Imaging of tissue damage and pain to assess animal burden

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Fabian Kießling

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+49 241 80 80117

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Project in the framework of the DFG Research Unit FOR 2591 Severity Assesment

Animal-based research has to adhere to the 3R principle (replace, refine, reduce), not only with regard to ethical justification of animal use but also to ensure quality in terms of standardization. Here, the assessment of severity experienced by animals has become a prerequisite for the project authorization in the EU (2010/63/EU) including the grading of pain, suffering, and distress animals experience during scientific procedures. However, scientifically sound and routinely applicable scales to grade severity in laboratory animals are not available or still inadequate, creating a deep gap between current regulations and scientific knowledge.

As part of the research unit FOR 2591 we are involved in a joint project with Prof. Felix Mottaghy. Within the 1st funding period, we collected important basic data on the influence of imaging on animal welfare and scientific outcome in healthy and tumour-bearing mice. Repeated anatomical magnetic resonance imaging (MRI) of animals did not influence behavioural and physiological characteristics related to animal wellbeing and tumour growth. Based on these data, we consider longitudinal MRI as a safe tool to obtain morphological and physiological information. Contrast-enhanced ultrasound (US) did not influence tumour growth and animal welfare as well, but increased tumour perfusion and leucocyte infiltration. Further investigations are necessary to unravel the mechanisms behind these effects and to provide a safety recommendation for US protocols.Even though, based on our severity tests, MRI and US can be considered well tolerated, this does not exclude the possibility that imaging modalities cause tissue damage. In this context, there is also uncertainty, whether the severity tests and clinical chemistry are sensitive enough to assess mild tissue responses. Especially during therapeutic interventions, advanced diagnostic technologies are needed to monitor not only vital signs, but also pain, stress and tissue damage. In that line, we employed [68Ga]Ga-NODAGA-duramycin positron emission tomography (PET) for the longitudinal evaluation of tissue damage after chemotherapy. The tracer binds to phosphatidylethanolamine, which is exposed upon oxidative stress, during apoptosis as well as necrosis. PET results were in line with clinical chemistry and histology and in some organs even more sensitive for tissue damage than the reference methods.Based on these results, in the second funding period, we intend to implement a new imaging concept for the simultaneous detection of pain and tissue damage. To achieve this, we will use a duramycin single photon emission computed tomography (SPECT) tracer for assessing tissue damage in combination with a novel PET tracer ([18F]FTC-146) that binds to the activated sigma-1 receptor for pain detection. The imaging protocol will be applied to different pain models (neuropathic, inflammatory, tumour-induced and post-operative pain) as well as the US imaging setup that caused local physiological and immunological responses. In case of the latter, we will also investigate the pathophysiological mechanisms of the observed changes and connect those to animal welfare. A comparison between histological analyses, results from PET/SPECT/CT measurements and behavioural tests will be performed to investigate the sensitivity of the different methods to detect pain and discomfort.