Lee Yi-Chen, Lu Shui-Chin, Hsieh Yu-Lin
Department of Anatomy, School of Medicine, College of Medicine, Kaohsiung Medical University; Department of Medical Research, Kaohsiung Medical University Hospital.
Department of Medical Research, Ultrastructural Laboratory, Kaohsiung Medical University Hospital.
J Vis Exp. 2018 Feb 13(132):56651. doi: 10.3791/56651.
Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to degeneration and injury of small-diameter sensory neurons, referred to as small fiber neuropathy. Present animal models of small fiber neuropathy affect both large- and small-diameter sensory fibers and thus create a neuropathology too complex to properly assess the effects of injured small-diameter sensory fibers. Therefore, it is necessary to develop an experimental model of pure small fiber neuropathy to adequately examine these issues. This protocol describes an experimental model of small fiber neuropathy specifically affecting small-diameter sensory nerves with resiniferatoxin (RTX), an ultrapotent agonist of transient receptor potential vanilloid type 1 (TRPV1), through a single dose of intraperitoneal injection, referred to as RTX neuropathy. This RTX neuropathy showed pathological manifestations and behavioral abnormalities that mimic the clinical characteristics of patients with small fiber neuropathy, including intraepidermal nerve fiber (IENF) degeneration, specifically injury in small-diameter neurons, and induction of thermal hypoalgesia and mechanical allodynia. This protocol tested three doses of RTX (200, 50, and 10 µg/kg, respectively) and concluded that a critical dose of RTX (50 µg/kg) is required for the development of typical small fiber neuropathy manifestations, and prepared a modified immunostaining procedure to investigate IENF degeneration and neuronal soma injury. The modified procedure is fast, systematic, and economic. Behavioral evaluation of neuropathic pain is critical to reveal the function of small-diameter sensory nerves. The evaluation of mechanical thresholds in experimental rodents is particularly challenging and this protocol describes a customized metal mesh that is suitable for this type of assessment in rodents. In summary, RTX neuropathy is a new and easily established experimental model to evaluate the molecular significance and intervention underlying neuropathic pain for the development of therapeutic agents.
糖尿病（DM）患者或经历化疗药物神经毒性作用的患者，可能会因小直径感觉神经元的退化和损伤而出现感觉障碍，即小纤维神经病变。目前的小纤维神经病变动物模型会影响大直径和小直径感觉纤维，因此产生的神经病理学过于复杂，无法正确评估受损小直径感觉纤维的影响。所以，有必要开发一种纯小纤维神经病变的实验模型，以充分研究这些问题。本方案描述了一种小纤维神经病变的实验模型，该模型通过单次腹腔注射树脂毒素（RTX，一种瞬时受体电位香草酸亚型1（TRPV1）的超效激动剂）特异性地影响小直径感觉神经，称为RTX神经病变。这种RTX神经病变表现出模仿小纤维神经病变患者临床特征的病理表现和行为异常，包括表皮内神经纤维（IENF）退化、小直径神经元的特异性损伤，以及热痛觉减退和机械性异常性疼痛的诱导。本方案测试了三种剂量的RTX（分别为200、50和10μg/kg），并得出结论，典型的小纤维神经病变表现的发展需要临界剂量的RTX（50μg/kg），并准备了一种改良的免疫染色程序来研究IENF退化和神经元胞体损伤。该改良程序快速、系统且经济。神经性疼痛的行为评估对于揭示小直径感觉神经的功能至关重要。在实验啮齿动物中评估机械阈值特别具有挑战性，本方案描述了一种适合此类啮齿动物评估的定制金属网。总之，RTX神经病变是一种新的且易于建立的实验模型，可用于评估治疗药物开发中神经性疼痛的分子意义和干预措施。
