Laboratory environmental factors and pain behavior: the relevance of unknown unknowns to reproducibility and translation

Perspective Focus on Reproducibility Laboratory environmental factors and pain behavior: the relevance of unknown unknowns to reproducibility and translation © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. Jeffrey S Mogil The poor record of basic-to-clinical translation in recent decades has led to speculation that preclinical research is “irreproducible”, and this irreproducibility in turn has largely been attributed to deficiencies in reporting and statistical practices. There are, however, a number of other reasonable explanations of both poor translation and difficulties in one laboratory replicating the results of another. This article examines these explanations as they pertain to preclinical pain research. I submit that many instances of apparent irreproducibility are actually attributable to interactions between the phenomena and interventions under study and “latent” environmental factors affecting the rodent subjects. These environmental variables—often causing stress, and related to both animal husbandry and the specific testing context—differ greatly between labs, and continue to be identified, suggesting that our knowledge of their existence is far from complete. In pain research in particular, laboratory stressors  can produce great variability of unpredictable direction, as stress is known to produce increases  (stress-induced hyperalgesia) or decreases (stress-induced analgesia) in pain depending on its parameters. Much greater attention needs to be paid to the study of the laboratory environment if replication and translation are to be improved. Ever since the publication in 2011–2012 of two front-of-themagazine pieces by Prinz et al.1 and Begley and Ellis2, the research world has been rocked by accusations of the “irreproducibility” of preclinical research. Both of these articles described, anecdotally, the inability of pharmaceutical preclinical testing units to reproduce experiments by academic laboratories published in the scientific literature. These surveys were widely reported on in the media (e.g., ref. 3), and their pessimistic message was embraced by funding agencies4 and journal editors5. The desire for a more systematic approach to the issue yielded high-profile “reproducibility projects” in both psychology6 and cancer7; the former has been published and confirmed that large proportions of published research findings cannot be clearly reproduced by independent laboratories. A recent survey of preclinical scientists revealed that 52% think there is a “significant crisis”8. This being said, it is hard to decide whether something in science has actually changed, or is “broken”, or whether the apparent epidemic of irreproducibility is simply due to more attention being paid to the topic against a backdrop of the (undeniably) poor translational success of recent decades in drug development9. Those commenting on irreproducibility have most often attributed it to poor experimental design, poor reporting of methods and results (for example, missing statements about blinding and randomization), various types of bias, investigator misconduct, and misuse of statistics10. I believe that all of these things occur. I would argue, however, that the current state of affairs is certainly no worse than when I started in science 25 years ago, and almost certainly better. At the beginning of my career, for example, methods sections would simply refer to “mice”; now at least we’re usually told that they are male—alas, always male—C57BL/6 mice. Thus, it seems to me that the true reasons for poor translation lie elsewhere. What is responsible for poor translation? What then is responsible for our current translational challenges? Some believe that the drugs that work in animals really do also work in people, but that it’s becoming harder and harder to prove this so. That is, the real problem may actually be the changing nature of clinical trials, in which fewer and fewer drug-naive participants are available, and in which the placebo response has steadily increased over time11. Others have pointed to shortcomings in the validity Departments of Psychology and Anesthesia and Alan Edwards Centre for Research on Pain, McGill University, Montreal, Canada. Correspondence should be addressed to J.S.M. (). 136    Volume 46, No. 4 | APRIL 2017 www.nature.com/laban © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. Focus on Reproducibility of animal models (and their implementation) used in preclinical studies12. I have previously argued that a major problem with the status quo in preclinical pain research is the animal models in common use; the inadequacies are related to all three facets of an animal model: subjects, assays, and measures13. The human sufferers of chronic pain are overwhelmingly female14, middle-aged or elderly15, and of heterogeneous genetic background, whereas the animal subjects in pain experiments are overwhelmingly young-adult, male Sprague Dawley rats or C57BL/6 mice13,16,17. Both quantitative and robust qualitative differences in pain processing have been documented between strains18 and the sexes19,20, confounding simple conclusions. The most common chronic pain syndromes in humans are low back pain, arthritis of the joints, and headache21, whereas the most common chronic pain assays in current use for animal subjects involve experimental ligations of afferent fibers serving, and injection of inflammatory substances into, the hind paw22. Finally, whereas the most prevalent (and bothersome) clinical symptoms of chronic pain are deep, spontaneous (ongoing) pain and numbness23,24, and comorbidities like sleep disruption21, preclinical pain researchers continue to focus almost exclusively on measuring mechanical and thermal pain hypersensitivity (allodynia and hyperalgesia)13,25. Whether currently popular animal models are close enough to clinical reality or not is hard to say (is the glass half-empty, or is it half-full?), and such discussions tend to quickly devolve into simply counting up recent efficacy successes (e.g., ziconotide, tanezumab) and failures (e.g., neurokinin-1 antagonists, fatty acid amide hydrolase inhibitors). Simply put, preclinical pain research with currently popular animal models performs more than adequately for backwards translation26,27; the forward translation jury is still out. The role of latent environmental factors in pain research I would suggest that another reason for apparent irreproducibility may be more important than is commonly appreciated: stealth or “latent” laboratory environmental factors28, the “unknown unknowns” of biology. Simply stated, any factor affecting stress levels in the testing environment may have huge effects on any biological phenomenon affected by stress, which includes essentially all of them, behavioral and non-behavioral alike. In turn, we are aware of only a few such factors, and even when we are aware of them they hardl (...truncated)