In the field of angiogenesis research considerable effort is put in the development of in vitro assays of angiogenesis to replace animal experiments. Unfortunately, reproducibility of these assays frequently fails depending on the particular batch of endothelial cells delivered by the distributor. This is due to the lack of reliable markers for the identification and isolation of angiogenic microvascular endothelial cells that have the capacity to perform all stages of the angiogenic cascade.This study was carried out to identify potential markers for angiogenic versus non-angiogenic endothelial cells. The protein expression profile of four capillary-derived human microvascular primary endothelial cell cultures of which only two batches could be stimulated to angiogenesis was investigated and compared by two-dimensional gel electrophoresis. Seven proteins were found to be expressed in the angiogenic batches only. One protein was detected exclusively in the non-angiogenic batches. These proteins might be verified as markers for angiogenic endothelial cells.

Eighty piglets aged 14, 28, 35 and 56 days -weaned at day 28 -were subjected to this investigation. Each age-group consisted of five animals which were fed an Enterococcus faecium NCIMB 10415 (Cylactin ) and Bacillus cereus var. toyoi (Toyocerin ) based diet. Five animals served as controls. Tissue samples were collected immediately after sacrifice at 8.30 h a.m. from duodenum, jejunum, ileum, cecum and colon to examine intestinal morphology and histochemistry. The results showed that with respect to villus height and crypt depth supplementation of probiotics in piglets feed seemed to influence the morphology and enlargement factor not at all or only to a certain extent. With respect to the number of goblet cells, the difference between probiotic fed animals and control animals was generally extremely low. The shape of the villi of the small intestinal segments greatly varied in all age groups of control and probiotic fed animals. However, this morphological variety does not depend on the mode of feeding.

The histological characterization of the intestinal mucus layer is important for many scientific experiments investigating the interaction between intestinal microbiota, mucosal immune response and intestinal mucus production. The aim of this study was to examine and compare different fixation protocols for displaying and quantifying the intestinal mucus layer in piglets and to test which histomorphological parameters may correlate with the determined mucus layer thickness. Jejunal and colonal tissue samples of weaned piglets (n=10) were either frozen in liquid nitrogen or chemically fixed using methacarn solution. The frozen tissue samples were cryosectioned and subsequently postfixed using three different postfixatives: paraformaldehyde vapor, neutrally buffered formalin solution and ethanol solution. After dehydration, methacarn fixed tissues were embedded in paraffin wax. Both, sections of cryopreserved and methacarn fixed tissue samples were stained with Alcian blue (AB)-PAS followed by the microscopically determination of the mucus layer thickness. Different pH values of the Alcian Blue staining solution and two mucus layer thickness measuring methods were compared. In addition, various histomorphological parameters of methacarn fixed tissue samples were evaluated including the number of goblet cells and the mucin staining area. Cryopreservation in combination with chemical postfixation led to mucus preservation in the colon of piglets allowing mucus thickness measurements. Mucus could be only partly preserved in cryosections of the jejunum impeding any quantitative description of the mucus layer thickness. The application of different postfixations, varying pH values of the AB solution and different mucus layer measuring methods led to comparable results regarding the mucus layer thickness. Methacarn fixation proved to be unsuitable for mucus depiction as only mucus patches were found in the jejunum or a detachment of the mucus layer from the epithelium was observed in the colon. Correlation analyses revealed that the proportion of the mucin staining area per crypt area (relative mucin staining) measured in methacarn fixed tissue samples corresponded to the colonal mucus layer thickness determined in cryopreserved tissue samples. In conclusion, the results showed that cryopreservation using liquid nitrogen followed by chemical postfixation and AB-PAS staining led to a reliable mucus preservation allowing a mucus thickness determination in the colon of pigs. Moreover, the detected relative mucin staining area may serve as a suitable histomorphological parameter for the assessment of the intestinal mucus layer thickness. The findings obtained in this study can be used for the implementation of an improved standard for the histological description of the mucus layer in the colon of pigs.

This paper describes the in situ presence and distribution of collagen types I, 111, IV and V in the bovine placenta. The objective was to determine whether there are qualitative and/or quantitative differences in the collagen content of placentomes originating from cows with retained placenta and cows with normal discharge of the placenta. Twelve h post partum discharge of the placenta or the incidence of retained placenta was monitored. From 57 cows one placentome per cow was collected within 1 h post partum.The cows were divided into three groups: retained placenta after caesarean section (Group l), retained placenta after spontaneous calving (Group 2) and normal discharge of placenta within 12 h post partum after spontaneous calving (Group 3). A pilot study was conducted to establish the technique of collecting the placentomes and to verify the applied immunohistological methods used in this work. In the following study, 32 placentomes were used to determine the amount of collagen (types I, 111, IV and V) with qualitative and semi-quantitative methods using immunohistochemical techniques. Collagen types I, 111, IV and V were found in large quantities in the maternal tissue. In the fetal connective tissue the amount of these collagen types was smaller. In the placentomes of the three groups, no qualitative or quantitative differences could be detected.

Mucins are of great interest in intestinal research and histochemical methods are often employed to identify them. Since it is in the nature of mucins that they are “hard to hold onto” once they come into contact with water, a frequently used medium in histochemistry, there are a number of challenges that may decrease diagnostic accuracy. As the outcome of methods published for microscopic detection of mucosubstances proved to be unsatisfactory in our hands, the aim was the establishment of a reliable and reproducible protocol. Tissue samples were available from pig feeding experiments. In the present study, we focus on a fixation / staining procedure without making comparisons between differently fed pigs. Several fixation and staining procedures were evaluated for their use in semiautomatic quantification and quality assessment of different mucus fractions simultaneous on one tissue section. Cryostat sectioning, subsequent fixation steps with heat, ethanol and modified Bouin’s solution, followed by triple staining with high iron diamine, alcian blue and periodic acid-Schiff turned out to be the best method to identify sulfomucin, sialomucin and neutral mucin simultaneous on one tissue section. This methodology resulted in very good morphology of goblet cells with intact mucin containing vesicles within the cells, which was comparable to ultrastructural electron microscopical observations. Semiautomatic quantification of different mucins was possible. In conclusion, reliable mucus quantification and assessment of mucus quality requires strictly tested procedures. According to our experience, the most important aim after cryosectioning is fast fixation of the mucosubstances, which requires a combination of different fixation steps.