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.
We propose to use a systematic approach for identifying the immune protein mediators that act on nociceptors to produce inflammatory pain. Discovering exactly how the immune system operates on the nervous system to produce pain is likely to reveal targets suitable for the generation of novel analgesics.
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