Dysbiosis of the intestinal microbiota is associated with Crohn's disease (CD). Functional evidence for a causal role of bacteria in the development of chronic small intestinal inflammation is lacking. Similar to human pathology, TNF(deltaARE) mice develop a tumour necrosis factor (TNF)-driven CD-like transmural inflammation with predominant ileal involvement.Heterozygous TNF(deltaARE) mice and wildtype (WT) littermates were housed under conventional (CONV), specific pathogen-free (SPF) and germ-free (GF) conditions. Microbial communities were analysed by high-throughput 16S ribosomal RNA gene sequencing. Metaproteomes were measured using LC-MS. Temporal and spatial resolution of disease development was followed after antibiotic treatment and transfer of microbial communities into GF mice. Granulocyte infiltration and Paneth cell function was assessed by immunofluorescence and gene expression analysis.GF-TNF(deltaARE) mice were free of inflammation in the gut and antibiotic treatment of CONV-TNF(deltaARE) mice attenuated ileitis but not colitis, demonstrating that disease severity and location are microbiota-dependent. SPF-TNF(deltaARE) mice developed distinct ileitis-phenotypes associated with gradual loss of antimicrobial defence. 16S analysis and metaproteomics revealed specific compositional and functional alterations of bacterial communities in inflamed mice. Transplantation of disease-associated but not healthy microbiota transmitted CD-like ileitis to GF-TNF(deltaARE) recipients and triggered loss of lysozyme and cryptdin-2 expression. Monoassociation of GF-TNF(deltaARE) mice with the human CD-related Escherichia coli LF82 did not induce ileitis.We provide clear experimental evidence for the causal role of gut bacterial dysbiosis in the development of chronic ileal inflammation with subsequent failure of Paneth cell function.
Severity assessment in animal models is a data-driven process. We therefore present a use case for building a repository for interlaboratory collaboration with the potential of uploading specific content, making group announcements and internal prepublication discussions. We clearly show that it is possible to offer such a structure with minimal effort and a basic understanding of web-based services, also taking into account the human factor in individual data collection. The FOR2591 Online Repository serves as a blueprint for other groups, so that one day not only will data sharing among consortium members be improved but the transition from the private to the persistent domain will also be easier.
BackgroundInfection may trigger clinically overt mucosal inflammation in patients with predisposition for inflammatory bowel disease. However, the impact of particular enteropathogenic microorganisms is ill-defined. In this study, the influence of murine norovirus (MNV) infection on clinical, histopathological, and immunological features of mucosal inflammation in the IL10-deficient (Il10−/−) mouse model of inflammatory bowel disease was examined.
This study presents a novel concept for a smart home cage design, tools, and software used to monitor the physiological parameters of mice and rats in animal-based experiments. The proposed system focuses on monitoring key clinical parameters, including heart rate, respiratory rate, and body temperature, and can also assess activity and circadian rhythm. As the basis of the smart home cage system, an in-depth analysis of the requirements was performed, including camera positioning, imaging system types, resolution, frame rates, external illumination, video acquisition, data storage, and synchronization. Two different camera perspectives were considered, and specific camera models, including two near-infrared and two thermal cameras, were selected to meet the requirements. The developed specifications, hardware models, and software are freely available via GitHub. During the first testing phase, the system demonstrated the potential of extracting vital parameters such as respiratory and heart rate. This technology has the potential to reduce the need for implantable sensors while providing reliable and accurate physiological data, leading to refinement and improvement in laboratory animal care.
Hintergrund: Bei der Pathogenese von chronisch entzündlichen Darmerkrankungen spielt eine überschießende Immunantwort auf die bakterielle Flora eine entscheidende Rolle. In früheren Untersuchungen konnten wir zeigen, dass eine CpG DNA Behandlung vor der Induktion einer experimentellen intestinalen Entzündung durch Induktion regulatorischer T-Zellen schützt. Um zu untersuchen, ob diese bisher in konventionell gehaltenen Tieren beobachtete CpG-DNA induzierte Toleranz spezifisch für Antigene der bakteriellen Flora ist, wurden keimfreie gehaltene Mäuse im SCID Transfer Modell auf einen prophylaktischen Effekt einer CpG Exposition untersucht.
In a recent paper, Nabekura et al. (1) provided compelling evidence that abrogating DNAM-1 (DNAX accessory molecule-1; CD226) activity resulted in milder development of graft-versus-host disease (GVHD). This is expected, because the role of CD226 as an important costimulatory receptor during T-cell activation is well-documented (2, 3). As shown in the paper (1), CD226 present on donor T cells contributed to the typical syndromes of GVHD. A milder course of GVHD was also achieved by treating transplanted mice with an antibody neutralizing CD226. The authors assumed that CD226 signaling is initiated by interaction of the allogeneic T cells with host cells expressing either CD112 or CD155, the two known ligands of CD226 (1). We analyzed the development of GVHD in mice deficient for CD155. Unexpectedly, the mice succumbed to GVHD within 1 wk, whereas WT mice survived for approximately 3 wk (Fig. 1A). Additional experiments suggested that the observed aggravation in the course of disease is mainly caused by CD4+ donor T cells (Fig. 1A). Additional analyses aimed at identifying the cause of premature death of CD155−/− recipients failed to reveal any differences to WT controls (Fig. 1B). Serum cytokine levels of IFNγ, IL-6, and TNFα as well as the extent of T-cell proliferation were virtually identical when investigated 3 d posttransplantation. We also subjected the intestine of recipients to a detailed histological examination 6 d posttransplantation, but again, significant differences in the degree of injury or infiltrating T cells between WT and CD155−/− mice were not detected. Of note, CD155−/− mice receiving T cell-depleted bone marrow only developed lethal GVHD after T cells differentiated from donor marrow. This corroborated that CD155−/− mice died of deleterious T-cell effects and not because of constitutional defects imposed by CD155 deficiency (Fig. 1A).
Fig. 1
(A) After lethal irradiation, WT BALB/c or CD155−/− (Pvrtm1Gbn) BALB/c (KO) mice received 5 × 106 C57BL/6 T cell-depleted bone marrow cells (BM) alone (n = 8) or BM supplemented with 1 × 106 T cells (CD4+ and CD8+ T cells ...
Even if the exact cause of the exacerbated course of GVHD in CD155−/− recipients remains elusive, our results suggest that presence of CD155 may attenuate otherwise devastating consequences of GVHD. Because the study by Nabekura et al. (1) clearly showed that CD226-triggered T-cell coactivation contributes to a worsening course of GVHD, CD155 possibly plays a Janus-faced role in GVHD development: its presence exerts protective effects but at the same time, aggravates GVHD by activating donor cells expressing CD226. Alternatively, as already discussed by Nabekura et al. (1), CD112 expressed by host cells in liver and intestine may be largely responsible for the observed CD226 effects. Future studies involving CD112−/− mice will help to clarify this point. Moreover, the GVHD model in use also matters. Whereas Nabekura et al. (1) chose an experimental strategy eliciting GVHD preferentially by CD8+ T cells, our investigations were based on an MHC fully mismatched model where CD4+ T cells mainly contribute to GVHD development (4). Nevertheless, we were unable to prolong survival time of CD155−/− recipients by treating mice with a CD226 neutralizing antibody (Fig. 1A) (5). Despite its importance in CD4+ T-cell stimulation and differentiation (3), CD226 may, thus, not always play a major role in the complex pathophysiology of GVHD.
Abstract Animal welfare and the refinement of experimental procedures are fundamental aspects of biomedical research. They provide the basis for robust experimental designs and reproducibility of results. In many countries, the determination of welfare is a mandatory legal requirement and implies the assessment of the degree of the severity that an animal experiences during an experiment. However, for an effective severity assessment, an objective and exact approach/system/strategy is needed. In light of these demands, we have developed the Relative Severity Assessment (RELSA) score. This comprehensive composite score was established on the basis of physiological and behavioral data from a surgical mouse study. Body weight, the Mouse Grimace Scale score, burrowing behavior, and the telemetry-derived parameters heart rate, heart rate variability, temperature, and general activity were used to investigate the quality of indicating severity during postoperative recovery. The RELSA scores not only revealed individual severity levels but also allowed a comparison of severity in distinct mouse models addressing colitis, sepsis, and restraint stress using a k -means clustering approach with the maximum achieved RELSA scores. We discriminated and classified data from sepsis nonsurvivors into the highest relative severity level. Data from mice after intraperitoneal transmitter implantation and sepsis survivor al were located in the next lower cluster, while data from mice subjected to colitis and restraint stress were placed in the lowest severity cluster. Analysis of individual variables and their combinations revealed model- and time-dependent contributions to severity levels. In conclusion, we propose the RELSA score as a validated tool for objective real-time applicability in severity assessment and as a first step towards a unified and accessible risk assessment tool in biomedical research. As an effective severity assessment system, it will fundamentally improve animal welfare, as well as data quality and reproducibility.
ObjectiveEthical decisions about an allowance for animal experiments need to be based on scientifically sound information about the burden and distress associated with the experimental procedure and models. Thereby, species differences need to be considered for recommendations regarding evidence-based severity assessment and refinement measures.MethodsA comprehensive analysis of behavioral patterns and corticosterone or its metabolites in serum and feces was completed in kindled mice. The impact of kindling via two different stimulation sites in the amygdala and hippocampus was determined. Data were compared to those from naive and electrode-implanted groups.ResultsAmygdala and hippocampus kindled mice exhibited comparable behavioral patterns with increased activity in the open field, reduced anxiety-associated behavior in the elevated-plus maze, and increased anhedonia-associated behavior in the saccharin preference test. In addition, repeated stimulation of the hippocampus caused a reduction in burrowing behavior and an increase in active social interaction. Levels of corticosterone and its metabolites were not altered in serum or feces, respectively. A comparison of mouse data with findings from amygdala kindled rats confirmed pronounced species differences in behavioral patterns associated with the kindling process.SignificanceTaken together the findings suggest a severity classification for the mouse kindling paradigms as moderate regardless of the stimulation site. The outcome of the species comparison provides valuable guidance for species selection for studies exploring behavioral comorbidities. In this context, it is emphasized that the mouse kindling paradigms seem to be well suited for studies exploring the link between ictal events and network alterations on the one hand, and hyperactivity and anhedonia-associated behavior on the other hand. Moreover, the underlying pathophysiological mechanisms and the impact of therapeutic interventions on these behavioral alterations can be studied in these paradigms providing guidance for the clinical management of respective psychiatric comorbidities in patients.
