Knockdown of TPI in human dermal microvascular endothelial cells and its impact on angiogenesis in vitro.

INTRODUCTION: Angiogenic behaviour has been shown as highly versatile among Endothelial cells (ECs) causing problems of in vitro assays of angiogenesis considering their reproducibility. It is indispensable to investigate influencing factors of the angiogenic potency of ECs. OBJECTIVE: The present study aimed to analyse the impact of knocking down triosephosphate isomerase (TPI) on in vitro angiogenesis and simultaneously on vimentin (VIM) and adenosylmethionine synthetase isoform type 2 (MAT2A) expression. Furthermore, native expression profiles of TPI, VIM and MAT2A in the course of angiogenesis in vitro were examined. METHODS: Two batches of human dermal microvascular ECs were cultivated over 50 days and stimulated to undergo angiogenesis. A shRNA-mediated knockdown of TPI was performed. During cultivation, time-dependant morphological changes were detected and applied for EC-staging as prerequisite for quantifying in vitro angiogenesis. Additionally, mRNA and protein levels of all proteins were monitored. RESULTS: Opposed to native cells, knockdown cells were not able to enter late stages of angiogenesis and primarily displayed a downregulation of VIM and an uprise in MAT2A expression. Native cells increased their TPI expression and decreased their VIM expression during the course of angiogenesis in vitro. For MAT2A, highest expression was observed to be in the beginning and at the end of angiogenesis. CONCLUSION: Knocking down TPI provoked expressional changes in VIM and MAT2A and a deceleration of in vitro angiogenesis, indicating that TPI represents an angiogenic protein. Native expression profiles lead to the assumption of VIM being predominantly relevant in beginning stages, MAT2A in beginning and late stages and TPI during the whole course of angiogenesis in vitro.

Expression of vimentin, TPI and MAT2A in human dermal microvascular endothelial cells during angiogenesis in vitro.

INTRODUCTION: In vitro assays of angiogenesis face immense problems considering their reproducibility based on the inhomogeneous characters of endothelial cells (ECs). It is necessary to detect influencing factors, which affect the angiogenic potency of ECs. OBJECTIVE: This study aimed to analyse expression profiles of vimentin (VIM), triosephosphate isomerase (TPI) and adenosylmethionine synthetase isoform type-2 (MAT2A) during the whole angiogenic cascade in vitro. Furthermore, the impact of knocking down vimentin (VIM) on angiogenesis in vitro was evaluated, while monitoring TPI and MAT2A expression. METHODS: A long-term cultivation and angiogenic stimulation of human dermal microvascular ECs was performed. Cells were characterized via VEGFR-1 and VEGFR-2 expression and a shRNA-mediated knockdown of VIM was performed. The process of angiogenesis in vitro was quantified via morphological staging and mRNA-and protein-levels of all proteins were analysed. RESULTS: While native cells ran through the angiogenic cascade chronologically, knockdown cells only entered beginning stages of angiogenesis and died eventually. Cell cultures showing a higher VEGFR-1 expression survived exclusively and displayed an upregulation of MAT2A and TPI expression. Native cells highly expressed VIM in early stages, MAT2A mainly in the beginning and TPI during the course of angiogenesis in vitro. CONCLUSION: VIM knockdown led to a deceleration of angiogenesis in vitro and knockdown cells displayed expressional changes in TPI and MAT2A. Cell populations with a higher number of stalk cells emerged as being more stable against manipulations and native expression profiles provided an indication of VIM and MAT2A being relevant predominantly in beginning stages and TPI during the whole angiogenic cascade in vitro.

Task force veterinary anatomy: joint efforts of the five German veterinary schools to ensure education during the COVID-19 pandemic.

At the start of the COVID-pandemic in March 2020, the Institutes of Veterinary Anatomy of the five German educational institutions were confronted with the challenge of digitalising all lectures for the second and fourth semesters of veterinary students. After an online kick-off event and a preliminary status quo meeting, available digital teaching material was exchanged for students to stream from learning platforms. Lectures were either synchronized or made available as audio recordings and connotated slides on the learning platforms. Fortunately, digital microscopic slides had already been in use, which made it easy for students to access them. Dissection exercises mostly consisted of self-study, using instructive videos and interactive exercises. In the second half of the semester, four of the educational institutions were able to offer a restricted number of in-person gross anatomy classes under reinforced conditions. Success monitoring took place online through different formats, and partially on a voluntary basis, via the learning platforms. Although the past two semesters had to almost exclusively take place online due to the unprecedented circumstances, and joint efforts of the five veterinary institutions, there is a general consensus that the practical education in anatomy, histology and embryology is essential to veterinary students. In fact, it is the only way they can obtain the necessary skills to successfully complete the rest of their degree.

Reliability of the Mouse Grimace Scale in C57BL/6JRj Mice.

To maintain and foster the welfare of laboratory mice, tools that reliably measure the current state of the animals are applied in clinical assessment. One of these is the Mouse Grimace Scale (MGS), a coding system for facial expression analysis. Since there are concerns about the objectivity of the MGS, we further investigated its reliability. Four observers (two experienced and two inexperienced in use of the MGS) scored 188 images of 33 female and 31 male C57BL/6JRj mice. Images were generated prior to, 150 min, and two days after ketamine/xylazine anesthesia. The intraclass correlations coefficient (ICC = 0.851) indicated good agreement on total MGS scores between all observers when all three time points were included in the analysis. However, interrater reliability was higher in the early post-anesthetic period (ICC = 0.799) than at baseline (ICC = 0.556) and on day 2 after anesthesia (ICC = 0.329). The best agreement was achieved for orbital tightening, and the poorest agreement for nose and cheek bulge, depending on the observers' experience levels. In general, experienced observers produced scores of higher consistency when compared to inexperienced. Against this background, we critically discuss factors that potentially influence the reliability of MGS scoring.

Cephalometric studies of the mandible, its masticatory muscles and vasculature of growing Göttingen Minipigs-A comparative anatomical study to refine experimental mandibular surgery.

Over many decades, the Göttingen Minipig has been used as a large animal model in experimental surgical research of the mandible. Recently several authors have raised concerns over the use of the Göttingen Minipig in this research area, observing problems with post-operative wound healing and loosening implants. To reduce these complications during and after surgery and to improve animal welfare in mandibular surgery research, the present study elucidated how comparable the mandible of minipigs is to that of humans and whether these complications could be caused by specific anatomical characteristics of the minipigs' mandible, its masticatory muscles and associated vasculature. Twenty-two mandibular cephalometric parameters were measured on CT scans of Göttingen Minipigs aged between 12 and 21 months. Ultimately, we compared this data with human data reported in the scientific literature. In addition, image segmentation was used to determine the masticatory muscle morphology and the configuration of the mandibular blood vessels. Compared to data of humans, significant differences in the mandibular anatomy of minipigs were found. Of the 22 parameters measured only four were found to be highly comparable, whilst the others were not. The 3D examinations of the minipigs vasculature showed a very prominent deep facial vein directly medial to the mandibular ramus and potentially interfering with the sectional plane of mandibular distraction osteogenesis. Damage to this vessel could result in inaccessible bleeding. The findings of this study suggest that Göttingen Minipigs are not ideal animal models for experimental mandibular surgery research. Nevertheless if these minipigs are used the authors recommend that radiographic techniques, such as computed tomography, be used in the specific planning procedures for the mandibular surgical experiments. In addition, it is advisable to choose suitable age groups and customize implants based on the mandibular dimensions reported in this study.

Recovery of strongylid third-stage larvae from herbage samples: standardisation of a laboratory method and its application in the field.

The estimation of parasitic nematode larvae present on pasture is an important tool applied in many epidemiological studies. In the face of climatic changes, there is increased interest in identifying parameters influencing the survival of free-living stages of parasites under different meteorological conditions. In order to predict possible risk factors for grazing livestock, reliable and reproducible methods to assess the density of larvae on pasture are required. A laboratory method for the recovery of strongylid third-stage larvae from herbage samples was developed, standardised and its efficacy assessed in controlled experiments as well as under field conditions. Grass samples free of any nematode larvae were used and inoculated with known numbers of third-stage larvae of Cooperia oncophora in different concentrations. The grass samples were inoculated with larvae over 24 h, followed by soaking for 4 h. The recovery process included washing over sieves and centrifugation of the obtained liquid. The total time required for the recovery process was about 5-7 h (excluding inoculation). Recovery rates range from 68% to 98% and a strong correlation between numbers of larvae added to the grass samples and numbers of larvae that could be recovered was observed (p < 0.001). The new method proved to be reproducible and provides high recovery rates combined with the potential to handle many samples simultaneously in a relatively short time, thus offering high throughput opportunities applicable to field experiments.