Structural and functional mapping of visceral pain afferent neurocircuitries of the colorectum and bladder in preclinical models

摘要 肠易激综合征(IBS)和间质性膀胱炎/痛性膀胱综合征(IC/PBS)是常见的慢性疾病 分别影响结肠和膀胱的内脏疼痛障碍。这两种情况都会带来巨大的健康 和经济负担。值得注意的是，IBS和IC/PBS表现出相当大的重叠症状，而 个别患者可能同时患有这两种疾病。结肠和膀胱的交叉敏感和 共享脊椎神经。然而，尽管对它们的症状和作用的了解增加了 疼痛传入自身的病理生理，目前尚无有效的治疗方法。为数不多的药理药物 包括阿片类药物在内，都会产生严重的不良影响。目前开发有效疗法的障碍 对于IBS和IC/PBS，包括：a)对二分背侧的结构和功能了解不足 远端结肠和膀胱的根神经节(DRG)神经元。B)缺乏对性的充分调查 跨器官敏化的差异，尽管这两种情况在女性中的患病率高于 男性(女性与男性之比：IBS为2：1，IC/PBS为5-10：1)c)缺乏感觉神经传入知识 与人类有较高翻译相关性的物种的神经支配和跨器官敏化。我们 建议通过同时监测结肠和膀胱痛的两个回路来解决这些差距 临床前模型，并在3个具体目标下。1)：结肠和膀胱痛传入的结构映射 神经回路：二分和非二分神经元，它们的空间分布和连通性。2)： 急、慢性跨器官损伤模型中结肠和膀胱传入神经的功能特征 敏化和3)：定位结肠和膀胱疼痛的脊髓部位和跨器官敏化通过 神经调节。我们将使用急性和慢性跨器官致敏模型，最先进的高科技 分辨率成像、3D映射、膀胱和结肠感觉神经元的双重逆行追踪、清晰度、例如 二分神经元的活体DRG钙离子成像，体外膀胱和膀胱的电生理记录 结肠传入和经皮脊髓刺激(TSCS)方法。小鼠模型将解开 肠-膀胱和膀胱-肠急性痛的传入/神经元分类及其功能环路 和慢性疼痛交叉敏感症。猪的研究将提供关于结构图的新信息 结肠和膀胱的二分神经元及其联系以及痛觉的交叉敏感化 回应。它还将允许我们深入了解TSCS的使用，以绘制脊髓回路和回溯 结肠和膀胱痛环及其人类使用的翻译可行性。多学科结合 方法将填补目前关于结肠/膀胱二分DRG神经元的知识空白，空间 敏化后痛觉回路的分布和联系以及可塑性。我们的发现将 为了解结肠和膀胱神经回路奠定基础，这将是紧急情况下的关键 对于IBS和IC/PBS等慢性内脏疼痛综合征，需要药物和/或神经调节剂治疗。

ABSTRACT Irritable Bowel Syndrome (IBS) and interstitial cystitis/painful bladder syndrome (IC/PBS) are common chronic visceral pain disorders that affect the colon and bladder respectively. Both conditions pose significant health and financial burden. Notably, IBS and IC/PBS present with considerable overlapping symptoms, whilst individual patients can suffer from both disorders concurrently. The colon and bladder cross-sensitize and share spinal innervations. However, despite increased understanding of their symptomatology and the role of pain afferents in their pathophysiology, there are no effective therapies. The few pharmacological drugs including opioids have significant adverse effects. Current barriers to the development of effective therapies for IBS and IC/PBS include: a) Inadequate structural and functional knowledge on the dichotomizing dorsal root ganglia (DRG) neurons of the distal colon and bladder. b) The lack of adequate investigation on sex differences in cross-organ sensitization, despite the prevalence of both conditions being higher in women than in men (female to male ratio: 2:1 for IBS, 5-10:1 for IC/PBS) c) Lack of knowledge of the sensory afferent innervation and cross-organ sensitization in species with higher translational relevance to humans. We propose to address these gaps through concurrent monitoring of the colon and bladder pain circuitries in two pre-clinical models and under 3 specific aims. 1): Structural mapping of colon and bladder pain afferent neurocircuits: Dichotomizing and non-dichotomizing neurons, their spatial distribution and connectivity. 2): Functional characterization of colon and bladder afferents in acute and chronic models of cross-organ sensitization and 3): Map spinal sites of colon and bladder pain and cross-organ sensitization through neuromodulation. We will use acute and chronic cross-organ sensitization models, state-of-the-art high- resolution imaging, 3D mapping, dual retrograde tracing of bladder and colon sensory neurons, CLARITY, ex vivo DRG Ca2+ imaging of dichotomizing neurons, ex vivo electrophysiological recordings from bladder and colon afferents and transcutaneous spinal stimulation (TSCS) approaches. The murine model will unravel the classes of pain afferents/neurons and their functional circuits in bowel-to-bladder and bladder-to-bowel acute and chronic pain cross sensitization. The porcine studies will provide novel information on the structural map of the colon and bladder dichotomizing neurons and their connections as well as cross sensitization of pain responses. It will also allow us to gain insight on the use of TSCS to map spinal cord circuits and backtrack colon and bladder pain circuits and its translational feasibility for human use. The combined multidisciplinary approaches will fill the gaps in current knowledge on the colon/bladder dichotomizing DRG neurons, spatial distribution and connections as well as the plasticity in the pain circuitries post sensitization. Our findings will contribute to the foundation for understanding colon and bladder neurocircuitries that will be critical for urgently needed drugs and/or neuromodulator therapies for chronic visceral pain syndromes such as IBS and IC/PBS.