The Pain Neural Transcriptome

疼痛神经转录组

• 批准号: 8336411
• 负责人: Michael J. Iadarola
• 金额: $ 75.22万
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8336411
• 关键词: Acute Pain; Address; Afferent Neurons; Analgesics; Anesthesiology; Animal Model; Autopsy; Axon; Behavior; Behavioral; Bioinformatics; Biological Models; C Fiber; Canis familiaris; Cells; Cerebral cortex; Characteristics; Complement; Computational Biology; Computer Workstations; Control Animal; Data; Data Set; Devices; Dissection; Doctor of Philosophy; Dorsal; Dose; Equipment; Esthesia; Evaluation; Excision; Face; Fiber; Fresh Tissue; Functional RNA; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic Crossing Over; Genomics; Goals; Head; Human; Human Resources; Hyperalgesia; Inflammation; Institutes; Interview; Intractable Pain; Knowledge; Laboratories; Left; Libraries; Lipids; Malignant Bone Neoplasm; Messenger RNA; Methods; MicroRNAs; Modeling; Molecular; Molecular Neurobiology; Monkeys; Motor Neurons; National Institute of Mental Health; Nerve; Neurons; Nociception; Opioid Receptor; Pain; Pain Disorder; Pain management; Pathway interactions; Pattern; Pennsylvania; Peripheral; Persistent pain; Persons; Population; Process; Protocols documentation; Pyramidal Tracts; RNA; RNA Sequences; RNA analysis; Rattus; Relative (related person); Research; Research Infrastructure; Research Methodology; Research Project Grants; Resiniferatoxin; Reverse Transcriptase Polymerase Chain Reaction; Ribosomal RNA; Role; Rome; Sampling; Scientist; Sensory; Sensory Ganglia; Sequence Alignment; Sequence Analysis; Services; Shapes; Side; Slice; Specific qualifier value; Speed; Spinal Cord; Spinal Ganglia; Staging; Stimulus; Structure; Structure of trigeminal ganglion; Synapses; System; TRPV1 gene; Testing; Time; Tissues; Training; Transcript; Trigeminal System; Universities; Visit; Work; base; cDNA Library Construction; chronic pain; cohort; comparative; dorsal column; dorsal horn; experience; human tissue; improved; injured; killings; meetings; nerve injury; nonhuman primate; osteosarcoma; patient population; programs; receptor; relating to nervous system; spinal nerve posterior root; tissue processing; transmission process; white matter

Summary: The purpose of this research project is to understand the molecular basis of pain sensation and transmission. The goals are to understand human chronic pain disorders, mechanisms of acute pain and to discover new ways to treat pain. The scope comprises comparative transcriptome analysis of nociceptive (pain-sensing) circuits using targeted model systems and, where possible, equivalent studies in human tissue. We aim the identify transcripts that are critical for pain transduction, that are conserved and convergent across species, that display characteristic expression in pain processing tissues relative to other tissues, clear differences in absolute expression levels or state dependent changes indicative of a role in pathological pain states. Several questions we address are: How does the nociceptive circuit meet the challenge of chronic pain? What genes are important to nociceptive processing in humans? What are the effects of analgesic drugs or other manipulations of the pain circuit on gene expression in persistent pain states. These systematic studies will help us understand and manipulate the pain system in a more informed and effective fashion. This project is in the beginning stages. At this point we have established several research methods and protocols, built the supporting infrastructure in terms of equipment, space in the lab and collaborative arrangements, and have hired and begun to train a team of scientists and support personnel to conduct the research project. In terms of infrastructure and personnel I have interviewed and hired a person with a Ph.D. in Bioinformatics and Computational Biology who is skilled at analysis of RNA data from next gen sequencing (RNA-seq) platforms. I have hired a post-BAC IRTA who has experience in RNA extraction, cDNA library construction and RT-PCR analysis and I have a visiting fellow from the Anesthesiology Department of the Catholic University of Rome (supported by his university) working on the project as well. The laboratory has obtained two dedicated computer workstations for high-speed sequence analysis and alignment of the transcriptome libraries with genomic structure. We have also upgraded our Bioanalyzer for RNA quality assessment and our 96 well real-time PCR device for independent verification of alterations seen with the RNA-seq methods and obtained two high-speed PCR devices. We shall use the sequencing services from the National Institutes Sequencing Center (NISC). At NISC we will use the Illumina High-Seq device, generally in multiplex mode to analyze 6 to 8 independent samples/lane. We have met with NISC personnel and will begin evaluation of mRNA library construction using either polyA+ selection or ribosomal RNA depletion to enrich for mRNA. We will also be analyzing microRNAs and long non-coding RNAs during the course of this study. We shall work with the University of Pennsylvania to obtain canine tissue for the RNA studies. These tissues will come from controls and animals with osteosarcoma euthanized because of inadequate pain control or treated with resiniferatoxin. Tissues are obtained at autopsy. The data generated will allow us to test for genes activated by nociceptive input from naturally occurring bone cancer and modulated by treatment. The data will also be used for comparison to parallel studies in rat monkey and human (although the exact models will be different). The transcriptome project will systematically investigate the first three steps in the pain pathway beginning with injured peripheral tissue, the dorsal root ganglion and the dorsal (sensory) spinal cord. The equivalent structures for the face and head are the trigeminal ganglia and the medulla. For human studies, it is relatively straightforward to obtain the trigeminal ganglia at autopsy and we have been doing this for nearly two years in conjunction with Dr. Joel Kleinman of NIMH. However, the second order synapse in the medulla is a bit more challenging. The trigeminal nociceptive primaryy sensory neurons synapse with second order neurons in the medulla, which is located at the top of the spinal cord. Thus, by dissecting this region we obtain both neural steps in the pain pathway. For the purpose of dissecting the pain sensing medullary dorsal horn from the surrounding tissue, we have conducted an in-depth neuroanatomical analysis of the human medulla. This region is quite different from the functionally equivalent regions of spinal cord due to the crossing over of the pyramidal tracts, which contain the axons of motor neurons from cerebral cortex. Our studies revealed that the dorsal horn is compressed into a tight circular shape by the fibers crossing over. This arrangement was very favorable for dissection of the medullary dorsal horn from fresh tissue because, due to its compactness, it can be removed in its entirety from a 3 mm slice of medulla. We take several such slices and dissect the dorsal horn from both left and right sides as well as white matter from the dorsal columns and pyramidal tracts for comparison. These same methods will be applied to rat, dog and monkey. Using the Bioanalyzer, we have analyzed total RNA extracted from the human medulla. Analysis yielded moderately good RNA integrity numbers and we are trying to improve the results using a method to remove lipids first. We have begun the initial study on inflammation induced genes in rats. This will include a careful analysis of behavioral hyperalgesia in each cohort using our A-delta and C-fiber selective stimuli and a systematic examination of the time course of gene regulation as it compares to the behavior. Comparisons will be made to several nerve injury models since these are distinct from the inflammation. We are also working with another NIMH laboratory to examine the dorsal root, trigeminal ganglia and spinal cord/medulla from non-human primates and have filed and taken the appropriate biosafety registrations and training courses, respectively.

摘要：该研究项目的目的是了解疼痛感觉和传播的分子基础。目标是了解人类的慢性疼痛障碍，急性疼痛的机制，并发现治疗疼痛的新方法。该范围包括使用靶向模型系统以及在可能的人类组织中等效研究的伤害性（疼痛）电路的比较转录组分析。我们瞄准的是对疼痛转导至关重要的识别转录本，这些转录物相对于其他组织，它们在疼痛加工组织中表现出特征性的表达，绝对表达水平的明显差异或状态依赖性变化，表明了病理疼痛状态的作用。我们解决的几个问题是：伤害感受电路如何应对慢性疼痛的挑战？哪些基因对于人类的伤害感受处理很重要？镇痛药或其他对疼痛回路的操纵对持续性疼痛状态中基因表达的影响。这些系统的研究将有助于我们以更加明智和有效的方式理解和操纵疼痛系统。 该项目处于开始阶段。 在这一点上，我们已经建立了几种研究方法和协议，以设备，实验室的空间和协作安排来建立支持基础设施，并雇用并开始培训一组科学家和支持人员来进行研究项目。 在基础设施和人员方面，我已经采访并雇用了博士学位的人。在生物信息学和计算生物学中，他们擅长从下一个一代测序（RNA-SEQ）平台的RNA数据分析。 我雇用了一名BAC IRTA，他在RNA提取，cDNA图书馆建设和RT-PCR分析方面都有经验，我也有一位来自罗马天主教大学麻醉学系（由他的大学支持）的来访者。 该实验室获得了两个专用的计算机工作站，用于高速序列分析和与基因组结构的转录组库对齐。我们还升级了生物分析仪进行RNA质量评估和96个良好的实时PCR设备，以独立验证RNA-Seq方法看到的变化，并获得了两个高速PCR设备。 我们将使用美国国立机构测序中心（NISC）的测序服务。 在NISC，我们将使用Illumina High-Seq设备，通常以多重模式分析6至8个独立的样品/泳道。 我们已经与NISC人员会面，并将使用Polya+选择或核糖体RNA耗竭来开始评估mRNA文库的构建，以富集mRNA。 在本研究过程中，我们还将分析microRNA和长期非编码RNA。 我们将与宾夕法尼亚大学合作获得RNA研究的犬组织。 这些组织将来自对照和动物，因为疼痛控制不足或用树脂毒素治疗，对骨肉瘤的动物。尸检时获得组织。产生的数据将使我们能够测试来自天然发生的骨癌的伤害感受性输入并通过治疗调节的基因。数据还将用于将大鼠猴和人的平行研究进行比较（尽管确切的模型将不同）。 转录组项目将系统地研究疼痛途径的前三个步骤，从受伤的外围组织，背根神经节和背侧（感觉）脊髓开始。 面部和头部的等效结构是三叉神经节和髓质。 对于人类的研究，在尸检时获得三叉神经节是相对简单的，我们已经与NIMH的Joel Kleinman博士一起进行了将近两年。 但是，髓质中的二阶突触更具挑战性。 三叉神经感受性的原发性感觉神经元与位于脊髓顶部的髓质中的二阶神经元突触。因此，通过解剖该区域，我们在疼痛途径中获得两个神经步骤。为了从周围的组织中剖析疼痛传感髓质角，我们对人髓质进行了深入的神经解剖分析。 该区域与脊髓的功能等效区域大不相同，这是由于锥体区的横穿，这些区域包含来自大脑皮层的运动神经元的轴突。 我们的研究表明，背角通过越过的纤维被压缩成紧密的圆形形状。这种布置非常有利于从新鲜组织中解剖髓质的背角，因为由于其紧凑性，它可以从3毫米髓质切片中全部清除。 我们采取几个这样的切片，从左右两侧剖析背角，以及从背柱和锥形区的白质进行比较。 这些相同的方法将应用于老鼠，狗和猴子。 使用生物分析仪，我们分析了从人髓质中提取的总RNA。 分析产生了适度的RNA完整性数，我们正在尝试使用一种方法首先去除脂质的方法来改善结果。 我们已经开始对大鼠炎症诱导基因的初步研究。 这将包括使用我们的A-DELTA和C纤维选择性刺激对每个队列中的行为痛觉过敏的仔细分析，以及对与行为相比的基因调节时间过程的系统检查。将与几种神经损伤模型进行比较，因为这些模型与炎症不同。 我们还与另一个NIMH实验室合作，检查了非人类灵长类动物的背根，三叉神经节和脊髓/髓质，并分别提交并采取了适当的生物安全注册和培训课程。