Repeated Social Defeat Stress Promotes Reactive Brain Endothelium and Microglia- Dependent Pain Sensitivity DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Caroline Maria Sawicki, B.S. Oral Biology Graduate Program The Ohio State University 2020 Dissertation Committee Dr. John F. Sheridan, Advisor Dr. Jonathan P. Godbout Dr. Brian L. Foster Dr. Michelle L. Humeidan 1 Copyright by Caroline Maria Sawicki 2020 2 ABstract Exposure to chronic stress disrupts the homeostatic communication pathways between the central nervous system (CNS) and peripheral immune system, leading to dysregulated and heightened neuroinflammation that contributes to the pathophysiology of anxiety and pain. Repeated social defeat (RSD) is a murine model of psychosocial stress that recapitulates many of the behavioral and immunological effects observed in humans in response to stress, including activation of the sympathetic nervous system (SNS) and hypothalamic-pituitary-adrenal (HPA) axis. In both humans and rodents, the brain interprets physiological stress within fear and threat appraisal circuitry. RSD promotes the trafficking of bone marrow-derived inflammatory monocytes to the brain within discrete stress-responsive brain regions where neuronal and microglial activation are observed. Notably, RSD induces the expression of adhesion molecules on vascular endothelial cells within the same brain regions where microglial activation and monocyte trafficking occur in response to stress (Chapter 2). Further studies demonstrated that these monocytes express high levels of Interleukin (IL)-1β, activating brain endothelial IL-1 receptors to promote the onset of anxiety-like behavior. In addition to promoting anxiety, psychological stress increases susceptibility to experience pain and exacerbates existing pain. Notably, clinical studies indicate that enhanced neuroimmune activation elicits adaptive changes in the nervous system that can contribute to exaggerated pain sensation. Therefore, the next series of experiments investigated whether stress-induced neuroimmune responses ii contribute to increased pain sensitivity in mice exposed to RSD (Chapter 3). RSD increased mechanical allodynia in an exposure-dependent manner that persisted for at least one week following cessation of the stressor. Additionally, we showed that blocking peripheral nociception was effective in inhibiting enhanced pain signaling without altering stress- induced innate immune or behavioral responses. Despite these findings, the mechanism by which stress caused increased pain sensitivity was unclear. Therefore, the next series of experiments mechanistically tested the role of microglia in promoting increased mechanical allodynia during RSD. We showed that microglia were activated selectively within the nociceptive neurocircuitry of the dorsal horn of the lumbar spinal cord during RSD independent of peripheral monocyte recruitment. Notably, pharmacological ablation of microglia prevented the development of mechanical allodynia during RSD that corresponded with a reduction in inflammatory mediators associated with nociceptive signaling. Taken together, these findings provide significant insight into the neuroimmune interactions that mediate stress-related psychiatric disorders and chronic pain states. Furthermore, these studies may lead to the development of novel therapeutic strategies for the management of behavioral complications associated with stress. iii This thesis is dedicated to my father, Jerzy Sawicki, without whom none of my success would have been possible. iv Acknowledgements First, I would like to extend my sincerest gratitude to my advisor, Dr. John Sheridan, for picking up a random phone call from me in 2013 asking to be a part of his laboratory. What intended to be just a summer research position turned into seven of the most challenging, inspiring, and best years of my life. I cannot thank you enough for your constant support, encouragement, and ability to challenge me to become the best scientist I can be. Second, I would like to thank my “unofficial” co-advisor, Dr. Jonathan Godbout, for teaching me how to take criticism, and use it as fuel to become better. It took nearly seven years for me to appreciate your unique methods of mentorship, but I would never have made it to this point without your guidance. Third, I am beyond grateful for the best group of fellow graduate students that I had throughout my seven years in lab. In no particular order, Dr. Eric Wohleb, Dr. Brenda Reader, Dr. Ashley Fenn, Dr. Brant Jarrett, Dr. Diana Norden, Dr. Dan McKim, Dr. Xiaoyu Liu, Dr. Mike Weber, Dr. Kristina Witcher, Megan Muccigrosso, Damon DiSabato, Shane O’Neil, Danny Nemeth, Ren Yin and Zoe Tapp, thank you for the innumerable life talks, the unforgettable happy hours, and the most enriching graduate school experience. None of these research projects would have been completed without the generous help of our lab managers, technicians, and undergraduates: Danny Shea, Yufen Wang, Dave Hammond, Victoria Wilson, Natalie Gallagher, Chelsea Bray, January Kim and Jenna Patterson. v Thank you for your constant willingness to help with experiments at any hour of the day, all while pretending to listen to my recap of The Bachelor each week. I would also like to acknowledge my committee members for their support and patience as I transitioned from a pre-candidacy student to a true scientist. Dr. Michelle Humeidan, I truly appreciate the unique experience of having a fellow female clinician- scientist to not only provide me with scientific guidance, but also to teach me how to juggle the challenges of work-life balance with a family. Dr. Brian Foster, I thank you for being so willing to be a part of my scientific journey, and for the unique insight you have provided me every step of the way. Last, I would like to thank my family for their continuous love and support. To my husband, Spencer, thank you for always being my constant throughout the chaos of dental school, graduate school, and life. To my parents, Ewa and Jerzy, thank you for always embracing my everchanging life plans, helping me to push through challenging times, and never letting me give up. And finally, I would like to thank my older brother and role model. Konrad, thank you for holding my hand throughout life and for always paving the perfect pathway for me to follow in your footsteps. vi Vita 2013 ………………………………………………………….……...…B.S. Pre-Dentistry, University of Dayton, Dayton, OH 2013 – present …………………………………………………Graduate Research Fellow, The Ohio State University, Columbus, OH PuBlications Sawicki CM, Humeidan ML, Sheridan JF. (in press). Neuroimmune Interactions in Pain and Inflammation—An Interdisciplinary Approach. The Neuroscientist. Wade SD, McKim DB, Sawicki CM, Blakeslee JJ, Wang H, Vasas M, Jatana CA, Kennedy KS, Dib P, Humeidan ML, Cornelius BW. (in press). Stability of Epinephrine in a Normal Saline Solution. Journal of Implant and Advanced Clinical Dentistry. Yin W, Gallagher NR, Sawicki CM, McKim DB, Godbout JP, Sheridan JF. Repeated social defeat in female mice induces anxiety-like behavior associated with enhanced myelopoiesis and increased monocyte accumulation in the brain. Brain Behav Immun. 2019 May;78:131-142. doi: 10.1016/j.bbi.2019.01.015. Epub 2019 Jan 23. PubMed PMID: 30684650; PubMed Central PMCID: PMC6488440. Sawicki CM, Kim JK, Weber MD, Faw TD, McKim DB, Madalena KM, Lerch JK, Basso DM, Humeidan ML, Godbout JP, Sheridan JF. Microglia Promote Increased Pain Behavior through Enhanced Inflammation in the Spinal Cord during Repeated Social Defeat Stress. J Neurosci. 2019 Feb 13;39(7):1139-1149. doi: 10.1523/JNEUROSCI.2785-18.2018. Epub 2018 Dec 17. 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Microglial recruitment of IL-1β-producing monocytes to brain endothelium causes stress-induced anxiety. Mol Psychiatry. 2018 Jun;23(6):1421-1431. doi: 10.1038/mp.2017.64. Epub 2017 Apr 4. PubMed PMID: 28373688; PubMed Central PMCID: PMC5628107. Sawicki CM, McKim DB, Wohleb ES, Jarrett BL, Reader BF, Norden DM, Godbout JP, Sheridan JF. Social defeat promotes a reactive endothelium in a brain region-dependent manner with increased expression of key adhesion molecules, selectins and chemokines associated with the recruitment of myeloid cells to the brain. Neuroscience. 2014 Oct 14;PubMed PMID: 25445193; NIHMSID: NIHMS639767; PubMed Central PMCID: PMC4397120. Lieblein-Boff JC, McKim DB, Shea DT, Wei P, Deng Z, Sawicki C, Quan N, Bilbo SD, Bailey MT, McTigue DM, Godbout JP. Neonatal E coli infection causes neuro-behavioral