Evaluation of sensory neuron plasticity following neonatal maternal separation

描述（由申请人提供）：内脏疼痛目前是美国患者就诊的主要原因，也是肠易激综合征（IBS）的主要衰弱方面。在儿童时期经历过不良事件（包括言语、身体和性虐待，以及后来受影响器官的疾病）的个体中，肠易激综合征的发病率更高。亲本K01提案的重点是一种新的新生儿结肠刺激（NCI）小鼠模型，该模型在没有组织病理学的情况下再现了肠易激综合征-结肠超敏反应的主要特征。主要假设是，脏器感觉系统的早期损伤在结肠、周围神经和DRG水平上产生了持久的伤害性加工改变。目前的提案旨在通过在正在进行的NCI研究中增加新生儿应激模型（新生儿产妇分离[NMS]）来扩大最初K01奖的重点，以期更全面地了解发育中的脏器感觉系统的脆弱性以及新生儿关键期扰动可能导致的长期有害影响。本应用程序中的实验有两个特定目的，旨在从功能上验证NMS在小鼠中产生终身内脏超敏反应的假设，即由于特定膜受体表达的变化，NMS通过提高周围感觉神经元的兴奋性，从而加剧对实验性结肠炎的反应。SA1：为了验证NMS在小鼠中产生长期结肠超敏反应和增加支配结肠远端周围神经的兴奋性/受体变化的假设。为了验证这一假设，a)将评估成年NMS小鼠对结肠膨胀的内脏运动反应（VMR）， b)将对结肠DRG神经元进行单细胞RT-PCR和钙成像，以确定TRPV1和TRPA1这两个调节内脏敏感性的通道的表达、功能和相互作用的变化。SA2：验证NMS增强实验性结肠炎易感性，并加剧炎症诱导的远端结肠及相关外周感觉神经元变化的假设。为了验证这一假设，将给药三硝基苯磺酸（TNBS）诱导结肠炎，a)炎症程度将通过评估髓过氧化物酶（MPO）活性和生长因子产生的增加来确定，b) VMR、单细胞RT-PCR和钙成像将重复进行，以确定结肠炎如何影响NMS小鼠已经改变的伤害感受加工。将NMS模型添加到正在进行的NCI研究中，可以对两种不同的新生儿侮辱模型进行比较，这些模型具有相同的行为结果，提高了内脏敏感性。利用这些模型所取得的发现将允许识别内脏过敏的共同和/或不同途径，这可能作为治疗肠易激综合征和其他功能性肠疾病的潜在治疗靶点。

DESCRIPTION (provided by applicant): Visceral pain is currently the leading cause for patient visits in the U.S and is the main debilitating aspect in irritable bowel syndrome (IBS). The incidence of IBS is higher among individuals who experienced adverse events during childhood, including verbal, physical and sexual abuse, as well as disease of later affected organs. The focus of the parent K01 proposal was a novel mouse model of neonatal colon irritation (NCI) that reproduced the principal characteristic of IBS - colon hypersensitivity in the absence of histopathology. The main hypothesis was that early insult in the viscerosensory system produces a long-lasting alteration in nociceptive processing at the level of the colon, peripheral nerve and DRG. The current proposal aims to broaden the focus of the original K01 award, by adding a model of neonatal stress (neonatal maternal separation [NMS]) to the ongoing studies of NCI in an attempt to gain a more complete understanding of the vulnerabilities of the developing viscerosensory system and the long-lasting deleterious effects that can result from perturbations during the neonatal critical period. The experiments in this application are outlined in two specific aims and are designed to functionally test the hypothesis that NMS produces lifelong visceral hypersensitivity in mice through heightened excitability of peripheral sensory neurons due to changes in expression of specific membrane receptors, which exacerbate the response to experimental colitis. SA1: To test the hypothesis that NMS in mice produces long-lasting colon hypersensitivity and increased excitability/receptor changes in peripheral nerves innervating the distal colon. To test this hypothesis, a) the visceromotor response (VMR) to colorectal distension will be evaluated in adult NMS mice and b) single cell RT-PCR and calcium imaging will be performed on colon DRG neurons to determine changes in expression, function and interactions between TRPV1 and TRPA1, two channels proposed to regulate visceral sensitivity. SA2: To test the hypothesis that NMS enhances susceptibility to experimental colitis and exacerbates inflammation-induced changes in the distal colon and associated peripheral sensory neurons. To test this hypothesis, trinitrobenzene sulfonic acid (TNBS) will be administered to induce colitis and a) the extent of inflammation will be determined by assessing increases in myeloperoxidase (MPO) activity and growth factor production, and b) VMR, single cell RT-PCR and calcium imaging will be repeated to determine how colitis affects the already altered nociceptive processing in the NMS mice. The addition of the NMS model to ongoing studies of NCI allows for comparisons to be made between two different models of neonatal insult with the same behavioral outcome, heightened visceral sensitivity. Discoveries made using these models will allow for the identification of common and/or divergent pathways that underlie visceral hypersensitivity, which could serve as potential therapeutic targets for treatment of IBS and other functional bowel disorders. PUBLIC HEALTH RELEVANCE: The focus of my research is on how adverse events during neonatal development can permanently alter pain processing in adulthood. As neonatal care improves, early gestational-age infant survival rates are increasing. However, these premature infants can spend weeks or months in the neonatal care unit where they are subjected to adverse conditions, both in terms of stress from maternal separation and from the multiple painful interventions required on a daily basis. The experiments included in this proposal will address neonatal stress- related outcomes in visceral pain processing, in terms of colon sensitivity, the expression of pain-related molecules on neurons innervating the colon and susceptibility to future colon insult (inflammation).