Hana Hünigen

Vascular endothelial growth factor receptor 2-specific microbubbles for molecular ultrasound detection of prostate cancer in a rat model.

Objectives:To investigate whether rat prostate cancer can be detected by means of molecular ultrasound (US) using target-specific microbubbles versus a nonspecific contrast agent. Materials and Methods:A total of 20 Copenhagen rats were randomly examined 75 to 104 days after orthotopic implantation of G-Dunning rat prostatic tumor cells was using a high-end US system and either 1.2 mL/kg of the nonspecific agent A or 0.1 mL/kg of the target-specific agent B, containing vascular endothelial growth factor receptor 2 binding peptide. Contrast inflow (areas under the curve analysis) was determined during the first 30s, and all microbubbles were destroyed in the scan plane by means of the flash technique 20 minutes after intravenous administration to investigate specific accumulation of individual bubbles in tumors. Differences between normal prostate tissue and tumor were analyzed using luminance images. Sonographically determined tumor localization and extent were compared with magnetic resonance imaging and histology. Results:The median tumor size in the 20 rats determined on US (2.3 mm) and magnetic resonance imaging (2.4 mm) showed a very high correlation (r = 0.92, P = 0.01). Both agent A and agent B demonstrated higher vascularization of tumor periphery compared with normal prostate (P < 0.05) based on contrast inflow and areas under the curve analysis. Twenty minutes after administration, luminance for agent B in the tumor was significantly higher (P = 0.003) compared with nonspecific agent A (11.8–0.1). In consensus reading, the increase in signal intensity of the tumor compared with normal prostate tissue was significantly higher for agent B (P = 0.005), whereas no significant difference was found for agent A (P = 0.180). Conclusions:The target-specific contrast agent was superior to the unspecific US contrast agent both with regard to early inflow analysis and specific accumulation in prostate cancer after 20 minutes.

Vascular dynamics in relation to immunolocalisation of VEGF-A, VEGFR-2 and Ang-2 in the bovine corpus luteum

Vascular dynamics during development and regression of the bovine corpus luteum were investigated morphometrically in relation to immunolocalisation of vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor receptor 2 (VEGFR-2) and angiopoietin 2 (Ang-2) in the different cell types. Angiogenesis and remodelling of the capillary network between day 8 and 17 of the oestrous cycle was driven by luteal cells, which were highly immunopositive for VEGF-A. Thereafter, immunoreactivity for VEGF-A and VEGFR-2 was mainly found in vascular smooth muscle cells, the predominant cell type in late luteolysis. During early regression, apoptosis of luteal and endothelial cells was closely correlated, resulting in a significant decrease of capillarity. In late regression, an increase in capillary density was found, suggesting that regression and transformation into the corpus albicans requires adequate perfusion. In the phases of vascular remodelling, i.e. mature and late regressing corpus luteum, high scores of Ang-2-immunopositive endothelial and smooth muscle cells were found. Therefore, it may be hypothesised that Ang-2 supports the angiogenic effects of VEGF-A in these luteal stages. Results emphasise that precise staging and a differentiated view on the cellular populations is important in evaluating the controlled regression and transformation of the corpus luteum.

Active participation instead of passive behaviour opens up new vistas in education of veterinary anatomy and histology.

Teaching morphology, a fundamental part of medicine curricula is traditionally based on lectures and practical trainings. We introduced peer‐assisted learning (PAL) and student expert teams to the courses to give the students the possibility to improve their free speech and self‐confidence. We involved students in active preparation of online materials such as labelled e‐slides and e‐pics. We offered online digital microscopy (ZoomifyTM) and dissection (CyberPrep) allowing repeating the learned material and studying veterinary morphology outside the dissection theatre. Over 60% of first and third semester students profited from being a peer or being taught by a peer and 50% said the expert teams were an excellent method to learn the topographic anatomy. Almost all students applied ZoomifyTM and CyberPrep and 75% of them found the digital microscopy and dissection to be a helpful or very helpful learning tool. In face of reduced contact hours, these forms of education compensated in part the lost teaching time. We observed improvement of rhetoric and presentation skills and self‐confidence. The approaches should therefore find their constant place in the veterinary medicine curricula.

Enhancement of immunohistochemical detection of Salmonella in tissues of experimentally infected pigs

Salmonella Typhimurium is one of the main pathogens compromising porcine and human health as well as food safety, because it is a prevailing source of foodborne infections due to contaminated pork. A prominent problem in the management of this bacteriosis is the number of subclinically infected carrier pigs. As very little is known concerning the mechanisms allowing Salmonella to persist in pigs, the objective of this study was to develop an immunohistochemical approach for the detection of salmonellae in tissue of pigs experimentally infected with Salmonella Typhimurium. Samples were obtained from a challenge trial in which piglets of the German Landrace were intragastrically infected with Salmonella enterica serovar Typhimurium DT104 (1.4-2.1×1010 CFU). Piglets were sacrificed on days 2 and 28 post infection. Tissue samples of jejunum, ileum, colon, ileocecal mesenteric lymph nodes (Lnn. ileocolici), and tonsils (Tonsilla veli palatini) were fixed in Zamboni’s fixative and paraffin-embedded. Different immunohistochemical staining protocols were evaluated. Salmonella was detected in varying amounts in the tissues. Brown iron-containing pigments in the lymph nodes interfered with the identification of Salmonella if DAB was used as a staining reagent. Detergents like Triton X-100 or Saponin enhanced the sensitivity. It seems advisable not to use a detection system with brown staining for bacteria in an experimental setup involving intestinal damage including haemorrhage. The use of detergents appears to result in a higher sensitivity in the immunohistochemical detection of salmonellae.

Structure and age-dependent development of the turkey liver: a comparative study of a highly selected meat-type and a wild-type turkey line

In this study the macroscopic and microscopic structure of the liver of a fast growing, meat-type turkey line (British United turkeys BUT Big 6, n=25) and a wild-type turkey line (Wild Canadian turkey, n=48) were compared at the age of 4, 8, 12, 16, and 20 wk. Because the growth plates of long bones were still detectable in the 20-week-old wild-type turkeys, indicating immaturity, a group of 8 wild-type turkeys at the age of 24 wk was included in the original scope of the study. Over the term of the study, the body and liver weights of birds from the meat-type turkey line increased at a faster rate than those of the wild-type turkey line. However, the relative liver weight of the meat-type turkeys declined (from 2.7 to 0.9%) to a greater extent than that of the wild-type turkeys (from 2.8 to 1.9%), suggesting a mismatch in development between muscle weights and liver weights of the meat-type turkeys. Signs of high levels of fat storage in the liver were detected in both lines but were greater in the wild-type turkey line, suggesting a better feed conversion by the extreme-genotype birds i.e., meat-type birds. For the first time, this study presents morphologic data on the structure and arrangement of the lymphatic tissue within the healthy turkey liver, describing two different types of lymphatic aggregations within the liver parenchyma, i.e., aggregations with and without fibrous capsules. Despite differences during development, both adult meat-type and adult wild-type turkeys had similar numbers of lymphatic aggregations.

The effect of prone versus supine positioning of Goettingen minipigs on lung density as viewed by computed tomography

Minipigs are frequently used for scientific research as they are easy to handle and the dimensions of their vascular system do not change after 20 months of age. Although surgical interventions under anaesthesia are often performed in the supine position the effects of this positioning on lung functionality in minipigs have not been systematically described. This study aimed to analyse the influence of supine positioning on the macrostructure of the lung and the pulmonary density by the use of computed tomography imaging in pre-adult Göttingen minipigs. Twelve pre-adult female minipigs were used in the study and lung density was investigated in both the prone and the supine positions. The time between the scans in prone and supine positions was less than 5 minutes (296 ± 6 sec). In the prone position lung density did not differ between the dorsal and ventral part of the lung (-641 ± 72 Hounsfield units [HU]). However in the supine position there was a ventrodorsal gradient of decreasing density (ventral part of the lung: -497 ± 106 HU, dorsal part of the lung: -723 ± 51 HU). The changes in lung density were not accompanied by changes in lung volume (829 ± 191 ml). These results suggest an influence of the body position on the ventilation/perfusion (V(A)/Q) matching of the lung which could possibly result in lowered lung oxygenation as well as in an increased heart activity in the supine position. Additionally, due to the steep course of the vena cava caudalis from the caval foramen in the diaphragm across to the heart (in contrast to the more shallow course in the prone position) the activity of the heart necessary to pump the venous blood to the right atrium has to be higher in the supine position than in the prone position. In pigs the capacity of the heart to increase frequency is limited due to a diastolic/systolic (D/S) ratio <1. Supine positioning may possibly increase their risk of cardiovascular complications.

Human microvascular endothelial cells displaying reduced angiogenesis and increased uptake of lipids during in vitro culture

Human microvascular ECs from the neonatal foreskin of two donors purchased from one distributor were used in an angiogenesis assay under the same culture conditions. Different angiogenic potency was apparent in these two batches (ECang and ECnon-ang). During the cultivation period of three weeks, ECang ran through all stages of angiogenesis starting from proliferation to migration up to the formation of three-dimensional capillary-like structures. Despite of expression of endothelial markers, ECnon-ang showed excessive intracellular storage of lipids in form of multilamellar bodies and decreased angiogenic potency in contrast to its counterpart, ECang. Results indicate that lipid metabolism differs in ECang versus ECnon-ang. This study points up that these differences are based on the different donors and presents a novel and valuable model for the study of mechanisms of atherosclerosis in endothelial cells in vitro.

Gross morphology and histology of the alimentary tract of the convict cichlid Amatitlania nigrofasciata.

The primary objectives of this study were to document the macroscopic and histological structure of the alimentary tract (AT) of the convict cichlid Amatitlania nigrofasciata, because there are no data available for this omnivorous freshwater fish of the family Cichlidae. The morphology of the AT of A. nigrofasciata resembles that of related species. While having morphological criteria of the AT typical of most omnivorous fishes, such as a blind sac stomach and medium length intestine, A. nigrofasciata also has some structural peculiarities: the oesophagus is lined by a uniform stratified squamous epithelial layer with interspersed goblet cells along its entire length. Additionally, it has well-developed layers of the tunica muscularis including muscle fibre bundles that ascend into its mucosal folds. Occasionally, taste buds are present. In the transitional area between oesophagus and stomach, a prominent torus-like closure device is present. The mucosa of the stomach cannot be divided into different regions according to mucosal and morphological properties. The simple pattern of intestinal loops of A. nigrofasciata has few variations, irrespective of sex, mass and length of the individual fish. The first segment of the intestine is characterized by the largest mucososerosal ratio and the most complex mucosal surface architecture. A distinction of midgut and hindgut was not possible in A. nigrofasciata due to lack of defining structural components as described for other fish species.

Comparative Quantitative Studies on the Microvasculature of the Heart of a Highly Selected Meat-Type and a Wild-Type Turkey Line

In this study the macroscopic and microscopic structure of the heart of a fast growing, meat-type turkey line (British United turkeys BUT Big 6) and a wild-type turkey line (Canadian Wild turkey) were compared. At 8 and 16 weeks of age, 10 birds of each genotype and sex were sampled. The body mass and heart mass of the meat-type turkey both increased at a faster rate than those of the wild-type turkey. However in both turkey lines, the relative heart mass decreased slightly with age, the decrease was statistically significant only in the male turkeys. Furthermore meat-type turkeys had a significantly (p < 0.01) lower relative heart mass and relative thickness of the left ventricle compared to the wild-type turkeys of the same age. The wild-type turkeys showed no significant change in the size of cardiomyocytes (cross sectional area and diameter) from 8 weeks to 16 weeks. In contrast, the size of cardiomyocytes increased significantly (p < 0.001) with age in the meat-type turkeys. The number of capillaries in the left ventricular wall increased significantly (p < 0.001) in wild-type turkeys from 2351 per mm2 at the age of 8 weeks to 2843 per mm2 at 16 weeks. However, in the meat-type turkeys there were no significant changes, capillary numbers being 2989 per mm2 at age 8 weeks and 2915 per mm2 at age 16 weeks. Correspondingly the area occupied by capillaries in the myocardium increased in wild-type turkeys from 8.59% at the age of 8 weeks to 9.15% at 16 weeks, whereas in meat-type turkeys this area decreased from 10.4% at 8 weeks to 9.95% at 16 weeks. Our results indicate a mismatch in development between body mass and heart mass and a compromised cardiac capillary density and architecture in the meat-type turkeys in comparison to the wild-type turkeys.

Influence of iodine-containing radiographic contrast media on the phenotype of erythrocytes from different laboratory animal species

It is well known that clinically relevant concentrations of iodine-containing radiographic contrast media (CM) induce morphological changes in human erythrocytes. However, there are only few reports about CM effects on erythrocytes of animals (e.g. mice, rats, rabbits, and pigs). Thus, two conventional iodine-containing CM (iodixanol, Visipaque™ 320; iomeprol, Iomeprol™ 350) were tested for their effects on the morphology of erythrocytes from these. After venous blood sampling and blood centrifugation, the autologous plasma was supplemented with 40 vol% CM. Then, a defined number of erythrocytes was incubated in this CM-supplemented plasma for 5 min at body temperature (37°C). Subsequently, 10 μL of the cell suspension were transferred to a purified glass slide and the number of discocytes, echinocytes, and acanthocytes was counted within a total number of 100 erythrocytes (40 fold primary magnification, transmitted light mode). Shape changes of the erythrocytes from all animal species strongly depended on the type of CM and compared to the effects which have already been described for human erythrocytes. Incubation in both CM resulted in morphological changes of the erythrocytes. Incubation in a iodixanol/plasma mixture induced the lowest echinocyte or acanthocyte formation. Porcine erythrocytes showed a much more distinct shape change than those of other animal species and humans. These results suggest erythrocytes from mice, rats, and rabbits are a suitable model system for a model system for human erythrocytes when CM effects on the cellular shape of erythrocytes have to be tested. The distinct deformation of the pig erythrocytes could be due to differences in the pig erythrocyte membrane or the physical and chemical constitution of pig erythrocytes.