Preclinical imaging and animal welfare: assessing risks and refinement approaches in rodent models

RWTH Publications (RWTH Aachen)

Abstract

Summary of the Dissertation: “Preclinical Imaging and Animal Welfare: Assessing Risks and Refinement Approaches in Rodent Models”Ultrasound (US) imaging and positron emission tomography/computed tomography (PET/CT) are extensively applied in preclinical research to non-invasively evaluate anatomical, physiological, and pathological processes in laboratory animals with high spatial and temporal resolution. US, including contrast-enhanced ultrasound (CEUS), allows dynamic visualization of tissues and vascular structures, while PET/CT imaging with radiolabeled tracers, such as [¹⁸F]fluspidine, provides molecular-level insights into biological functions, including pain perception. These imaging modalities enable the acquisition of detailed experimental data and offer opportunities to explore their potential for refining animal experiments, improving welfare, and enhancing the reliability of preclinical models. The first study systematically evaluated the effects of US and CEUS on the welfare of both healthy and tumor-bearing mice with and without regorafenib treatment. In healthy mice, repeated US and CEUS examinations over four weeks did not alter severity parameters, although isoflurane anaesthesia alone slightly reduced spleen size. In tumor-bearing mice, CEUS and molecular CEUS under isoflurane anaesthesia did not affect general welfare parameters but resulted in reduced leukocyte counts and spleen weights, suggesting potential immunomodulatory effects. Notably, these changes were not observed in mice receiving regorafenib. The findings highlight the importance of monitoring potential systemic effects of imaging procedures, particularly when combined with necessary anaesthesia or therapeutic interventions, to refine preclinical protocols and ensure reliable interpretation of experimental outcomes.The second study introduced a novel approach of PET/CT imaging to assess postoperative pain (partial liver resection) in female rats under analgesia and compare the results with established behavioral tests. This approach addressed potential limitations of conventional behavioral tests in prey animals. Pain-associated molecular changes were detected via sigma-1 receptor (σ1R) expression using the PET/CT tracer [¹⁸F]fluspidine. Despite analgesic treatment, elevated σ1R expression was observed at the incision site on the first postoperative day, particularly in liver-resected animals, likely due to the additional peritoneal opening, indicating underlying pain-related molecular activity. σ1R expression returned to baseline by day 4, while behavioral indicators of pain remained minimal. Transient mechanical hypersensitivity detected on day 4, including in control animals, was likely attributable to analgesic side effects. By enabling the localization of pain-associated receptor upregulation without discontinuing analgesia, [¹⁸F]fluspidine PET/CT provides a sensitive and objective tool to potentially complement behavioral testing, refine postoperative pain monitoring, and improve severity grading in preclinical models. It also offers insight into the temporal and spatial patterns of σ1R expression, which may help re-evaluate existing models, optimize analgesia protocols, and enhance mechanistic understanding of pain.In conclusion, these studies illustrate how preclinical imaging can contribute to methodological refinement of preclinical studies. Assessing side effects of US and CEUS enables safer, more reliable imaging protocols, while innovative PET/CT approaches offer sensitive tools to monitor physiological and pathological processes, such as pain, that may be missed by behavioral tests alone. By combining careful evaluation of procedural impacts with the development of advanced imaging techniques, these approaches advance preclinical research, improve animal welfare, and enhance the translational relevance and ethical standards of biomedical studies.

Authors 1

1. Renée Michèle Girbig corresponding Aachen

   RWTH Aachen University

   Affiliation as printed

   RWTH Aachen