A wide variety of lipid mediators have been implicated in the processing of pain signals at the spinal level. Many of these lipids are derived from the reaction catalyzed by phospholipase A2 (PLA2). The lipid mediators derived from the action of PLA2 include one of the direct products arachidonic acid (AA) as well as the secondary metabolites including the prostaglandins (PG), leukotrienes, platelet activating factor (PAF), and lysophosphatidic acid (LPA). Microglia and astrocytes, which are two fundamental cellular constituents of the spinal cord, have been suggested to play a central role in pathological pain through their ability to facilitate spinal pain processing. The overall focus of our research on facilitated spinal pain processing is directed toward the bioactive lipid mediators derived from the action of PLA2. In extensive preliminary results, we have identified a number of prostaglandins that are produced in cerebrospinal fluid (CSF) of rats challenged with several pain models, and we have conducted numerous experiments implicating these products as important players in central pain processing. Our first hypothesis is that PLA2 derived lipid mediators play a crucial role in pain processing which we will test by intrathecal injection of these agents into rat cerebrospinal fluid (CSF) and measurement of the changes in pain response. We will also measure the changes in response when these agents are administered in conjunction with two models of persistent pain, i.e. inflammation and nerve-injury. Time courses and inhibitor profiles will be obtained for all of these systems. In separate studies, we will repeat the above studies with pain causing agents, such as NMDA, substance P or other lipid mediators, directly administered into the CSF via intrathecal injection. Our second hypothesis is that microglia and/or astrocytes are responsible for the in vivo release of lipid mediators. We will explore this hypothesis by correlating the eicosanoid release profiles, the time courses of release, and the inhibition profiles in primary cultures of microglia and astrocytes. Our third hypothesis is that the inhibition of lipid mediator production in microglia or astrocytes will lead to improved methods for treating pain. To this end, we will employ chemical inhibitors and antisense/siRNAs that we have found effective at suppressing lipid mediator release in P388D1 and RAW264.7 macrophage cells in work on the current grant. We will suppress the activity of these enzymes in both the in vivo rat and ex vivo cellular systems. We will then correlate the effects on lipid mediator release in the two systems with changes in the pain response measured in the in vivo experiments. We will also examine these cells to determine if any of the post translational control mechanisms that we have demonstrated to exist in P388D1 and RAW264.7 cells are operative in microglia and astrocytes. Established LC/mass spectrometry protocols/procedures will be employed to identify and quantitate the lipid mediators produced in all of these experiments. The development of therapeutics to manage pain secondary to injury and inflammation has been and remains a principal target of drug development. This grant explores the role of prostaglandins in this pain process and how the levels of these compounds are controlled. This will lead to a better understanding of pain processing and to the development of new classes of drugs for treating pathological pain.
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