Characterization and Plasticity of Visceral Nociceptors

描述(申请人提供)：慢性内脏高敏感性是一些衰弱的人类综合征的共同特征，通常发生在炎症之后。这种超敏反应被认为是由于炎症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发展起来的。在前一个资金阶段，我们发现内脏器官(结肠、膀胱、胰腺和胃)密集地受到表达TRPV1和/或TRPA1的传入神经的支配，这两个通道已被证明是发展炎性痛觉过敏所必需的。我们还发现，GDNF家族的所有成员都可以像NGF一样敏化初级感觉神经元，并且这些生长因子在较低剂量下比NGF产生更大的增强作用。为这一应用产生的初步数据显示，皮肤或内脏结构的炎症导致GDNF家族成员的mRNA显着增加。使用我们的体外生理准备，我们发现结肠至少由两个功能离散的神经元群体支配，TRPV1仅在一个先前被确定为机械敏感、高阈值、伤害性感受器的群体中表达。因此，这些研究的一般假设是，内脏痛觉过敏是由内脏伤害性感受器中表达TRPV1/TRPA1的群体中选择性增加的敏感性启动的，这至少部分是由炎症组织中生长因子的变化调节的。我们的一般假设将在三个具体目标中进行检验：SA1：检验以下假设：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敏化已识别的、分离的、内脏传入。实时定量聚合酶链式反应和Western分析将用于证实炎性损伤后结肠和膀胱中生长因子表达的变化。脊髓感觉神经节将检测TRPV1、TRPA1、TrkA、ret和GFR11-3的变化。钙成像将被用来确定GDNF家族成员如何影响从NAOVE小鼠和器官发炎的小鼠分离的神经元中识别的、分离的结肠和膀胱传入的反应特性。SA2：测试新生儿或成人炎症导致超敏反应的假设，特别是在TRPV1阳性的传入细胞。我们已经开发了一种新的体外制剂，可以从完整的内脏传入细胞内记录。我们将使用这个范例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导致另一个器官的超敏反应，以及新生儿损伤是否会导致成年神经元的长期变化。SA3：验证体内内脏高敏感性诱导需要青蒿素反应性传入的假设。我们实验室最令人兴奋的发现之一是，有大量的内脏伤害性感受器表达GFR13、TrkA、TRPV1和TRPA1的组合。我们将使用一种专门针对这些神经元的细胞毒素来确定它是否可以用来消融这些神经元，以防止或逆转慢性内脏疼痛。与公共卫生相关：在美国，腹部、胸部和盆腔器官疼痛是患者去医生办公室就诊的首要原因。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特定类型的神经元是否对持续的痛感负责，以及是否可以在不影响正常器官功能的情况下对这些感觉进行消融。

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.