Regulation of Nerve Injury-induced Gene Expression in Neuropathic Pain

摘要 手术或创伤后神经损伤引起的神经性疼痛具有显著的经济负担， 影响生活质量。尽管鉴定出了许多与肿瘤发生有关的分子靶点， 神经性疼痛、手术后和创伤后神经性疼痛通常是难治的。不像其他 神经性疼痛病症，如糖尿病性神经病变，在手术期间发生神经损伤的时间，或 创伤后的症状是明确的。因此，无论是在战争期间， 围手术期或创伤后不久，可以防止持续性疼痛的发展。事实上，当地 已经开发了基于麻醉剂的超前镇痛技术。然而，尽管这些 虽然这些技术在管理术后即刻疼痛方面非常好，但它们只能提供有限的长期益处。 在一个神经病理性疼痛的小鼠模型中，坐骨神经被手术损伤，我们最近证明， 损伤诱导的细胞因子集落刺激因子的从头合成和释放的关键贡献 1（CSF 1，也称为巨噬细胞集落刺激因子），来自背根神经节（DRG）感觉 神经元我们的研究表明，CSF 1是神经损伤诱导激活的必要和充分条件 脊髓小胶质细胞，CNS的组织巨噬细胞，以及损伤后机械超敏反应/ 神经性疼痛行为在这里，在具体目标1和2中，我们提出了实验，首先将定义 在受损的DRG神经元中触发CSF 1诱导的转录机制。我们初步分析了 来自B细胞淋巴瘤的圣克鲁斯基因组浏览器ChIP-Seq数据揭示了19种转录因子， CSF 1启动子。在这19个基因中，使用定量RT-PCR，我们确定c-Myc也是 在神经损伤后的DRG中诱导。有趣的是，c-Myc的诱导发生在神经损伤后的几个小时内， 在诱导CSF 1之前。免疫组化证实了c-Myc的诱导及其共表达 与CSF 1在DRG神经元中的表达。值得注意的是，c-Myc是癌症中基因表达的通用放大器， 胚胎发育，但其在成人非增殖细胞中的功能在很大程度上是未知的。更重要的是， 在损伤前对坐骨神经进行预先局部麻醉治疗并不能阻止c-Myc的诱导 在DRG神经元中，表明其诱导不是活动依赖性的。基于这些发现，我们 假设c-Myc是损伤的DRG神经元中CSF 1基因诱导的主要贡献者， 靶向c-Myc可能阻止CSF 1基因诱导，从而阻止神经性疼痛的发展。 最后，由于CSF 1的表达在神经损伤后持续数周，我们还将检验以下假设： CSF 1不仅有助于神经性疼痛表型的起始，而且有助于其持续性。因此 在具体目标3中，我们将在损伤发生后从DRG神经元中删除或阻断CSF 1。最后，鉴于最近 在雄性和雌性小鼠中小胶质细胞对神经性疼痛表型的贡献的差异的证据， 我们的研究还将评估这些机制在雄性和雌性小鼠中的作用。

Abstract Nerve injury-induced neuropathic pain following surgery or trauma has a significant economic burden that impairs quality of life. Despite the identification of a host of molecular targets implicated in the development of neuropathic pain, post-surgical and post-trauma neuropathic pains are often intractable. Unlike other neuropathic pain conditions, such as diabetic neuropathy, the time that nerve injury occurs during surgery or following trauma is well defined. As a result, it is possible that a pre-emptive intervention, either during the perioperative period or shortly after the trauma, could prevent the development of persistent pain. Indeed, local anesthetic based pre-emptive analgesia techniques have been developed. However, although these techniques are very good in managing immediate post-surgical pain, they only offer limited long-term benefit. In a mouse model of neuropathic pain in which the sciatic nerve is surgically injured, we recently demonstrated a critical contribution of injury-induced de novo synthesis and release of the cytokine, colony stimulating factor 1 (CSF1, also known as macrophage colony stimulating factor) from dorsal root ganglion (DRG) sensory neurons. Our studies established that CSF1 is both necessary and sufficient for nerve injury-induced activation of spinal cord microglia, the tissue macrophages of the CNS, and for post-injury mechanical hypersensitivity/ neuropathic pain behavior. Here, in Specific Aims 1 and 2, we propose experiments that will first define the transcriptional mechanism that triggers CSF1 induction in injured DRG neurons. Our preliminary analysis of the Santa Cruz Genome Browser ChIP-Seq data from B-cell lymphoma revealed 19 transcription factors that bind to CSF1 promoter. Among these 19 genes, using quantitative RT-PCR we determined that c-Myc is also induced in DRG after nerve injury. Interestingly, the c-Myc induction occurred within hours of nerve injury, well before the induction of CSF1. Immunohistochemistry confirmed the induction of c-Myc and its co-expression with CSF1 in DRG neurons. Of note, c-Myc is a universal amplifier of gene expression in cancer and embryonic development, but its function in adult non-proliferating cells is largely unknown. More importantly, pre-emptive local anesthetic treatment of sciatic nerve prior to the injury did not prevent the induction of c-Myc in DRG neurons, indicating that its induction is not activity dependent. Based on these findings, we hypothesize that c-Myc is a major contributor to CSF1 gene induction in the injured DRG neurons, and that targeting c-Myc might prevent the CSF1 gene induction and thus prevent neuropathic pain development. Finally, as the expression of CSF1 persists for weeks after nerve injury, we will also test the hypothesis that CSF1 not only contributes to the initiation of the neuropathic pain phenotype, but also to its persistence. Thus in Specific Aim 3, we will delete or block CSF1 from DRG neurons after injury occurs. Finally, given recent evidence for differences in microglial contribution to the neuropathic pain phenotype in male and female mice, our studies will also assess the contribution of these mechanisms both in male and female mice.