项目来源

美国卫生和人类服务部基金(HHS)

项目主持人

OSHINSKY, MICHAEL L.

项目受资助机构

BOSTON CHILDREN'S HOSPITAL

立项年度

2017

立项时间

未公开

项目编号

5R37NS039518-16

研究期限

未知 / 未知

项目级别

国家级

受资助金额

717563.00美元

学科

Neurosciences; Pain Conditions - Chronic; Pain Research;

学科代码

未公开

基金类别

Non-SBIR/STTR RPGs

关键词

未公开

参与者

WOOLF, CLIFFORD J

参与机构

NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE

项目标书摘要：DESCRIPTION (provided by applicant): The overall question to be addressed by this proposal is which immune protein mediators produced during peripheral inflammation contributes to the generation of inflammatory pain by altering ion channel function in nociceptors. To achieve this objective three aims are proposed. In Aim 1 we will first phenotype two models of peripheral inflammation in the adult mouse; a pathogen-based model (intraplantar complete Freund's adjuvant) and a model of sterile tissue injury (surgical incision of the plantar surface of the paw) by examining the temporal pattern of recruitment of different specific immune cells in the two models, to be detected by flow cytometry and Q-RT-PCR, and the time course of the behavioral manifestations of mechanical and thermal hypersensitivity, to be detected by measures of evoked and spontaneous pain-like behavior. We will then determine the relative contribution of different immune cells to the two inflammatory model pain phenotypes using a mixture of mice where a genetic mutation has resulted in loss of a particular immune cell, and validated strategies for depleting specific immune cells. The second specific aim is to use unbiased proteomic and bioinformatics techniques to identify all the immune protein mediators expressed in the two models at the site of inflammation using tryptic digestion, isobaric TMT peptide tagging, fractionation and mass spectrometry. In addition, we will determine which receptors for the induced immune protein mediators are expressed by purified dorsal root ganglion (DRG) neuronal membrane preparations using flow cytometry and biotinylation to label membrane proteins, affinity purification, trypsinization and liquid chromatography/mass spectrometry to identify peptides. The final aim will be test which of the immune protein mediators produced by one or both of the inflammatory models and with a corresponding receptor expressed by DRG neurons activates the JAK- STAT, NFKB, ras/MAPK (ERK and p38), PLC-DAG, IP3 and PI3K signal transduction pathways in DRG neurons or causes calcium influx. We will them perform whole cell patch electrophysiology in current and voltage clamp mode to identify if a candidate immune protein mediator alters excitability, and if so by what changes in TRP and voltage-gated sodium ion channel threshold and kinetics.