Postoperative pain is a major clinical concern, as it can delay patient recovery and increase probability ofchronic pain syndromes. More than half of patients report moderate to severe pain after surgery, thus, it isimportant to investigate the mechanisms that generate and maintain this condition. Plantar incision in the rathind paw is a model of postoperative pain resulting in spontaneous pain and hyperalgesia, or increased painin response to stimulation; symptoms that are observed in patients. Spinal glutamate receptor pharmacologyof this model is distinct. Spinally administered conventional antagonists to NMDA receptors have no effect onany aspect of incision-evoked pain in the rat. However, NR2B-NMDA and calcium-permeable AMPA/KAglutamate receptor subtypes, appear to mediate separate aspects of post-operative pain behavior, althoughthis is not certain. No studies have addressed the role of spinal signal transduction cascades occurringdownstream from calcium-permeable AMPA/KA and NR2B-NMDA receptor activation after incision.This application examines postoperative pain behaviors by targeting the facilitation of various elements ofthese cascades.
Aim 1 will measure increases in incision-evoked activation of protein kinase A (PKA),conventional protein kinase C (PKC) and calcium/calmodulin-dependent protein kinase II alpha (CaMKIIalpha) over a 5-day period. Administration of selective kinase antagonists will demonstrate if these changesare necessary for the manifestation of pain behavior.
Aim 2 will determine if pain behavior and/or CaMKIIalpha activation are downstream of N-type calcium channel activation.
Aim 3 will focus on eventsdownstream of calcium-permeable AMPA/KAand NR2B-NMDA receptors. Receptor-specific antagonists willbe administered to determine, if indeed, separate aspects of post-operative pain are diminished and, if so,whether each antagonist produces a distinct pattern of kinase inhibition.
Aim 4 will measure phosphorylationof AMPA GLUR1 and NMDA NR1 and NR2B receptor subunits and AMPA GLUR1 receptor subunit insertioninto the synaptic membrane after incision. These events are associated with synaptic strengthening andincreased spinal sensitization. Treatment with calcium-permeable AMPA/KA and NR2B-NMDA receptorantagonists will be used to ascertain if these events occur downstreamof receptor activation. Western blotsand protein kinase activity assays will be used to measure activation and/or phosphorylation of specificagents. Subcellularfractionation combined with Western blots will be used to demonstrate receptormovement from cytosol to membrane, while immunohistochemistry in tandem with confocal microscopy willbe used to determine cellular or regional localization of phosphorylated proteins. Delineation of theseimportant spinal signaling mechanisms and identification of relevant receptor subtypes involved inpostoperative pain will increase our ability to develop effective treatments for pain after surgery.
| Jones, Toni L; Hefferan, Michael P; Marsala, Martin et al. (2007) Low-speed subcellular fractionation method for determining noxious stimulus-evoked spinal neurokinin-1 receptor internalization. J Neurosci Methods 161:23-31 |
