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Notes: 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.

Notes: 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.

Notes: The aim of this study was to establish a co-culture of bovine hoof keratinocytes and fibroblasts in different types of perfusion chambers under defined conditions. The perfusion chamber PCS3c (Oligene, Berlin) was used to culture dermal fibroblasts and epidermal keratinocytes separated by a Millicell® Insert (Millipore, Schwalbach). In addition novel perfusion chambers developed by Dirk Hoffmann were used in combination with S & S membrane filters (Schleicher & Schuell, Dassel). First, fibroblasts were seeded onto one side of the insert/membrane and allowed to grow for 4 days. Subsequently keratinocytes were seeded onto the other side and allowed to adhere for one day. Then the chambers were connected to the tubing system with an attached 8-channel pump and a gas permeable media bag. We worked with different media and flow rates ranging from 0.035 ml/min to 0.35 ml/min. The system was run for up to 20 days. After a few days in culture the cells had grown to confluence. Then keratinocytes began to differentiate and built up stratified colonies. Within these colonies the cells showed the characteristic morphology of a stratified squamous epithelium. The use of perfusion chambers allows three-dimensional cultures to grow and survive for weeks, because of an unlimited medium supply and gas exchange. Additional, perfusion chamber systems enable the co-cultivation of fibroblasts and keratinocytes separated by membranes, which permit the exchange of molecules like growth factors. Furthermore, it is possible to perfuse the two different parts of the perfusion chamber with various and even different types of media. This provides the opportunity to add a growth factor to the medium for only one cell type and to study the effect of this factor to the other cell type. 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.

Notes: Introduction:  Interactions between horn quality and flooring systems significantly impact on claw health. Poor horn quality is associated with reduced milk yield, painful lesions or slaughtering. Horn quality is determined by horn cells, intercellular cement and hoof horn architecture, i.e. the structure and arrangement of the horn tubules. Soft and elastic flooring systems have been reported to have a positive influence on the microstructure of hoof horn.Aims:  We wanted to correlate the microstructure of claw horn to the horn quality in a large number of samples and detect influences of the flooring system on hoof horn microstructure.Methods:  Samples were obtained from two different studies: 40 cows from Hohenheim/Germany kept on slatted floor and subsequently on rubber mats for 4 months; 1000 cows from 11 farms in Luxembourg, kept on concrete floor. From all samples 7 μm-sections were cut and stained with the PAS-reaction. Morphometry was done with the LuciaG©-software (Nikon GmbH, Düsseldorf, Germany) and statistical analysis was performed with SPSS® (SPSS Inc., Chicago, IL, USA). Parameters measured were area, minimal and maximal diameter of both medulla and cortex of horn tubules. Statistical analysis included: tubules per visual field, area per visual field, total medulla area, total cortex area and the limiting values of diameters.Results:  Luxembourg samples showed consistency of tubule-parameters between both dates; all farms showed an average number ranging from 13.36 to 14.79 tubules per visual field. Hohenheim horn from the hard flooring showed 19.9 and horn of the same animals kept on rubber floor showed 20.5 tubules per visual field. Maximum diameter of hard-floor horn is 55 μm whereas 45 μm in animals kept on rubber floor.Conclusions:  The results show that genetic/individual differences take smaller influence on horn quality than housing systems. These outcomes provide evidence for the economical and animal welfare benefit of soft and elastic flooring systems. Further research will focus on correlation of biodata, such as physiological state, lactation, age, and claw horn microstructure.This work was supported by Biopsytec/Rheinbach.

Notes: Introduction and aims:  Claw diseases in dairy cows not only lead to high economic losses, but also cause pain and reduce animal welfare. Former studies pointed out that flooring properties influence the microstructure of hoof horn. In this study, we looked for correlations between different flooring systems and horn quality. We obtained horn samples from an experimental farm in Alnarp/Sweden from cows that were first kept on slatted floor and subsequently on mastic asphalt respectively on rubber mats.Methods:  Untreated horn samples were glued onto wooden blocks: 7 μm sections were prepared with a Polycut-S (Leica) microtome and afterwards stained with the PAS reaction. For morphometrical analysis we used a Zeiss Axioskope, a digital camera (Progress 3012) and the Lucia 32 G software (Nikon). These data were exported into EXCEL (Microsoft) and statistical analysis was carried out with SPSS (SPSS Inc. [[Au: please provide manufacturer details for ‘SPSS’: city, state code (if USA) and country]]). We measured area, minimal and maximal diameter of medulla and cortex of the horn tubules. Statistical evaluation included number of tubules and tubular area per visual field, total area of medulla and cortex and the limiting diameters.Results:  We found significant alterations of the hoof horn after moving the cows from hard to soft floor. The diameter of the horn tubules decreased significantly. The area of medulla and the ratio of medulla to cortex decreased as well.Conclusions:  Our findings indicate a higher quality of claw horn and an increased resistance against mechanical, chemical and microbial environmental influences in animals kept on soft floors. The functional implications of these observations are discussed with regard to claw health as well as the arising consequences for dairy housing systems.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.

Notes: 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.

Notes: The aim of this study was to characterize the ultrastructure of three-dimensional keratinocyte colonies in relation to ex vivo bovine hoof epidermis. Keratinocytes were isolated from the claw and cultured in equal parts of Dulbecco's modified Eagle's medium (Sigma, Taufkirchen) supplemented with 10% fetal bovine serum (Sigma, Taufkirchen) and serum-free MCDB 153 complete medium (Biochrom, Berlin). Cells were allowed to grow until formation of colonies occurred. Then the colonies were isolated by removing the surrounding cells with a cell scraper every 3 days. The colonies were harvested when the reached diameters of 6–8 mm and were fixed in Karnovsky's solution. They were routinely processed for the embedding in Agar 100. Semithin and ultrathin sections were prepared and examined under the light and transmission electron microscope.These colonies revealed the morphological properties and tissue architecture, which are specific for claw epidermis in vivo. We detected up to 22 layers of keratinocytes and could discover between three stages of differentiation: a basal cell layer, followed by several cell layers corresponding morphologically to the cells of the stratum spinosum and a few cornified superficial cell layers. Throughout all layers cells were connected by desmosomes, which were linked to the cytoskeleton by dense bundles of filaments. In addition we observed the formation of desmosomes. The cells of the upper un-cornified layers generated an cornified envelope. In the following layers cell death and subsequent cornification accompanied by the characteristic alterations of the nuclei occurred. The cells at the superficial layers were filled by electron dense, solid horn masses. Additionally we detected intercellular cementing substance in the intercellular space between the upper layers. Our electron microscopical characterization of the colonies provides evidence that the bovine hoof keratinocytes invitro undergo basically a similar pattern of proliferation and differentiation like in the claw in vivo. These findings provided a basis for further studies like the long-term culture and differentiation of bovine hoof keratinocytes in perfusion chambers.

Notes: 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.