Mechanisms of Neuroinflammation after Fracture

描述（由申请人提供）：肢体创伤可导致复杂区域性疼痛综合征（CRPS）的发展，这是一种潜在的使人衰弱的慢性疼痛状态。目前对CRPS的病理生理和治疗尚无共识，明确需要进行转化研究以确定治疗靶点和有效的治疗方法。我们已经建立了一个胫骨骨折的啮齿动物模型，该模型与CRPS的临床场景非常相似，后肢体重减轻，异常性疼痛，发热，水肿，骨质流失，神经炎症反应加剧，受影响皮肤中角质形成细胞表达的炎症介质（TNF， IL-1和NGF）水平升高。本研究验证了骨折和固定可增强皮肤角质形成细胞和背根神经节（DRG）胶质细胞的感觉和交感神经传出信号，引起细胞增殖和炎症介质释放，导致慢性炎症和疼痛发生的假设。本提案的主要目标是使用已批准或即将批准的药物确定针对CRPS的靶向治疗。具体目标是：1)确定感觉神经肽和肾上腺素能激动剂在培养的角化细胞、雪旺细胞和DRG细胞中的促炎作用；2)确定小鼠骨折和三周石膏固定后感觉和交感神经输出信号是否上调，并确定这种神经信号在骨折后后肢crps样变化和损伤肢体皮肤和DRGs中炎症介质表达的发展中所起的作用。3)表征固定和再活动对骨折后crps样变化、感觉和交感信号以及炎症介质表达的影响。角化细胞、雪旺细胞和DRG细胞系的体外细胞培养研究将用于鉴定翻译小鼠骨折研究的候选神经递质和受体亚型。骨折CRPS模型将用于缺乏特定神经递质或其受体的小鼠品系，以确定感觉和交感神经递质信号在骨折引起的疼痛、炎症和骨质流失的发展中所起的作用。其他研究将确定1)完整小鼠的石膏固定效果，2)骨折和髓内固定小鼠的早期再活动，以及3)骨折和石膏小鼠的日常跑步机运动治疗，观察这些干预措施对疼痛行为、水肿、温暖和皮肤角化细胞和DRG卫星胶质中炎症介质表达的影响。我们预计这些实验将有助于识别调节骨折后炎症和疼痛的神经系统、递质和外周细胞靶点，发现创伤后活动和运动的新型抗炎作用，并为未来的CRPS试验产生识别特定分子靶点的转化数据。

DESCRIPTION (provided by applicant): Limb trauma can lead to the development of a complex regional pain syndrome (CRPS), a potentially debilitating chronic pain state. Currently there is no consensus on either the pathophysiology or treatment for CRPS and translational studies are clearly needed to identify therapeutic targets and effective treatments. We have developed a tibia fracture rodent model that closely replicates the CRPS clinical scenario with hindlimb unweighting, allodynia, warmth, edema, bone loss, exaggerated neuroinflammatory responses and increased levels of inflammatory mediators (TNF, IL-1, and NGF) expressed by keratinocytes in the affected skin. This proposal tests the hypothesis that fracture and immobilization can enhance sensory and sympathetic efferent signaling in skin keratinocytes and dorsal root ganglion (DRG) glial cells, causing cellular proliferation and inflammatory mediator release leading to the development of chronic inflammation and pain. The primary objective of this proposal is to identify target-specific treatments for CRPS using approved or soon-to-be-approved medications. The specific aims are: 1) to identify the pro-inflammatory effects of sensory neuropeptides and adrenergic agonists in cultured keratinocyte, Schwann, and DRG cells, 2) to determine whether sensory and sympathetic efferent signaling is up-regulated after fracture and three- week cast immobilization in mice and to establish the role this neuronal signaling plays in the development of post-fracture CRPS-like changes in the hind limb and inflammatory mediator expression in the skin and DRGs innervating the injured limb, and 3) to characterize the effects of immobilization and remobilization on post-fracture CRPS-like changes, sensory and sympathetic signaling, and inflammatory mediator expression. In vitro cell culture studies in keratinocyte, Schwann, and DRG cell lines will be utilized to identify candidate neurotransmitters and receptor subtypes for translational mouse fracture studies. The fracture CRPS model will be utilized in mouse strains deficient for specific neurotransmitters or their receptors to determine the role played by sensory and sympathetic neurotransmitter signaling in the development of fracture induced pain, inflammation, and bone loss. Additional studies will determine the effects of 1) cast immobilization in intact mice, 2) early remobilization in fractured and intramedullary pinned mice, and 3) daily treadmill exercise therapy in fracture and casted mice, looking at the effects of these interventions on the development of pain behaviors, edema, warmth, and inflammatory mediator expression in the skin keratinocytes and the DRG satellite glia. We anticipate that these experiments will help identify the neural systems, transmitters, and peripheral cellular targets that modulate post-fracture inflammation and pain, discover novel anti- inflammatory effects for mobilization and exercise after trauma, and generate translational data identifying specific molecular targets for future CRPS trials. PUBLIC HEALTH RELEVANCE: We anticipate that the proposed studies in this application will provide a sound experimental foundation supporting the neuroinflammatory basis of complex regional pain syndrome (CRPS). Identifying the relevant inflammatory signaling pathways leading to the development of post-traumatic CRPS could readily translate into more effective CRPS drug treatments. If funded, this work would be an important step towards the ultimate goal of improving the clinical management of this debilitating condition.