Investigating sex differences in the role of microglia in a rat model of osteroarthritic pain

Background & Rationale: Six million Canadian adults are diagnosed with arthritis costing the health care system an average of $33 billion annually. According to the Arthritis Society of Canada, 67% of those affected are women. In the clinic, women afflicted with Osteoarthritis (OA) report higher levels of pain based on multiple diagnostic tests despite having similar levels of radiographic degradation in the joints. A meta-analysis study concluded that women have greater risk, prevalence, incidence, and severity of OA in all joints. Contrary to claims describing OA as a disease due to wear and tear, OA has both inflammatory and neuropathic components. The neuropathic components of OA will be the primary focus of this project. In mouse models of neuropathic pain, it has been reported that spinal microglia contribute to pain hypersensitivity in males, whereas T-cells serve a similar function in females. In males, microglial signaling downregulates the expression of the potassium-chloride co-transporter 2 (KCC2) in spinal cord neurons, leading to spinal disinhibition. However, the signaling cascade that leads to KCC2 downregulation in females remains unknown. Given that OA has neuropathic features, we aim to explore if similar sexual dimorphisms exist in the pain associated with this disease. Hypothesis and Aims: We hypothesize that immune cells have differential roles in male and female OA pain. Specifically, that microglia mediate pain behaviour in males but not in females. To address this hypothesis, three aims were explored:AIM 1 - Investigate the onset and intensity of pain-related behaviour in male and female rats using the monoiodoacetate (MIA) model of OA in the ankle joint. AIM 2 - Investigate neuropathic features of OA pain in male and female rats. AIM 3 - Investigate the efficacy of pharmacologically inhibiting microglia in reducing or reversing pain behavior in both sexes. Methods: AIM 1 - Animals underwent weekly behavioral testing utilizing common pain-related assays for mechanical hypersensitivity (pain-like response to a non-painful mechanical stimulus), cold allodynia (pain-like response to a non-painful cold stimulus), and heat hyperalgesia (pain-like response to noxious thermal stimulus). Additionally, they underwent biweekly testing of joint function using a treadmill. Female estrous cycles were synchronized to minimize potential hormonal confounds. AIM 2 - To investigate the role of microglia in the central nervous system (CNS) an anti-Iba1 antibody and bright-field microscopy were used to detect microglial immunoreactivity in the superficial dorsal horn. Additionally, changes in KCC2 expression were analyzed by immunofluorescence using an anti-KCC2 antibody and IMARIS software. AIM 3 - Five weeks after the induction of OA, microglia function was inhibited by performing an intrathecal injection of minocycline and mechanical hypersensitivity and cold allodynia were re-assessed.Results: No sex differences in pain onset or severity were detected. At 5 weeks, the number of microglia increased and KCC2 downregulation was observed in all lamina of the ipsilateral dorsal horn of both sexes in excitatory and inhibitory neurons. Furthermore, when a single dose of minocycline was administered at 5 weeks, mechanical allodynia was reversed in male but not female rats. Discussion: Although there is clinical evidence of a distinction between osteoarthritic pain in males and females, the mechanisms underlying these differences are still unknown. These OA pain mechanisms are important to investigate, as the elucidation of sexually dimorphic mechanisms could provide insight into improved and personalized treatment plans. Our preliminary findings suggest a male specific role of microglia in OA pain encouraging further investigation. It is our wish that this project also initiates a new discussion in the pain field in the importance of considering both sexes when studying pain

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