An animal’s capacity to suffer is a prerequisite for any animal welfare concern, and the minimization of suffering is a key aim of refinement research. In contrast to the traditional focus on avoiding or reducing negative welfare states, modern animal welfare concepts highlight the importance of promoting positive welfare states in laboratory animals. Reliable assessments of affective states, as well as the knowledge of how to elicit positive affective states, are central to this concept. Important achievements have been made to assess pain and other negative affective states in animals in the last decades, but it is only recently that the neurobiology of positive emotions in humans and animals has been gaining more interest. Thereby, the need for promotion of positive affective states for laboratory animals is gaining more acceptance, and methods allowing the assessment of affective states in animals have been increasingly introduced. In this overview article, we present common and emerging methods to assess affective states in laboratory rodents. We focus on the implementation of these methods into applied refinement research to identify achieved progress as well as the future potential of these tools to improve animal welfare in animal-based research.

Regeneration of injured tissues requires effective therapeutic strategies supporting vasculogenesis. The lack of instantly available autologous cell sources and immunogenicity of allogeneic endothelial (progenitor) cells limits clinical progress. Based on the immunosuppressive potency of mesenchymal stem/progenitor cells (MSCs), we investigated whether crosstalk between endothelial colony-forming progenitor cells (ECFCs) and MSCs during vasculogenesis could lower allogeneic T cell responses against ECFCs allowing long-term engraftment in vivo. Immunodeficient mice received subcutaneous grafts containing human ECFCs alone, or pairs of human ECFCs/MSCs from the same umbilical cord (UC) to study vasculogenesis in the presence of human leukocyte antigen (HLA)-mismatched human peripheral blood mononuclear cells (PBMCs). In vitro, cell surface marker changes due to interferon gamma (IFNγ) stimulation during ECFC/MSC coculture were determined and further effects on allostimulated T cell proliferation and cytotoxic lysis were measured. IFNγ-induced HLA-DR expression on ECFCs and MSCs, but both cell types had significantly less HLA-DR in cocultures. ECFC-induced T cell proliferation was abolished after MSC coculture as a result of HLA-DR downregulation and indolamin-2,3-dioxygenase activation. Additionally, allospecific CD8 T cell-mediated lysis of ECFCs was reduced in cocultures. ECFC/MSC coapplication in immunodeficient mice not only promoted the generation of improved blood vessel architecture after 6 weeks, but also reduced intragraft immune cell infiltration and endothelial HLA-DR expression following PBMC reconstitution. Crosstalk between UC-derived ECFCs and MSCs after combined transplantation can lower the risk of ECFC rejection, thus enabling their coapplication for therapeutic vasculogenesis. Stem Cells 2017;35:1233-1245.

Supplemental material:Figure S1: Slightly increasing body weight during habituation period Body weight [g] at day one and six of habituation period per strain were presented as box plot with median and whiskers [2.5, 97.5 %] (n = 38 C57BL/6J: p-value = 0.0018, n = 15 BALB/cJ: p-value = 0.0004 and n = 15 129S1/SvImJ: p-value < 0.0001, Wilcoxon matchedpairs signed rank test, two-tailed).

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From the preference of one good over another, the strength of the preference cannot automatically be inferred. While money is the common denominator to assess the value of goods in humans, it appears difficult at first glance to put a price tag on the decisions of laboratory animals. Here we used consumer demand tests to measure how much work female mice expend to obtain access to different liquids. The mice could each choose between two liquids, one of which was free. The amount of work required to access the other liquid, by contrast, increased daily. In this way, the value of the liquid can be determined from a mouse's microeconomic perspective. The unique feature is that our test was carried out in a home-cage based setup. The mice lived in a group but could individually access the test-cage, which was connected to the home-cage via a gate. Thereby the mice were able to perform their task undisturbed by group members and on a self-chosen schedule with minimal influence by the experimenter. Our results show that the maximum number of nosepokes depends on the liquids presented. Mice worked incredibly hard for access to water while a bitter-tasting solution was offered for free whereas they made less nosepokes for sweetened liquids while water was offered for free. The results demonstrate that it is possible to perform automated and home-cage based consumer demand tests in order to ask the mice not only what they like best but also how strong their preference is.

Boredom is an emotional state that occurs when an individual has nothing to do, is not interested in the surrounding, and feels dreary and in a monotony. While this condition is usually defined for humans, it may very well describe the lives of many laboratory animals housed in small, barren cages. To make the cages less monotonous, environmental enrichment is often proposed. Although housing in a stimulating environment is still used predominantly as a luxury good and for treatment in preclinical research, enrichment is increasingly recognized to improve animal welfare. To gain insight into how stimulating environments influence the welfare of laboratory rodents, we conducted a systematic review of studies that analyzed the effect of enriched environment on behavioral parameters of animal well–being. Remarkably, a considerable number of these parameters can be associated with symptoms of boredom. Our findings show that a stimulating living environment is essential for the development of natural behavior and animal welfare of laboratory rats and mice alike, regardless of age and sex. Conversely, confinement and under-stimulation has potentially detrimental effects on the mental and physical health of laboratory rodents. We show that boredom in experimental animals is measurable and does not have to be accepted as inevitable.

The cytochrome P450 (CYP) enzyme superfamily is the most important enzyme system for phase I biotransformation. For toxico- and pharmacokinetic studies, use of liver-based microsomes, including those of mice, is state-of-the-art to study CYP-dependent metabolism. However, reproducibility and interpretation of these data is still very variable, partly because current testing guidelines do not cover details on organ sampling and potential liver lobe differences. Hence, we analyzed CYP activity, CYP protein content, mRNA expression of CYP1A, CYP2C, CYP2D and CYP3A isozymes, and cytochrome P450 reductase (CPR) activity of the four different liver lobes and processus papillaris of male C57BL/6J mice in comparison to whole liver. Additionally, we used whole liver of Balb/cJ and 129S1/SvImJ for strain comparison. Our data show significant differences in CYP activity, being most prominent in lobus sinister lateralis and lobus medialis, and lowest in processus papillaris. These differences were not caused by varying Cyp gene expression or CYP protein level, but partly correspond with lobe specific CPR activities. We also observed significant strain differences in CYP mRNA expression and activities with overall high activities in 129S1/SvImJ mice and low activities in Balb/cJ mice compared to C57BL/6J mice. In addition, strain specific differences in CYP2C and CYP2D activity seem to be reflected in strain dependent differences in CPR activity. In summary, our results indicate that in mice CYP activity and gene expression are strain dependent and may vary highly between liver lobes. To ensure reproducibility and comparability of different probes and studies, this should be taken into account when liver samples are collected for the analysis of CYP-dependent metabolism.

AbstractReliability of data has become a major concern in the course of the reproducibility crisis. Especially when studying animal behavior, confounding factors such as novel test apparatus can lead to a wide variability of data. At worst, effects of novelty or stress related behavior can mask treatment effects and the behavioral data may be misinterpreted. Habituation to the test situation is a common practice to circumvent novelty induced increases in variance and to improve the reliability of the respective measurements. However, there is a lack of published empirical knowledge regarding reasonable habituation procedures and a method validation seems to be overdue.This study aimed at setting up a simple strategy to increase reliability of behavioral data. Therefore, exemplary data from mice tested in an Open Field (OF) arena were used to elucidate the potential of habituation and how reliability of measures can be confirmed by means of a repeatability analysis using the software R. On seven consecutive days, male C57BL/6J, BALB/cJ and 129S1/SvImJ mice were tested in an OF arena once daily and individual mouse behavior (distance travelled, average activity) was recorded. A repeatability analysis was conducted in order to estimate the reliability of measured animal behavior with regard to repeated trials of habituation.Our data analysis revealed that monitoring animal behavior during habituation is important to determine when individual differences of the measurements are stable. Here, the mixed effect model framework proved to be a powerful tool for estimating repeatability values. Repeatability values from distance travelled and average activity increased over the habituation period, revealing that around 60 % of the variance of the data can be explained by individual differences between mice. The first day of habituation was statistically significantly different from the following 6 days in terms of distance travelled and average activity. A habituation period of three days appeared to be sufficient in this study. Overall these results emphasize the importance of habituation and in depth analysis of habituation data to define the correct starting point of the experiment for improving the reliability and reproducibility of experimental data.