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SummaryMicroglia contribute to the initiation of pain, however, a translationally viable approach addressing how or when to modulate these cells remains elusive. We used a targeted, inducible, genetic microglial depletion strategy at both acute and acute-to-chronic transition phases in the clinically-relevant tibial fracture/casting pain model to determine the contribution of microglia to the initiation and maintenance of pain. We observed complete resolution of pain after transient microglial depletion at the acute-to-chronic phase, which coincided with the timeframe of full repopulation of microglia. These repopulated microglia were morphologically distinct from control microglia, signifying they may exhibit a unique transcriptome. RNA sequencing of repopulated spinal cord microglia identified genes of interest using weighted gene co-expression network analysis (WGCNA). We intersected these genes with a newly-generated single nuclei microglial dataset from human spinal cord dorsal horn and identified human-relevant genes that may ultimately promote pain resolution after injury. This work presents a novel approach to gene discovery in pain and provides comprehensive datasets for the development of future microglial-targeted therapeutics.

The tibial fracture-pin model is a mouse model of orthopedic trauma and surgery that recapitulates the complex muscle, bone, nerve, and connective tissue damage that manifests with this type of injury in humans. This model was developed because previous models of orthopedic trauma did not include simultaneous injury to multiple tissue types (bone, muscle, nerves) and were not truly representative of human complex orthopedic trauma. The authors therefore modified previous models of orthopedic trauma and developed the tibial fracture-pin model. This modified fracture model consists of a unilateral open tibial fracture with intramedullary nail (IMN) internal fixation and simultaneous tibialis anterior (TA) muscle injury, resulting in mechanical allodynia that lasts up to 5 weeks post injury. This series of protocols outlines the detailed steps to perform the clinically relevant orthopedic trauma tibial fracture-pin model, followed by a modified hot plate assay to examine nociceptive changes after orthopedic injury. Taken together, these detailed, reproducible protocols will allow pain researchers to expand their toolkit for studying orthopedic trauma-induced pain.

Chronic pain is a common and often debilitating problem that affects 100 million Americans. A better understanding of pain's molecular mechanisms is necessary for developing safe and effective therapeutics. Microglial activation has been implicated as a mediator of chronic pain in numerous preclinical studies; unfortunately, translational efforts using known glial modulators have largely failed, perhaps at least in part due to poor specificity of the compounds pursued, or an incomplete understanding of microglial reactivity. In order to achieve a more granular understanding of the role of microglia in chronic pain as a means of optimizing translational efforts, we utilized a clinically-informed mouse model of complex regional pain syndrome (CRPS), and monitored microglial activation throughout pain progression. We discovered that while both males and females exhibit spinal cord microglial activation as evidenced by increases in Iba1, activation is attenuated and delayed in females. We further evaluated the expression of the newly identified microglia-specific marker, TMEM119, and identified two distinct populations in the spinal cord parenchyma after peripheral injury: TMEM119+ microglia and TMEM119- infiltrating myeloid lineage cells, which are comprised of Ly6G + neutrophils and Ly6G- macrophages/monocytes. Neurons are sensitized by inflammatory mediators released in the CNS after injury; however, the cellular source of these cytokines remains somewhat unclear. Using multiplex

Complex regional pain syndrome (CRPS) is a chronic pain condition that usually follows minor extremity trauma and is characterized by sensory, vasomotor, sudomotor, and motor/trophic changes. While epidemiological studies are limited, CRPS occurs with an estimated incidence between 5.5 and 29.0 cases per 100,000 person-years and is more common in female patients. The pathophysiology underlying CRPS remains an active area of study, likely varying between patients and even over time in a single patient. Proposed mechanisms include sympathetically maintained pain, peripheral and central sensitization, oxidative stress, autoimmunity, and psychological factors. Changes in presentation are common with increasing duration of symptoms and often include a shift from a warm, edematous, and erythematous limb to a cool, atrophic, and blue/purplish limb with non-dermatomal spreading. This more chronic phase coincides with increased refractoriness to treatment and suggests a transition from peripheral to central mechanisms over time.

Complex regional pain syndrome (CRPS) is a chronic pain disorder with a clear acute-to-chronic transition. Preclinical studies demonstrate that toll-like receptor 4 (TLR4), expressed by myeloid-lineage cells, astrocytes, and neurons, mediates a sex-dependent transition to chronic pain; however, evidence is lacking on which exact TLR4-expressing cells are responsible. We used complementary pharmacologic and transgenic approaches in mice to more specifically manipulate myeloid-lineage TLR4 and outline its contribution to the transition from acute-to-chronic CRPS based on three key variables: location (peripheral vs central), timing (prevention vs treatment), and sex (male vs female). We demonstrate that systemic TLR4 antagonism is more effective at improving chronic allodynia trajectory when administered at the time of injury (early) in the tibial fracture model of CRPS in both sexes. In order to clarify the contribution of myeloid-lineage cells peripherally (macrophages) or centrally (microglia), we rigorously characterize a novel spatiotemporal transgenic mouse line,Cx3CR1-CreERT2-eYFP;TLR4fl/fl(TLR4 cKO) to specifically knock out TLR4 only in microglia and no other myeloid-lineage cells. Using this transgenic mouse, we find that early TLR4 cKO results in profound improvement in chronic, but not acute, allodynia in males, with a significant but less robust effect in females. In contrast, late TLR4 cKO results in partial improvement in allodynia in both sexes, suggesting that downstream cellular or molecular TLR4-independent events may have already been triggered. Overall, we find that the contribution of TLR4 is time- and microglia-dependent in both sexes; however, females also rely on peripheral myeloid-lineage (or other TLR4 expressing) cells to trigger chronic pain.SIGNIFICANCE STATEMENTThe contribution of myeloid cell TLR4 to sex-specific pain progression remains controversial. We used complementary pharmacologic and transgenic approaches to specifically manipulate TLR4 based on three key variables: location (peripheral vs central), timing (prevention vs treatment), and sex (male vs female). We discovered that microglial TLR4 contributes to early pain progression in males, and to a lesser extent in females. We further found that maintenance of chronic pain likely occurs through myeloid TLR4-independent mechanisms in both sexes. Together, we define a more nuanced contribution of this receptor to the acute-to-chronic pain transition in a mouse model of complex regional pain syndrome.

We present a case series of patients treated with hydroxychloroquine for complex regional pain syndrome and demonstrated analgesia and reduced spinal cord autoinflammation in a preclinical model of complex regional pain syndrome., Introduction: Complex regional pain syndrome (CRPS) is a condition that occurs after minor trauma characterized by sensory, trophic, and motor changes. Although preclinical studies have demonstrated that CRPS may be driven in part by autoinflammation, clinical use of immune-modulating drugs in CRPS is limited. Hydroxychloroquine (HCQ) is a disease-modifying antirheumatic drug used to treat malaria and autoimmune disorders that may provide benefit in CRPS. Objectives: To describe the use of HCQ in patients with refractory CRPS and investigate possible mechanisms of benefit in a mouse model of CRPS. Methods: We initiated HCQ therapy in 7 female patients with refractory CRPS undergoing treatment at the Stanford Pain Management Center. We subsequently undertook studies in the mouse tibial fracture–casting model of CRPS to identify mechanisms underlying symptom reduction. We evaluated behavior using mechanical allodynia and spinal cord autoinflammation by immunohistochemistry and enzyme-linked immunosorbent assay. Results: We treated 7 female patients with chronic, refractory CRPS with HCQ 200 mg twice daily for 2 months, followed by 200 mg daily thereafter. Two patients stopped HCQ secondary to lack of response or side effects. Overall, HCQ significantly improved average numerical rating scale pain from 6.8 ± 1.1 before HCQ to 3.8 ± 1.9 after HCQ treatment. In the tibial fracture–casting mouse model of CRPS, we observed reductions in allodynia, paw edema, and warmth following daily HCQ treatment starting at 3 weeks after injury. Spinal cord dorsal horn microglial activation and cytokine levels were also reduced by HCQ treatment. Conclusion: Together, these preclinical and clinical results suggest that HCQ may benefit patients with CRPS at least in part by modulating autoinflammation and support further investigation into the use of HCQ for CRPS.