The Pain Neural Transcriptome

概述：该项目的目标是了解(a)在转录组水平上痛觉神经元和受损外周组织的分子生物学，(b)急性和慢性疼痛模型中转录组参数的调节，以及(c)使用来自人类患者、器官供体或死后病例的组织扩展和验证动物模型。除了这些问题之外，我们正在开发的经验框架为我们的转化项目（CL090033-07疼痛和疼痛控制的综合和分子研究和CL090034-07：疼痛和免疫过程的机制）和一些树脂化毒素（RTX）的人体临床试验提供了基础知识。为了实现这些目标，我们建立了研究协议、硬件和软件基础设施、分析管道，以及数据分析、实验模型和人类协议的协作安排。我们利用细胞生物学和体内行为测量，结合深度RNA-Seq，多重荧光原位杂交和单核测序来探索多种物种的基本伤害机制。由此产生的疼痛转录组包括炎症、手术切口和其他伤害感觉模型中的初级传入和脊髓神经元以及外周组织。所有这些都在一系列出版物中进行了深入分析。

Overview: The objectives of this project are to understand (a) the molecular biology of pain-sensing neurons and damaged peripheral tissues at the transcriptome level, (b) the modulation of transcriptomic parameters in acute and chronic pain models and (c) to extend and verify the animal models using tissue from human patients, organ donors or post-mortem cases. Beyond these questions, the empirical framework we are developing forms the foundational knowledge for our translational projects (CL090033-07 Integrative and Molecular Studies of Pain and Pain Control and CL090034-07: Mechanisms of Pain and Immune Processes) and several human clinical trials of resiniferatoxin (RTX). To meet these objectives, we established research protocols, hardware and software infrastructure, analytical pipelines, and collaborative arrangements for data analysis, experimental models, and human protocols. We utilize cell biological and in vivo behavioral measurements in combination with deep RNA-Seq, multiplex fluorescent in situ hybridization and single nucleus sequencing to probe basic nociceptive mechanisms in multiple species. The resulting pain transcriptome encompasses primary afferent and spinal cord neurons and peripheral tissues during inflammation, surgical incision, and other models of nociception. All have been intensively analyzed in a series of publications. Another objective is to extend our studies to include human nociceptive neurons. In this cycle we performed an extensive set of multiplex in situ hybridization studies of human dorsal root ganglion for transcripts in pain sensing and non-nociceptive neurons. What emerged was a new synthesis of pain neurons in which we can divide the population of nociceptors into those that contain the mu opioid receptor and those that do not. The data suggest that the Mu+ nociceptors transmit noxious sensations following tissue damage and that this pain can be controlled by opioid agonists. Pain sensations transmitted by the Mu- nociceptors is not responsive to are not responsive to opioids and we hypothesize that this population is active during neuropathic nerve injury types of pain. Pain in neuropathic disorders is notoriously insensitive to control by opioid agonists. This is a remarkable level of new insight into the molecular repertoire of pain control and which neuronal population to target with a specific analgesic for a specific pain indication. Thus, extensive foundational data acquired this cycle greatly facilitates formation of incisive hypotheses regarding pain physiology and the choice and design of effective interventions. The TRPV1 Transcriptome: One important focus is the subpopulation of DRG neurons expressing the thermo-, chemo-, pH-, and lipid-responsive ion channel called TRPV1. This ion channel is also gated by capsaicin, the active ingredient in hot pepper. We have demonstrated that the potent capsaicin analog resiniferatoxin (RTX) can control cancer pain in canine and human patients by inactivating TRPV1 axons or nerve terminals, indicating a crucial role for TRPV1+ neurons in transmission of clinically relevant pain. We published several comprehensive transcriptomic profiles of these clinically important nociceptive neurons and extended the analysis to DRGs obtained at autopsy from two of our human cancer pain patients who had been treated with RTX. We are now extending this to a much broader number of human individuals via organ donor programs. The data from the clinical trial demonstrate that, with intrathecal administration, the centrally projecting axon is the most vulnerable neuronal component, whereas the cell bodies in DRG are comparatively resistant to RTX. This important mechanistic insight is being used, in part, to fine-tune drug administration in our human clinical trial (low volume, slow rate of infusion). The strong efficacy of RTX implicates TRPV1+ DRG neurons as a crucial population of nociceptive neurons for transmitting human clinical pain. One objective is to identify new molecular routes within the two TRPV1 nociceptor populations to control various types of pain. This rational, data-based targeting is a much more informed route to personalized pain control than currently available. The Spinal Pain Transcriptome: In the present cycle we continued to delve into the spinal dorsal horn nociceptive processing systems. Multiplex in situ hybridization is a key tool for revealing the complex network of neurons and ligands in the dorsal horn with different sets of combinatorial expression in neurons in the superficial laminae. We are exploring the meaning of the observations with additional anatomical probes. Peripheral Inflammatory and Surgical Incision Transcriptomes: Our objective is to understand peripheral processes following tissue damage at a molecular level. In the last cycle we conducted RNA seq on peripheral tissues in the rat after experimental inflammation. We were able to delineate the tissue damage secretome through identification of multiple, temporally distinct gene induction profiles and cellular recruitment responses related to resident cells and infiltrating leukocytes. We are now nearing completion of a human study to verify and extend the rat results. We worked with a team of surgeons from the NCI to obtain skin from the edge of the incision site at various intervals out to wound closure (between 6 and 8 hours) and have performed RNA-Seq, in situ hybridization and LC-MS-MS lipidomic profiling on the samples. Interesting the human time course is short compared to the 12-day rat incision study, and we are revising the human protocol to obtain wound tissue at later time points. Nonetheless, the results provide a transcriptomic roadmap of objective biochemical readouts that may inform a patients pain and wound healing status, may guide therapeutic interventions, and forms a consistent basis for targeted interventions. Anesthesia Transcriptome: In this cycle we published our work on ketamine infusion and its acute and more prolonged actions on cortical, hippocampal and amygdala transcriptomes. We hypothesize that mechanistic insight into the acute effects and the recovery therefrom can be obtained by understanding molecular-level transcriptome changes.