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The EU Directive 2010/63/EU changed the requirements regarding the use of laboratory animals and raised important issues related to assessing the severity of all procedures undertaken on laboratory animals. However, quantifiable parameters to assess severity are rare, and improved assessment strategies need to be developed. Hence, a Sheep Grimace Scale (SGS) was herein established by observing and interpreting sheep facial expressions as a consequence of pain and distress following unilateral tibia osteotomy. The animals were clinically investigated and scored five days before surgery and at 1, 3, 7, 10, 14 and 17 days afterwards. Additionally, cortisol levels in the saliva of the sheep were determined at the respective time points. For the SGS, video recording was performed, and pictures of the sheep were randomized and scored by blinded observers. Osteotomy in sheep resulted in an increased clinical severity score from days 1 to 17 post-surgery and elevated salivary cortisol levels one day post-surgery. An analysis of facial expressions revealed a significantly increased SGS on the day of surgery until day 3 post-surgery; this elevated level was sustained until day 17. Clinical severity and SGS scores correlated positively with a Pearson´s correlation coefficient of 0.47. Further investigations regarding the applicability of the SGS revealed a high inter-observer reliability with an intraclass correlation coefficient of 0.92 and an accuracy of 68.2%. In conclusion, the SGS represents a valuable approach for severity assessment that may help support and refine a widely used welfare assessment for sheep during experimental procedures, thereby meeting legislation requirements and minimizing the occurrence of unrecognized distress in animal experimentation. [ABSTRACT FROM AUTHOR]

The phenotype of an organism results from its genotype and the influence of the environment throughout development. Even when using animals of the same genotype, independent studies may test animals of different phenotypes, resulting in poor replicability due to genotype-by-environment interactions. Thus, genetically defined strains of mice may respond differently to experimental treatments depending on their rearing environment. However, the extent of such phenotypic plasticity and its implications for the replicability of research findings have remained unknown. Here, we examined the extent to which common environmental differences between animal facilities modulate the phenotype of genetically homogeneous (inbred) mice. We conducted a comprehensive multicentre study, whereby inbred C57BL/6J mice from a single breeding cohort were allocated to and reared in 5 different animal facilities throughout early life and adolescence, before being transported to a single test laboratory. We found persistent effects of the rearing facility on the composition and heterogeneity of the gut microbial community. These effects were paralleled by persistent differences in body weight and in the behavioural phenotype of the mice. Furthermore, we show that environmental variation among animal facilities is strong enough to influence epigenetic patterns in neurons at the level of chromatin organisation. We detected changes in chromatin organisation in the regulatory regions of genes involved in nucleosome assembly, neuronal differentiation, synaptic plasticity, and regulation of behaviour. Our findings demonstrate that common environmental differences between animal facilities may produce facility-specific phenotypes, from the molecular to the behavioural level. Furthermore, they highlight an important limitation of inferences from single-laboratory studies and thus argue that study designs should take environmental background into account to increase the robustness and replicability of findings., Competing Interests: The authors have declared that no competing interests exist.

The liver is known to possess extensive regenerative capabilities, the processes and pathways of which are not fully understood. A necessary step towards a better understanding involves the analysis of regeneration on the microscopic level in the in vivo environment. We developed an evaluation method combining longitudinal imaging analysis in vivo with simultaneous manipulation on single cell level. An abdominal imaging window was implanted in vivo in Balb/C mice for recurrent imaging after implantation. Intravenous injection of Fluorescein Isothiocyanate (FITC)-Dextran was used for labelling of vessels and Rhodamine 6G for hepatocytes. Minimal cell injury was induced via ablation with a femtosecond laser system during simultaneous visualisation of targeted cells using multiphoton microscopy. High-resolution imaging in vivo on single cell level including re-localisation of ablated regions in follow-up measurements after 2-7 days was feasible. Targeted single cell manipulation using femtosecond laser pulses at peak intensities of 3-6.6 μJ led to enhancement of FITC-Dextran in the surrounding tissue. These reactions reached their maxima 5-15 minutes after ablation and were no longer detectable after 24 hours. The procedures were well tolerated by all animals. Multiphoton microscopy in vivo, combined with a femtosecond laser system for single cell manipulation provides a refined procedure for longitudinal evaluation of liver micro-regeneration in the same region of interest. Immediate reactions after cell ablation and tissue regeneration can be analysed., Competing Interests: The authors have declared that no competing interests exist.

The fine-scale grading of the severity experienced by animals used in research constitutes a key element of the 3Rs (replace, reduce, and refine) principles and a legal requirement in the European Union Directive 2010/63/EU. Particularly, the exact assessment of all signs of pain, suffering, and distress experienced by laboratory animals represents a prerequisite to develop refinement strategies. However, minimal and noninvasive methods for an evidence-based severity assessment are scarce. Therefore, we investigated whether voluntary wheel running (VWR) provides an observer-independent behaviour-centred approach to grade severity experienced by C57BL/6J mice undergoing various treatments. In a mouse model of chemically induced acute colitis, VWR behaviour was directly related to colitis severity, whereas clinical scoring did not sensitively reflect severity but rather indicated marginal signs of compromised welfare. Unsupervised k-means algorithm-based cluster analysis of body weight and VWR data enabled the discrimination of cluster borders and distinct levels of severity. The validity of the cluster analysis was affirmed in a mouse model of acute restraint stress. This method was also applicable to uncover and grade the impact of serial blood sampling on the animal's welfare, underlined by increased histological scores in the colitis model. To reflect the entirety of severity in a multidimensional model, the presented approach may have to be calibrated and validated in other animal models requiring the integration of further parameters. In this experimental set up, however, the automated assessment of an emotional/motivational driven behaviour and subsequent integration of the data into a mathematical model enabled unbiased individual severity grading in laboratory mice, thereby providing an essential contribution to the 3Rs principles., Competing Interests: The authors have declared that no competing interests exist.

The role of the spleen in the induction of an immune response to orally administered antigens is still under discussion. Although it is well known that after oral antigen administration specific germinal centres are not only formed in the Peyers patches (PP) and the mesenteric lymph nodes (mLN) but also in the spleen, there is still a lack of functional data showing a direct involvement of splenic B cells in an IgA immune response in the gut. In addition, after removal of mLN a high level of IgA+ B cells was observed in the gut. Therefore, in this study we analysed the role of the spleen in the induction of IgA+ B cells in the gut after mice were orally challenged with antigens. Here we have shown that antigen specific splenic IgM+ B cells after in vitro antigen stimulation as well as oral immunisation of donor mice were able to migrate into the gut of recipient mice, where they predominantly switch to IgA+ plasma cells. Furthermore, stimulation of recipient mice by orally administered antigens enhanced the migration of the splenic B cells into the gut as well as their switch to IgA+ plasma cells. Removal of the mLN led to a higher activation level of the splenic B cells. Altogether, our results imply that splenic IgM+ B cells migrate in the intestinal lamina propria, where they differentiate into IgA+ plasma cells and subsequently proliferate. In conclusion, we demonstrated that the spleen plays a major role in the gut immune response serving as a reservoir of immune cells that migrate to the site of antigen entrance., Competing Interests: The authors have declared that no competing interests exist.

Severity assessment in laboratory animals is an important issue regarding the implementation of the 3R concept into biomedical research and pivotal in current EU regulations. In mouse models of inflammatory bowel disease severity assessment is usually undertaken by clinical scoring, especially by monitoring reduction of body weight. This requires daily observance and handling of each mouse, which is time consuming, stressful for the animal and necessitates an experienced observer. The time to integrate to nest test (TINT) is an easily applicable test detecting disturbed welfare by measuring the time interval mice need to integrate nesting material to an existing nest. Here, TINT was utilized to assess severity in a mouse DSS-colitis model. TINT results depended on the group size of mice maintained per cage with most consistent time intervals measured when co-housing 4 to 5 mice. Colitis was induced with 1% or 1.5% DSS in group-housed WT and Cd14-deficient mice. Higher clinical scores and loss of body weight were detected in 1.5% compared to 1% DSS treated mice. TINT time intervals showed no dose dependent differences. However, increased clinical scores, body weight reductions, and increased TINT time intervals were detected in Cd14-/- compared to WT mice revealing mouse strain related differences. Therefore, TINT is an easily applicable method for severity assessment in a mouse colitis model detecting CD14 related differences, but not dose dependent differences. As TINT revealed most consistent results in group-housed mice, we recommend utilization as an additional method substituting clinical monitoring of the individual mouse.

P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid rafts to gain its full functionality.