Post-operative pain is a major clinical concern, as it can delay patient recovery and increase probability of chronic pain syndromes. More than half of patients report moderate to severe pain after surgery, thus, it is important to investigate the mechanisms that generate and maintain this condition. Plantar incision in the rat hind paw is a model of post-operative pain resulting in spontaneous pain and hyperalgesia, or increased pain in response to stimulation, symptoms that are observed in patients. Spinal glutamate receptor pharmacology of this model is distinct. Spinally administered conventional antagonists to NMDA receptors have no effect on any aspect of incision-evoked pain in the rat. However, NR2B-NMDA and calcium-permeable AMPA/KA glutamate receptor subtypes appear to mediate separate aspects of post-operative pain behavior, although this is not certain. No studies have addressed the role of spinal signal transduction cascades occurring downstream from calcium-permeable AMPA/KA and NR2B-NMDA receptor activation after incision. ? ? This application examines post-operative pain behaviors by targeting the facilitation of various elements of these 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 (CaMKII alpha) over a 5-day period. Administration of selective kinase antagonists will demonstrate if these changes are necessary for the manifestation of pain behavior.
Aim 2 will determine if pain behavior and/or CaMKII alpha activation are downstream of N-type calcium channel activation.
Aim 3 will focus on events downstream of calcium-permeable AMPA/KA and NR2B-NMDA receptors. Receptor-specific antagonists will be 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 phosphorylation of AMPA GLUR1 and NMDA NR1 and NR2B receptor subunits and AMPA GLUR1 receptor subunit insertion into the synaptic membrane after incision. These events are associated with synaptic strengthening and increased spinal sensitization. Treatment with calcium-permeable AMPA/KA and NR2B-NMDA receptor antagonists will be used to ascertain if these events occur downstream of receptor activation. Western blots and protein kinase activity assays will be used to measure activation and/or phosphorylation of specific agents. Subcellular fractionation combined with Western blots will be used to demonstrate receptor movement from cytosol to membrane, while immunohistochemistry in tandem with confocal microscopy will be used to determine cellular or regional localization of phosphorylated proteins. Delineation of these important spinal signaling mechanisms and identification of relevant receptor subtypes involved in post-operative 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 |
