Mechanisms of Neuroinflammation after Fracture

描述(申请人提供)：肢体创伤可导致复杂区域疼痛综合征(CRPS)的发展，这是一种可能使人虚弱的慢性疼痛状态。目前，对于CRPS的病理生理学或治疗方法尚无共识，显然需要翻译研究来确定治疗靶点和有效的治疗方法。我们建立了一种胫骨骨折啮齿动物模型，该模型与CRPS的临床情景非常相似，表现为后肢重量减轻、疼痛、发热、水肿、骨质丢失、神经炎性反应加重，以及受影响皮肤角质形成细胞表达的炎症介质(肿瘤坏死因子、白介素1和神经生长因子)水平增加。这一建议验证了骨折和制动可以增强皮肤角质形成细胞和背根神经节(DRG)神经胶质细胞的感觉和交感传出信号，导致细胞增殖和炎症介质释放，从而导致慢性炎症和疼痛的假说。这项提案的主要目标是使用已批准或即将批准的药物来确定CRPS的靶向特异性治疗。其具体目的是：1)确定感觉神经肽和肾上腺素能激动剂对培养的角质形成细胞、雪旺细胞和DRG细胞的促炎作用；2)确定小鼠骨折和石膏固定三周后，感觉和交感传出信号是否上调，并建立这些神经元信号在骨折后后肢CRPS样变化和支配受损肢体的皮肤和DRG中炎症介质表达中的作用；3)表征制动和再动员对骨折后CRPS样变化、感觉和交感信号以及炎症介质表达的影响。角质形成细胞、雪旺细胞和背根神经节细胞系的体外细胞培养研究将被用来确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，以确定感觉和交感神经递质信号在骨折引起的疼痛、炎症和骨丢失发展中所起的作用。其他研究将确定1)完好小鼠的石膏固定，2)骨折和髓内钉住的小鼠的早期再活动，3)骨折和铸型小鼠的日常跑步机运动疗法的效果，观察这些干预措施对皮肤角质形成细胞和DRG卫星胶质细胞中疼痛行为、水肿、温暖和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，发现创伤后动员和锻炼的新的抗炎效果，并为未来的CRPS试验生成识别特定分子靶点的翻译数据。

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.