Characterization and Plasticity of Visceral Nociceptors

描述（由申请人提供）：慢性内脏高敏感性是许多使人衰弱的综合征的共同特征，通常发生在炎症之后。这种超敏反应被认为是由于发炎组织和支配这些结构的初级感觉神经元之间的相互作用而产生的。在之前的资助期间，我们发现内脏器官（结肠，膀胱，胰腺和胃）受到表达TRPV 1和/或TRPA 1的传入神经的密集支配，这两个通道已被证明是炎症性痛觉过敏发展所必需的。我们还发现，GDNF家族的所有成员的生长因子，如神经生长因子，敏感的初级感觉神经元，这些生长因子产生更大的增效作用，在较低的剂量比神经生长因子。本申请产生的初步数据显示，皮肤或内脏结构的炎症诱导GDNF家族成员mRNA的显著增加。使用我们的离体生理制剂，我们发现结肠受至少两个功能离散的神经元群体的神经支配，并且TRPV 1仅在先前被鉴定为机械敏感的高阈值伤害感受器的群体中表达。因此，所提出的研究的一般假设是内脏痛觉过敏是由TRPV 1/TRPA 1表达的内脏伤害感受器群体中选择性增加的敏感性引发的，并且这至少部分地由发炎组织中生长因子的变化调节。我们的一般假设将在三个具体目标中进行测试：SA 1：测试结肠和膀胱的超敏反应伴随着GDNF家族生长因子的上调以及这些生长因子可以使识别的、解离的内脏传入敏感的假设。实时PCR和Western分析将用于确认炎症损伤后结肠和膀胱中生长因子表达的变化。将测定脊髓感觉神经节中TRPV 1、TRPA 1、trkA、ret和GFR 11 -3的变化。钙成像将用于确定GDNF家族成员如何影响从naove小鼠和具有发炎器官的小鼠分离的神经元中鉴定的、解离的结肠和膀胱传入的响应特性。SA 2：检验新生儿或成人炎症在TRPV 1阳性传入中特异性产生超敏反应的假设。我们已经开发了一种新的离体制备，允许从完整的内脏传入的细胞内记录。我们将使用这种范式来研究幼稚和发炎的结肠和膀胱传入。我们将确定一个器官的炎症是否会在另一个器官产生超敏反应，以及新生儿损伤是否会在成年神经元中产生长期变化。SA 3：检验artemin反应性传入是体内内脏高敏感性诱导所需的假设。我们实验室最令人兴奋的发现之一是，有大量内脏伤害感受器表达GFR 13，trkA，TRPV 1和TRPA 1的组合。我们将使用一种专门针对这些神经元的细胞毒素，以确定它是否可以用来消融这些神经元，以预防或逆转慢性内脏疼痛。公共卫生相关性：在美国，腹部、胸部和盆腔器官疼痛是患者就诊的头号原因。这些器官由不同类型的感觉神经元支配。这些研究的目的是确定是否有一种特定类型的神经元负责持续的疼痛感觉，以及是否可以在不影响正常器官功能的情况下消融这些神经元。

DESCRIPTION (provided by applicant): Chronic visceral hypersensitivity is a common feature of a number of debilitating human syndromes, often occurring following inflammation. This hypersensitivity has been proposed to develop due to interactions between inflamed tissues and the primary sensory neurons that innervate these structures. In the previous funding period we found that visceral organs (colon, bladder, pancreas and stomach) are densely innervated by afferents expressing TRPV1 and/or TRPA1, two channels that have been shown to be required for development of inflammatory hyperalgesia. We also found that all members of the GDNF family of growth factors could, like NGF, sensitize primary sensory neurons and that these growth factors produced greater potentiation at lower doses than NGF. Preliminary data generated for this application show that inflammation of cutaneous or visceral structures induces significant increases in mRNA for GDNF family members. Using our ex vivo physiological preparation we found that the colon is innervated by at least two functionally discrete populations of neurons and that TRPV1 was exclusively expressed in a population that had previously been identified as mechanically sensitive, high threshold, nociceptors. Thus, the general hypothesis of the proposed studies is that visceral hyperalgesia is initiated by increased sensitivity selectively in the TRPV1/TRPA1- expressing population of visceral nociceptors and that this is regulated, at least in part, by changes in growth factors in the inflamed tissues. Our general hypothesis will be tested in three Specific Aims: SA1: Test the hypothesis that hypersensitivity of colon and bladder are accompanied by upregulation of the GDNF family of growth factors and that these growth factors can sensitize identified, dissociated, visceral afferents. Real-time PCR and Western analysis will be used to confirm changes in growth factor expression in colon and bladder following inflammatory insult. Spinal sensory ganglia will be assayed for changes in TRPV1, TRPA1, trkA, ret and GFR11-3. Calcium imaging will be used to determine how GDNF family members affect response properties of identified, dissociated colonic and bladder afferents in neurons isolated from naove mice and mice with inflamed organs. SA2: Test the hypothesis that neonatal or adult inflammation produces hypersensitivity specifically in TRPV1-positive afferents. We have developed a novel ex vivo preparation that allows intracellular recordings from intact visceral afferents. We will use this paradigm to study naive and inflamed colon and bladder afferents. We will determine if inflammation of one organ produces hypersensitivity in another, and whether neonatal injury produces long-term changes in adult neurons. SA3: Test the hypothesis that artemin responsive afferents are required for induction of visceral hypersensitivity in vivo. One of the most exciting findings from our lab is that there is a significant population of visceral nociceptors that express a combination of GFR13, trkA, TRPV1 and TRPA1. We will use a cell toxin that specifically targets these neurons to determine if it can be used to ablate these neurons to prevent or reverse chronic visceral pain. PUBLIC HEALTH RELEVANCE: Abdominal, thoracic and pelvic organ pain is the number one reason for patient visits to doctor's offices in the US. These organs are innervated by different types of sensory neurons. The goal of these studies is to determine if a specific type of neuron is responsible for persistent pain sensations and whether these can be ablated without affecting normal organ function.