Despite numerous studies and literature reviews, the social housing of laboratory mice is still under debate. Aggression and social stress can lead to physiological and behavioral alterations, especially in male mice, whereas single housing is a potential source of discomfort, particularly in female mice. This multicenter study aimed to assess the impact of single versus group housing on severity in both sexes and to examine whether a general recommendation could be made. C57BL/6NRj mice from the same litters of a commercial breeder were housed either singly or in groups of four across four different laboratory sites. Here we assessed fecal corticosterone metabolite concentration, aggressive behavior, wound scores and observed behavioral readouts on locomotion, stress coping in the forced swim test, burrowing, nesting and anxiety. Differences due to social housing conditions were not very pronounced. Instead, we found that, despite harmonization efforts, the differences in outcomes across the four laboratory sites were substantial. Furthermore, the individuality of the mice appears to have an additional influence on the results. These findings suggest that optimal housing is not generalizable but depends on the specific laboratory conditions. Beyond meeting the minimum housing standards as set by legislation, we should actively embrace refinement as a guiding principle and strive to create housing conditions that not only meet scientific needs but also continuously improve the welfare of the animals entrusted to our care. This study compares single and group housing in male and female laboratory mice across four research sites, finding that site-specific conditions and individual variability have a greater impact than housing type.
Complex diseases such as progressive cardiomyopathies are often insufficiently recapitulated in small animals or in vitro models. Large animal species such as pigs provide a valuable alternative, but constitutive genetic manipulation has not yet been applied to pigs in an effective manner, mainly due to biological and logistical limitations. Here we describe the generation of a humanized pig model for phospholamban-mediated cardiomyopathy and compare different methods for activating a pathogenic R14del mutation by Cre-mediated recombination. Both Cre treatment of pig primary cells before somatic cell nuclear transfer as well as microinjection of Cre-encoding mRNA into zygotes were similarly efficient in delivering piglets with an activated R14del mutation. Alternatively, administration of Cre-encoding adeno-associated virus into piglets was sufficient, albeit to a varying extent. Together, we describe a highly effective process to establish complex inducible genetic traits in pig and demonstrate that the lack of Cre-driver lines can be compensated by various interventions during reproduction or postnatally. The study describes the generation of a humanized pig model carrying an inducible phospholamban R14del mutation for cardiomyopathy modeling and demonstrates Cre-based activation strategies as efficient alternatives to germline Cre-driver lines in large animals.
Understanding an animal's capacity to experience negative sensations, such as pain and distress, during impaired states of consciousness is critical in many contexts, particularly when animals are being euthanized. Mice remain the predominant species used for scientific research globally, where a prerequisite requires them to be humanely killed at the end of the scientific work. However, whether some of the existing practices and methodologies (for example, exposure to a rising concentration of carbon dioxide) provide these animals with a humane death has been questioned, thus requiring the development of novel, refined methodologies. Here we highlight the potential of hypobaric hypoxia to provide a humane alternative to hypercapnic hypoxia by combining behavioral and neurophysiological assessment (using electroencephalography) to characterize brain activity during the induction of unconsciousness. We found different patterns of brain activity between hypobaric and hypercapnic hypoxia, suggestive of key mechanistic differences in the transition toward unconsciousness. Unlike hypercapnic hypoxia, hypobaric hypoxia resulted in an early shift in peak power to lower delta frequency bands (<4 Hz), suggestive of early cognitive impairment and an anesthetic-like state before loss of posture. As such, mice are likely to have a reduced capacity to have negatively valenced sensory-affective experiences. Hypobaric hypoxia could provide the basis for major refinement to essential killing practices for laboratory mice worldwide.
Post-traumatic osteoarthritis (PTOA) frequently arises following knee injury, such as anterior cruciate ligament (ACL) rupture. Small animal models are critical for studying PTOA pathology and translating therapeutic interventions. Here our objective was to identify key structural PTOA pathologies and evaluate their associations with aberrant pain and function after a clinically relevant ACL injury in a small animal model. This work leveraged a suite of quantitative cartilage and bone analysis techniques using high-resolution contrast-enhanced microcomputed tomography to investigate the associations between pain-related behaviors and structural osteoarthritis pathogenesis throughout the knee joint in rats. We hypothesized that ACL rupture would increase pain sensitization and limb dysfunction and that this would correlate with the development of clinically relevant osteophytes and cartilage lesions at moderate (4 weeks) and severe disease stages (8 weeks). We showed that ACL rupture induced hyperalgesia and prolonged hindlimb weight-bearing dysfunction; these pain-related behaviors were strongly correlated with changes in the tibiofemoral subchondral bone plate, but not with osteophyte formation. Moreover, we show that patella bone pathology correlates strongly with pain sensitivity and limb function. Joint degeneration initially manifested in the posterior aspect of the medial tibiofemoral compartments, as indicated by full-thickness femoral cartilage lesions and tibial osteophytes at 4 weeks and corresponding tibial cartilage hypertrophy and subchondral sclerosis by 8 weeks. The established outcome parameters and association of structural pathogenesis with pain and dysfunction provide a foundation for studying fundamental PTOA disease etiology and testing therapeutic efficacy in a rodent preclinical model.
New approach methodologies (NAMs) can greatly contribute to the replacement and reduction of animal experiments in biomedical science. However, while NAMs have seen considerable investment and adoption in toxicology, this growth has not found a parallel in basic and translational biomedical research. Here we examine the opportunities and the challenges of systematic implementation of NAMs in biomedical research. We demonstrate that NAMs offer considerable scientific value by enabling tailored investigations into biological mechanisms and disease processes, addressing aspects of human biology that may be difficult to capture or extrapolate from animal models, and harnessing cutting-edge technological innovations. We highlight key areas, such as neuroscience, cardiovascular research and oncology, where NAMs have a distinct advantage and the potential to drive major advancements. However, realizing their full potential requires overcoming critical barriers, including technological limitations, gaps in education and training and insufficient funding. To facilitate broader adoption of NAMs, we propose adapting clinical outcome pathways, originally developed to link mechanistic biology to clinical outcomes, as a framework to guide the selection, combination and interpretation of NAMs in biomedical research. This approach could help to address a central limitation of individual NAMs, namely their restricted ability to capture whole-organism complexity, by enabling the meaningful combination of complementary models. Finally, we outline a stepwise strategy to accelerate the adoption of NAMs, emphasizing interdisciplinary collaboration, parallel studies alongside existing animal models and sustained investment in infrastructure and education. This Perspective describes how new approach methodologies (NAMs) can advance biomedical research and reduce animal use, and proposes clinical outcome pathways to guide their selection, integration and interpretation.
