Rats were subject to either MRI restraint (Restrained) and loud scanner noise, or exposure (Exposed) to the apparatus and quiet scanner noise for 30 minutes per day over 3 days. Another group was subject to the same treatment, but on separate weeks (Controls, 12 animals in each group). Restrained animals were placed in a fabric snuggle with head and tail protruding, and strapped into a Perspex MRI cradle, and Exposed and Control animals were exposed to and allowed to explore the restraint apparatus. Thermal and mechanical sensitivity was measured with the Hargreaves and von Frey apparatus respectively 24 hours before, and immediately after every restraint session, as well as 24 hours after the final restraint. Sensory sensitivity was tested again at 12 days after treatment, and 24 hours later the formalin pain test was performed on all animals. Rats were given 50ul of 1% formalin injected into the right hindpaw and behavior observed for one hour. Immediately afterwards, rats were transcardially perfused and brain tissue was recovered for histological processing. 70ul of blood was taken at each time point for later analysis of corticosterone. We found that restraint causes long-lasting alterations in stress responses, pain behaviors and brain responses to pain. Animals do not gain weight at the same rate as controls, and show large stress hormone responses to physical restraint. When tested on a pain test two weeks after three days of restraint, both restrained and previously non-restrained animals that were exposed to olfactory cues from restrained animals (exposed) showed a decreased response to the formalin test compared to controls, suggesting a form of long-lasting stress-induced analgesia. Restrained animals showed a large increase in stress hormone response post-formalin that wasnt seen in exposed animals, suggesting different stress-related mechanisms mediate these behavioral responses. This is supported by data showing an increased number of Fos-positive cells (and by extension cell activation) in the nociceptive central nucleus of the amygdala in restrained animals, but in neither exposed nor control animals. Interestingly, a form of stress-induced hyperalgesia was seen in the olfactory exposed group, where thermal hypersensitivity increased over the course of three days of testing, with increased corticosterone levels seen in this group relative to controls too. All animals received an acute thermal pain stressor on day three of restraint/exposure/control treatment. All animals showed a spike in stress hormones afterwards, and all animals showed drops in withdrawal thresholds to mechanical and thermal stimuli, suggesting these tests are sensitive to acute stressors. However, the lack of behavioral differences between the clearly stressed restrained group versus controls suggests that these tests lack sensitivity for some forms of stress. Further experiments were required to complete this project January-July 2015, but these are now complete and data is being prepared for publication.
| Low, Lucie A; Bauer, Lucy C; Pitcher, Mark H et al. (2016) Restraint training for awake functional brain scanning of rodents can cause long-lasting changes in pain and stress responses. Pain 157:1761-72 |
