , Ramírez J. A. , Ramírez G. , Vázquez J. M.
Published: November 2005

The anatomical and clinical studies of computed tomography (CT) in Camelidae are scarce. The use of CT in large animal medicine is currently limited by the logistic problems of acquiring computed tomographic images. Several CT studies exist on adult llamas, but not in camels. Accurate interpretation of the planimetric CT normal anatomy is necessary for the study and evaluation of pathological tissues. The purpose of our work was to evaluate the thorax of the newborn camel and related structures by means of CT images and macroscopic sections. One newborn camel of one week was used. It was euthanized for medical reasons unrelated to disease of the thorax. CT images were obtained and detailed anatomy of the thorax was acquired. Different CT windows, soft‐tissue and pulmonary windows, were applied in order to obtain detailed attenuation shades of the thoracic structures. The camel was frozen and sectioned using an electric saw, and we obtained high quality images of the thorax compared with CT images. Clinically relevant anatomic structures of the thorax cavity were identified and labelled in the corresponding CT and gross‐section photographs. The information presented in this paper should serve as an initial reference to evaluate CT images of the newborn camel thorax.
Regulation of Cellular Proliferation and Differentiation in Claw Epidermis by Epidermal–Dermal Interactions
Authors: Mülling Ch. K. W. , Nebel U. , Budras K. ‐D.
Published: November 2005

Introduction:  Epidermal–dermal interactions play an important role in regulation of proliferation and differentiation of keratinocytes and also in disease and regeneration of skin. A double paracrine regulation of keratinocyte growth and differentiation has been postulated and described in vitro recently. Interleukin‐1 (Il‐1) and keratinocyte growth factor (KGF/FGF‐7) and their receptors are major mediators in this epidermal‐dermal signalling.

Aim:  We wanted to generate evidence for a reciprocal regulation of epidermal proliferation and differentiation in the bovine claw involving Il‐1 and KGF.

Methods:  Tissue samples from claws of slaughtered cows, co‐cultures and three‐dimensional cultures of bovine hoof keratinocytes and fibroblasts were used. Immunohistochemistry for Il‐1 and Il‐1 receptor, KGF and KGF receptor were carried out on frozen and paraffin sections from tissue and cell culture. RT‐PCR was used to study regulation at the mRNA level in dermis and epidermis (after micro separation) and in fibroblasts and keratinocytes from the cultures respectively.

Results:  In claw epidermis Il‐1 was accumulated and KGF receptor was detectable. Interleukin‐1 receptor and KGF were present in dermal cells. Il‐1 stimulated fibroblasts responded with KGF expression in vitro. Neutralization of Il‐1 reduced or stopped this effect. Keratinocytes co‐cultured with fibroblasts showed a significant better growth. Presence of keratinocytes increased KGF expression in the co‐cultured fibroblasts.

Conclusion:  Our results suggest that Il‐1 is leading to stimulation of KGF expression. KGF seems to be an important stimulator of keratinocyte proliferation in our co‐culture system. So far our results provide initial evidence for a reciprocal regulatory mechanism present in the bovine claw involving Il‐ 1 produced in the epidermis and KGF originating from fibroblasts. Future in vitro studies including mRNA analysis will provide deeper insight into the epidermal‐dermal cross‐talk and the involved transcriptional/translational regulation in the bovine claw.

This work was supported by the European Communities under the Lame cow project QLK5‐CT‐2002‐00969. The authors are solely responsible and the work does not necessarily represent the opinion of the European Communities.
In Vitro Infectioion of Bovine Hoof Cells and Skin Explants with Treponema Brennaborense and Treponema Denticola
Authors: Nebel U. , Mülling Ch. , Nordhoff M. , Budras K. ‐D.
Published: November 2005

The aim of this study was to establish an in vitro model that permits in vitro infection of bovine skin with Treponema spp. and enables to study the role of treponemes in the pathogenesis of digital dermatitis (DD). In all experiments, incubation with T. denticola or T. brennaborense was carried out simultaneously. Keratinocytes obtained from the claw were cultivated on cover glasses without antibiotics until they reached sub‐confluence. Then they were incubated with OMIZ Pat medium containing treponemes for up to 96 h. Every 24 h two cover glasses were fixed and stained with the DAPI method. Skin explants were obtained from typical sites of DD lesions. First the explants were maintained in medium with antibiotics to eliminate bacterial contamination. Subsequently, they were rinsed thoroughly with medium without antibiotics and incubated with Treponema suspensions for 48 h. The treponemes stayed vital under culture conditions for even up to 96 h. They were still showing their typical spiral shaped morphology and adhered to the cultured keratinocytes at all time points. With prolonged incubation time cultured cells began to show morphologic damage and some cells detached from the cover glasses. Light and electron microscopical investigations of the explants revealed that treponemes were adhering to the surface of the epidermis. They were visible in often‐enlarged intercellular spaces. In addition treponemes could be detected in deep epidermal layers and in very high concentrations in the dermis. In periodontal disease, spirochetes were observed in enlarged intercellular spaces. Our results support these findings suggesting that treponemes invade the deeper claw tissue via the intercellular spaces of the epidermis. We suggest that the enlargement of the intercellular spaces can improve an increased infection of deeper tissue layers and facilitates the way for infection with other anaerobic bacteria.