Regulation of spinal NK1R expression by chronic stress

描述(由申请人提供)：越来越多的证据支持持续应激与肠易激综合征症状的开始或加重之间的相关性。一些研究表明，强烈的应激与内脏敏感性的持续改变之间存在相关性。更好地了解应激可导致持续性内脏痛觉过敏的机制，可能对功能性肠病的治疗方法具有重要意义。我们先前的研究表明，慢性心理应激导致内脏伤害性感觉持续增加，涉及脊髓中NK1受体(NK1R)的激活。目前的建议是基于一个普遍的假设，即在该模型中观察到的NK1Rs的脊髓上调是由外周免疫激活和通过初级传入纤维、脊髓小胶质细胞和脊髓下行通路向脊髓背角(DH)神经元发出的信号增加的相互作用所介导的。我们将重点介绍初级传入纤维来源的信号分子在调节脊髓背根神经细胞NK1R基因表达中的作用。我们将使用实时定量聚合酶链式反应、免疫组织化学、凝胶迁移率改变分析、Western印迹和体内药理学方法相结合的方法来确定参与这一调控的介质。在目标1中，我们将评估应激诱导的结肠免疫激活、初级传入纤维刺激以及随后在DH神经元中NK1R表达增加之间的时程相关性。在目标2中，我们将研究应激诱导的脊髓NK1R上调与同一神经元中转录因子CREB和NFkappaB激活之间的潜在相关性。在目标3中，我们将通过药物治疗，评估初级传入神经元在应激诱导的CREB和NFkappaB激活以及NK1R表达增加中的功能贡献。CGRP和脊髓肿瘤坏死因子α在这一调节作用中的具体作用将被评估。这一建议有望为应激影响内脏伤害性感受的机制提供新的见解。

DESCRIPTION (provided by applicant): Converging evidence supports a correlation between sustained stress and the onset or exacerbation of symptoms for Irritable Bowel Syndrome. Some studies indicate a correlation between intense stress and persistent alteration of visceral sensitivity. A better understanding of the mechanisms by which stress can lead to sustained visceral hyperalgesia may have important implications for therapeutic approach of functional bowel disorders. We previously showed that chronic psychological stress in rat leads to sustained increase of visceral nociception that involves NK1 receptors (NK1R) activation in the spinal cord. The current proposal is based on the general hypothesis that spinal up-regulation of NK1Rs observed in this model is mediated by the interplay of peripheral immune activation and increased signaling to spinal dorsal horn (DH) neurons via primary afferent fibers, spinal microglia and descending spinal pathways. We will focus on the role of signaling molecules derived from primary afferent fibers in the regulation of NK1R gene expression in spinal DH neurons. We will use a combination of quantitative real time PCR, immunohistochemistry, electrophoretic mobility shift assay, Western blot and in vivo pharmacological approach to identify the mediators involved in this regulation. In aim 1, we will evaluate the time course correlation between stress-induced colonic immune activation, primary afferent fibers stimulation and subsequent increase of NK1Rexpression in DH neurons. In aim 2, we will address the potential correlation between stress-induced up-regulation of spinal NK1R and activation of the transcription factors CREB and NFkappaB in the same neurons. In aim 3, we will evaluate, through pharmacological treatment, the functional contribution of primary afferent neurons in stress-induced activation of CREB and NFkappaB, and increased expression of NK1R. The specific role of CGRP and spinal TNFalpha in this modulatory effect will be assessed. This proposal is expected to provide new insights into the mechanisms by which stress affects the processing of visceral nociception.