A novel application tunnel in combination with medical training reduces stress induced by frequent intraperitoneal injections and blood sampling in mice

Abstract Handling-induced stress represents a major burden on laboratory mice and is heavily influenced by the handling technique. As such, tail-handling induces much higher stress than cup- or tunnel-handling, which is further aggravated by interventions, e.g. injections, which require even harsher fixation methods. Previous studies demonstrated that habituation protocols can improve animal welfare during handling and interventions. Here, we developed a medical training program and an alternative fixation method using an application tunnel in the context of a liver cancer model, where frequent intraperitoneal injections over many weeks are needed to induce tumor development. The training regimen consisted of 5 sessions over 2 weeks, which gradually introduced the animals to being touched by handlers and restrained for procedures. The training program was completed once before the start of any interventions, additional training sessions were performed biweekly during the entire course of the experiments. The animals were randomized to receive injections either in the novel application tunnel or using conventional fixation. Training effect was continuously monitored by measuring the latency to interact with the experimenter, the surface body temperature and by movement tracking. The latency to interact rapidly decreased during initial training sessions, and this effect was sustained throughout the course of treatment. Movement tracking demonstrated that mice injected inside the tunnel were more active and returned to their normal behavior after injections faster than conventionally restrained mice. Those mice also showed less signs of defecation and urination. Furthermore, the tunnel had a positive influence on the well-being of mice during blood sampling demonstrated by reduced signs of pain and faster willingness to interact with the handler after the procedure. In conclusion, habituation of the mice to the interventions with medical training and the improved handling procedure during ip injections and blood draws durably reduces stress levels and improves welfare of mice. Competing Interest Statement A.S. and L.H. own a utility model for the injection tunnel in Germany (Registration No. 20 2024 100 448). All other authors declare no conflicts of interest. Footnotes Conflict of interests A.S. and L.H. own a utility model for the injection tunnel in Germany (Registration No. 20 2024 100 448). All other authors declare no conflicts of interest. Data availability This study protocol has been preregistered on animalstudyregistry.org and will be made publicly available upon publication. A free downloadable 3D-printing file for the application tunnel as well as a video demonstrating the injection procedure can be found at this link (view-only for peer review, will be made publicly available upon acceptance/publication of manuscript): https://osf.io/vsjnb/?view_only=ff970c7b95104b0888e26b00f43cc30c. The data that support the findings of this study are not openly available due to reasons of sensitivity and are available from the corresponding author upon reasonable request. Data are located in controlled access data storage at Charité Universitätsmedizin Berlin.