A refinement approach in a mouse model of rehabilitation research. Analgesia strategy, reduction approach and infrared thermography in spinal cord injury.

The principles of Refinement, Replacement and Reduction (3R's) should be taken into account when animals must be used for scientific purpose. Here, a Reduction / Refinement approach was applied to the procedure of spinal cord injury (SCI), an animal model used in rehabilitation medicine research, in order to improve the quality of experiments, avoiding unnecessary suffering. The aims of this investigation were 1- to assess acute surgical pain in mice subjected to SCI, 2- to compare the efficacy of commonly used analgesia (three buprenorphine subcutaneous injection in 48 hours, 0,15 mg/kg each) with a combination of opioid and NSAID (one subcutaneous injection of 5 mg/kg carprofen before surgery followed by three buprenorphine subcutaneous injection in 48 hours, 0,15 mg/kg each) and 3- to test if Infrared Thermography (IRT) could be a potential new Refinement method to easily assess thermoregulation, an important metabolic parameter. Finally, we aimed to achieve these goals without recruiting animals on purpose, but using mice already scheduled for studies on SCI. By using behaviours analysis, we found that, despite being commonly used, buprenorphine does not completely relieve acute surgical pain, whereas the combination of buprenorphine and carprofen significantly decreases pain signs by 80%. IRT technology turned out to be a very useful Refinement tool being a non invasive methods to measure animal temperature, particularly useful when rectal probe cannot be used, as in the case of SCI. We could find that temperatures constantly and significantly increased until 7 days after surgery and then slowly decreased and, finally, we could observe that in the buprenorphine and carprofen treated group, temperatures were statistically lower than in the buprenorphine-alone treated mice. To our knowledge this is the first work providing an analgesic Refinement and a description of thermoregulatory response using the IRT technology, in mice subjected to SCI.

Microwave-assisted synthesis of TEMPO-labeled hydrogels traceable with MRI.

Polymer functionalization strategies have recently attracted considerable attention for several applications in biomaterials science. In particular, technological advancements in medical imaging have focused on the design of polymeric matrices to improve non-invasive approaches and diagnostic accuracy. In this scenario, the use of microwave irradiation of aqueous solutions containing appropriate combinations of polymers is gaining increasing interest in the synthesis of sterile hydrogels without using monomers, eliminating the need to remove unreacted species. In this study, we developed a method for the in situ fabrication of TEMPO-labeled hydrogels based on a one-pot microwave reaction that can then be tracked by magnetic resonance imaging (MRI) without using toxic compounds that could be hostile for the target tissue. Click chemistry was used to link TEMPO to the polymeric scaffold. In an in vivo model, the system was able to preserve its TEMPO paramagnetic activity up to 1 month after hydrogel injection, showing a clear detectable signal on T1-weighted MRI with a longitudinal relaxivity value of 0.29 mM s-1, comparable to a value of 0.31 mM s-1 characteristic of TEMPO application. The uncleavable conjugation between the contrast agent and the polymeric scaffold is a leading point to record these results: the use of TEMPO only physically entrapped in the polymeric scaffold did not show MRI traceability even after few hours. Moreover, the use of TEMPO-labeled hydrogels can also help to reduce the number of animals sacrificed being a longitudinal non-invasive technique.