A. Molecular and Cellular Biology A01 - Immune Mediators (600/Paper 312) Systemic morphine administration reduces local cytokine expression after incision D Clark, D Liang, Y Qiao, X Li, X Shi, M Angst, D Yeomans; Stanford University, Palo Alto, CA The enhanced production of cytokines is a hallmark of acute inflammation including inflammation occurring in the setting of surgical incision. Recent studies demonstrate that several cytokines participate in the enhancement of nociception which occurs after incision. Since opioids like morphine interact with neutrophils, macrophages and other immunocytes, it is possible that morphine exerts some of its antinociceptive action by altering the vigor of the local inflammatory response. In these studies we used a murine hind paw incisional model to study the role of morphine in modulating incisional in- flammation. We measured the effects of pre-incisional morphine administra- tion on nociceptive thresholds and cytokine production in incised skin. We also followed neutrophil infiltration in these wounds. In other experiments mice were treated with daily morphine injections for 4 days followed by hind paw incision and cytokine analysis in order to study the effects of chronic morphine administration. As expected, the incised mouse hind paws dis- played profound allodynia within 30 minutes of incision and this effect was stable for several hours. Morphine dose-dependently reduced or reversed this sensitization. Skin samples harvested from these mice showed enhanced lev- els of 5 cytokines: IL-1beta, IL-6, tumor necrosis factor  (TNF), granulocyte colony stimulating factor (G-CSF) and keratinocyte-derived cytokine (KC). Morphine dose-dependently reduced these incision-stimulated cytokine lev- els as well. Separate analyses measuring myeloperoxidase (MPO) and using immunohistochemistry demonstrated that morphine strongly and dose-de- pendently reduced the infiltration of neutrophils into the peri-incisional tis- sue. Chronic morphine pretreatment caused mechanical allodynia, but had no overall effect on incision-stimulated skin cytokine levels. The administration of morphine prior to incision reduces the expression of multiple locally pro- duced cytokines possibly by reducing acute phase neutrophil infiltration. Chronic morphine treatment sensitizes skin, but does not have major effects on incision-stimulated cytokine production. These findings have implications surrounding pain management, risk of infection and wound healing. (601) Blockage of receptor-1 reduces neuropathic nociception and spinal cord c-Fos expression after tibia fracture in a rat model of complex regional pain syndrome type I I Sabsovich, T Guo, T Wei, X Li, D Clark, C Sommer, W Kingery; Physical Medicine and Rehabilitation Service, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA Tibia fracture in rats evokes chronic hindpaw warmth, edema, allodynia, and periarticular bone loss, a syndrome resembling complex regional pain syn- drome type I (CRPS I). Previous studies suggest that the pathogenesis of some of these changes is attributable to an exaggerated regional inflammatory response to injury. We postulated that the pro-inflammatory cytokine tumor necrosis factor  (TNF ), an important mediator of chronic neuropathic and inflammatory pain, might also mediate the development of CRPS-like changes after tibia fracture. RT-PCR and EIA assays were used to evaluate changes in expression and content in skin, nerve, and bone at 4 weeks post-fracture. Bilateral hindpaw thickness, temperature, and nociceptive thresholds were determined, and bone microarchitecture was measured using microcom- puted tomography (CT). In addition, lumbar spinal cord c-Fos immonostain- ing was performed for quantification of Fos positive neurons. After baseline behavioral testing the distal tibia was fractured and the hindlimb casted for 4 weeks. The rats were subcutaneously injected either with a receptor antago- nist (soluble receptor type 1, s-R1, 5mg/kg/d) or saline every 3 days, over 28 days and then were retested at 4-weeks post-fracture. Tibia fracture chroni- cally upregulated expression and protein levels in the sciatic nerve, hindpaw skin, and proximal tibia. After fracture the rats developed hindpaw mechan- ical allodynia and unweighting, and these nociceptive behaviors were re- versed by s-R1 treatment. Consistent with the behavioral data, spinal Fos increased after fracture and this effect was inhibited by s-R1 treatment. Con- versely, s-R1 treatment had no effect on the hindpaw warmth, edema, or periarticular bone loss that developed after tibia fracture. Collectively, these data suggest that facilitated signaling in the hindlimb is an important medi- ator of chronic regional nociceptive sensitization after fracture, but is not the major contributor to the hindpaw warmth, edema, and bone loss observed in this CRPS I model. (602) Effects of pro-nociceptive cytokines on genes related to nociception and neuroimmune functions in cultured DRG neurons K Mitchell, J Keller, H Yang, M Iadarola; Neurobiology and Pain Therapeutics Section, Laboratory of Sensory Biology, National Institute of Dental and Cranio- facial Research, NIH, Bethesda, MD 20892, Bethesda, MD Cytokines are involved with the development and maintenance of in- flammatory and neuropathic pain. These molecules, which can be re- leased from numerous cell types including leukocytes and endothelial cells, exert their effects directly or indirectly on nerve terminals located in the periphery, on neurons of sensory ganglia and on nociceptive sensing neurons in the spinal cord. To better understand the mechanism by which these molecules influence nociception at the level of the sen- sory ganglia, we treated embryonically cultured DRG neurons with the pro-nociceptive cytokines , monocyte chemoattractant protein-1 (MCP- 1/CCL2) or macrophage inflammatory protein- (MIP-/CCL3) and exam- ined the expression of numerous genes involved in nociceptive process- ing after 2 or 24 h. Among the genes induced by TNF- are cox-2, interleukin-1&Beta, S100A8 and CCL2. TNF-induced CCL2 upregulation is also associated with a release of MCP-1/CCL2 into the medium, as determined by ELISA. Treatment with MCP-1/CCL2 induced fewer genes than TNF, but it dramatically induced CCL2 in neurons as determined by RT-PCR and in situ hybridization. In contrast to TNF and MCP-1/CCL2, MIP-1  /CCL3 did not induce the expression of any of the genes tested (including CCL3). Moreover, CCL3 was not induced by TNF or MCP-1/ CCL2. Thus data from these experiments suggests mechanisms by which these cytokines contribute to pain and highlight cytokines that may be involved in the maintenance of chronic pain at the level of the sensory ganglia. Importantly, hind paw administration of TNF, MCP-1/CCL2 or MIP-/CCL3 has been reported to induce hyperalgesia in rats. We are currently investigating whether this hyperalgesic response is associated with an increase in genes involved in nociceptive processing in the sen- sory ganglia. A02 - Mechanisms of Opioid Action (603/Paper 312) Postsynaptic spinal mu opioid receptor-ex- pressing neurons are required for morphine anti-hyperalgesia R Kline IV, R Wiley; VA TVHS and Vanderbilt University, Nashville, TN Lumbar intrathecal (i.t.) injection of Dermorphin-saporin (500 ng) in rats has been shown to selectively destroy spinal cord dorsal horn neurons expressing the mu-opioid receptor (MOR) with resulting attenuation of antinociceptive effects of systemic and i.t. morphine to low intensity (44 C) but not high (52 C) heat and increased responding in the formalin test. The current study examined the effects of lumbar i.t. Derm-sap on morphine antinociception during capsaicin-induced thermal hyperalge- sia and in the formalin test. Effects of Derm-sap on primary afferent thermal nociceptors were assessed in baseline hotplate and tail flick testing and after systemic loperamide, a peripherally restricted mu opi- oid. Sixteen male rats and twenty female rats received a single lumbar intrathecal injection of 10 ul of PBS or Derm-sap (500 ng). Sensitivity to mu opioids was evaluated on the 44 C hotplate test under baseline conditions in the female rats and three hours after topical plantar cap- saicin cream (0.94%) in the male rats. Effects of morphine (10 mg/kg, s.c.) on formalin behavior (25 ul of 5%) was evaluated in female rats for 90 min after plantar formalin injection. Immunohistochemical staining for GIRK2, a postsynaptic potassium channel essential for morphine analge- sia, was evaluated in lumbar dorsal horns of PBS and Derm-sap rats. Derm-sap produced: 1) no change in baseline tail flick responses, 2) reduced antinociceptive effects of systemic morphine in the 44 C hot- plate test after plantar capsaicin; 3) reduced antinociceptive effects of systemic morphine, but not loperamide, in the 44 C hotplate test; 4) reduced antinociceptive effects of morphine in the formalin test and 4) decreased GIRK2 staining in the dorsal horn. These observations dem- onstrate an important role for dorsal horn MOR-expressing neurons in analgesic and anti-hyperalgesic actions of morphine. The Journal of Pain, Vol 8, No 4 (April), Supplement 1, 2007: pp S1-S87 Available online at www.sciencedirect.com A BSTRACTS FOR P OSTER P RESENTATIONS S1 View publication stats View publication stats