Neurotrophic factor regulation of regenerated sensory neuron response properties

描述(申请人提供)：已知周围神经损伤可引起初级感觉神经元和背角神经元的各种分子和生理变化。靶向神经营养信号可能在损伤反应中发挥重要作用。这些研究的长期目标是确定传入靶区的变化如何改变神经损伤后背根神经节(DRG)神经元的反应特性。我们首先提出的假设是，随着时间的推移，神经挤压和隐神经传入的再生会导致各种神经营养因子及其受体在皮肤和背根节中的水平和定位发生变化。我们的初步证据表明，周围神经切断导致皮肤中几种神经营养因子水平的变化，包括NGF和青蒿素，这与DRG中TRPV1等受体的增加有关。反过来，这可能支持我们最近的发现，神经损伤导致含有TRPV1的C纤维神经元的百分比发生变化，这些神经元在再生后只对热(CH)做出反应。例如，高水平的NGF在体外调节分离/切断的DRG神经元中TRPV1的表达，而皮肤中青蒿素的过表达在体内增强了DRG中TRPV1mRNA的表达。鉴于TRPV1、NGF受体TrkA和青蒿素受体GFRaS在背根节内的免疫染色广泛重叠，并且各自与疼痛反应有关，我们建议测试GFRaS和/或TrkA介导的神经营养因子信号是否调节损伤诱导的CH神经元的变化。我们将能够用免疫细胞化学的方法将已知的再生感觉神经元对神经营养因子信号转导变化的反应特性关联起来，并使用RT-PCR和Western blotts在皮肤再支配前后进行相关，我们将能够使用体内siRNA介导的GFRaS和/或TrkA被击倒后的体外皮肤-神经-DRG-脊髓制备方法，从功能上测试两种特定途径在建立损伤后CH神经元电生理反应特性中的作用。这些研究将使我们进一步了解皮肤中过量的生长因子信号对受损神经元的功能影响，以及这些变化与神经损伤引起的痛觉过敏的关系。这项工作还可能引导我们找到更好的方法来治疗与伤害相关的慢性疼痛状态。

DESCRIPTION (provided by applicant): Peripheral nerve injury is known to induce a variety of molecular and physiological changes in primary sensory and dorsal horn neurons. Target derived neurotrophic signaling may play an important role in the injury response. The long-term goal of these studies is to determine how changes in the afferent target regions alter the response properties of dorsal root ganglion (DRG) neurons after nerve injury. We first propose to test the hypothesis that nerve crush and regeneration of saphenous afferents induces altered levels and localization of various neurotrophic factors and their receptors in the skin and DRGs, respectively, over time. Our preliminary evidence suggests that peripheral axotomy induces changes in several neurotrophic factor levels, including NGF and artemin, in the skin, and this correlates to increases in receptors like TRPV1 in the DRGs. In turn, this may underlie our recent finding that nerve injury results in changes in the percentage of TRPV1 containing C-fiber neurons that only respond to heat (CH) after regeneration. For example, high levels of NGF regulate the expression of TRPV1 in dissociated/ axotomized DRG neurons in vitro, and overexpression of artemin in the skin enhances TRPV1 mRNA in the DRGs in vivo. Given that TRPV1, NGF receptor trkA and artemin receptor GFRaS immunostaining overlap extensively in the DRG and each have been linked to pain responses, we then propose to test if GFRaS and/or trkA mediated neurotrophic factor signaling regulates the injury induced changes in the CH neurons. We will be able to correlate the known response properties of regenerated sensory neurons to changes in neurotrophic factor signaling immunocytochemically, and using RT-PCR and western blots before and after reinnervation of the skin, and we will be able to functionally test the role of two particular pathways in establishing the changes in electrophysiological response properties of CH neurons after injury using an ex vivo skin-nerve-DRG-spinal cord preparation after in vivo siRNA mediated knockdown of GFRaS and/or trkA. These studies will allow us to further understand the functional implications of excess growth factor signaling in the skin on injured neurons and how these changes relate to nerve injury induced hyperalgesia. This work may also lead us to better ways to treat injury related chronic pain states.