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First Name Dominika Burek, Washington University in St. Louis


Pain and opioid use disorder prevalence, diagnoses of mental health disorders, and rate of suicide have all skyrocketed over the last 30 years as a matrix of public health crises. Each reduces quality of life and installs risk of the others; together, they have burdened our country with nearly unmanageable healthcare challenges. Although the critical demand for novel and more effective therapeutics has been clear for decades, relatively little progress has been made to address pain and its comorbidities. Repeated clinical trial failures have called into question the preclinical evidence upon which they are based. Preclinical models of pain-induced negative affect have been adapted from those of chronic stress and their corresponding classical tests of behavior, which featured predictive validity for antidepressants and anxiolytics. Now, animal models of pain and pain-induced behavioral comorbidities are described in extensive, highly variable, and inconclusive literature. The work presented in this dissertation aims to address these limitations by defining the behavioral outcomes of hind-paw inflammatory pain, using extensive phenotyping, systematic review, and meta-analysis. I hypothesized that a rigorous and reproducible model of inflammatory pain-induced negative affect would have similar behavioral outcomes to preclinical models of chronic stress. In the first study presented in this dissertation, I use systematic behavioral phenotyping and analysis following hind-paw injection of Complete Freund’s Adjuvant (CFA) in male C57BL/6J mice. CFA did not reduce exploratory behavior in the open field test (OFT) or elevated zero maze (EZM) and did not induce a passive stress coping strategy in the forced swim test (FST). Behavioral differences between saline controls and CFA mice were not apparent at four or six weeks post-injection, regardless of testing order and combination. Behavioral measures did not correlate between phenotyping assays, and composite behavioral scores did not correlate to severity of thermal hyperalgesia. A focused and limited meta-analysis of similar studies with male C57BL/6 mice revealed a modest but significant effect of CFA on exploratory behavior in the OFT and EPM/EZM, but not on stress coping in the FST. In the second study presented in this dissertation, I conduct a comprehensive systematic review and meta-analysis of the effect of CFA-induced hind-paw inflammation in rodents on multiple indicators of exploratory, stress coping, naturalistic, and rewarding behaviors. This meta-analysis broadens the one previously described by extending the considered population to include male and female mice and rats of all strains, and by expanding the considered outcomes to include anhedonic and ethologically relevant behavior. I hypothesized that CFA exposure in rodents would have little to no significant effect on exploratory behavior and stress coping, and moderate effect on naturalistic and rewarding behaviors, when evaluating dozens of studies together. Secondarily, I hypothesized that any statistically significant global summary effects would be moderated by methodological variables, such as species, strain, sex, injection laterality, age, amount of CFA injected, and interval between injection and testing. A systematic search of four databases in 2020 returned 46 studies evaluating performance in the elevated plus or zero maze (EPM/EZM), open field test (OFT), light/dark box (LDB), place escape/avoidance paradigm (PEAP), forced swim test (FST), tail suspension test (TST), sucrose preference test (SPT), wheel running, and burrowing assay after hind-paw CFA injection. Discrete meta-analyses for each of these nine behavioral measures indicated that CFA (relative to saline exposure) modestly but significantly decreases exploratory behavior, increases passive stress coping in the TST but not the FST, and significantly decreases preference for sucrose and naturally rewarding activity. Heterogeneity among included studies was moderate to high across all nine behavioral tests. Sub-group analyses and meta-regressions revealed that species and animal sourcing were significant moderators of those effects, but not duration of inflammation, sex, or paw laterality. Quality assessments identified multiple aspects of internal validity that future studies can improve on to achieve consistency and reproducibility in this field. These meta-analyses also identified significant publication bias for the most commonly used tests, the EPM/EZM and OFT. These findings together indicate that CFA-induced hind-paw inflammation does not have consistent or substantial effects on exploratory behavior and stress coping, and suggest that tests such as the EPM and FST exhibit poor face and construct validity for modeling pain and mental health comorbidities. The results of this dissertation provoke thoughtful reflection on whether such tests are appropriate for modeling any aspect of mental health, as they evoke the same symptoms they aim to measure and rely on subjective interpretations. Future research agendas may leverage the information presented in this dissertation to optimize the models of pain-induced negative affect used in mechanistic studies. Subsequent studies on the effects of inflammatory pain on central nervous system physiology are encouraged to focus on naturalistic and ethologically relevant, hedonically rewarding, and motivated behaviors, which will better align with the current paradigm shift of studying individual symptoms and ethologically relevant behaviors.