Background & Aims
Preclinical pain research has been criticised for its lack of translational value. A body of work from our lab suggests that confounds within the testing environment may be at least partially responsible for this deficiency in translatability.
Research shows that the testing environment can produce hypersensitive states in both humans and mice, with painful stimulus and context pairing leading to an exacerbated pain response via classical (Pavlovian) conditioning (Martin et al, 2019). Data from our lab suggests that this effect can occur in a number of mouse pain assays and in both sexes (Skvortsova et al, 2024).
Given that most behavioural preclinical pain studies involve multiple testing sessions in the same room, we wondered whether conditioning hypersensitivity might be conflated with hypersensitivity from an injury, thus confounding the interpretation of results.
Methods
Hind paw mechanical withdrawal thresholds (MWT) of male and female CD-1 mice were assessed using von Frey fibers pre- and post-chronic constriction injury (CCI), under the following experimental designs:
1)Baseline MWTs were measured in room A. After CCI, MWTs were assessed every 3 days in either the same context (A), alternating contexts (A/B) or with a switch in contexts at day 10 (A-to-B).
2)MWTs were tested prior to and post-CCI at 3-day intervals in room A (within-subjects condition). Separate groups of mice (between-subjects condition) were baselined and tested at single time points along the course of the testing paradigm, in room A.
A complementary review of published studies was conducted using a manual inspection of all preclinical papers (various assays and measures) published in the journal PAIN between 2016-2020 featuring multiple post-injury testing days (n=150). Number of post-injury testing days and the approximate duration of pain hypersensitivity were recorded
Results
When tested on a 3-day interval schedule in room A only, CCI mice presented with mechanical hypersensitivity lasting up to 35 days. Alternating context testing A/B, however, resulted in a shortened allodynic state, with mice returning to baseline approximately 21 days after CCI induction. An A-to-B switch at CCI day 10 led to a similarly reduced period of hypersensitivity, with MWTs also returning to baseline by day 21. Mice tested at a single time point only displayed a drastically shortened CCI-induced hypersensitive state, with mechanical hypersensitivity returning to baseline in all groups tested from day 7 post-CCI onward.
Furthermore, the analysis of the preclinical pain literature revealed a significant positive correlation (r=30, p<0.001) between the number of post-injury testing trials and the time taken to return to baseline pain sensitivity.
Conclusions
Our results suggest the existence of significant conditioning confounds in preclinical pain testing. Specifically, we find that reducing the classical conditioning (by switching rooms or by testing mice only once) inherent in pain testing greatly reduces the apparent duration of CCI allodynia. That is, it appears that a significant portion of the observed pain hypersensitivity after injury is not actually attributable to the injury, but rather to a classically conditioned response to the testing context associated with repeated pain-inducing stimulation.
Future experiments will involve the use of inflammatory pain models and alternative evoked threshold measures, such as radiant heat paw withdrawal, as well as inhibitory experiments that aim to block the formation of conditioning during this process.
References
Martin, L. J., Acland, E. L., Cho, C., Gandhi, W., Chen, D., Corley, E., Kadoura, B., Levy, T., Mirali, S., Tohyama, S., Khan, S., MacIntyre, L. C., Carlson, E. N., Schweinhardt, P., & Mogil, J. S. (2019). Male-Specific Conditioned Pain Hypersensitivity in Mice and Humans. Current biology : CB, 29(2), 192–201.
Presenting Author
Oakley B Morgan
Poster Authors
Topics
- Models: Chronic Pain - Neuropathic