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  • For this reason downstream components of the inflammatory pa


    For this reason, downstream components of the inflammatory pathway have been investigated in the search for alternative targets. Targeting such downstream elements might result in safer and more tolerable analgesics. PGE mediates its effects through binding to four G-protein-coupled receptors (EP1-4) . Although work with subtype-specific agonists has suggested that several of these EP receptors interfere with neural excitability during baseline- and inflammatory conditions either at spinal or at peripheral sites , most drug discovery and development studies have focused on the EP1 receptor . Use of EP1 mice demonstrated a role of this receptor in mediating especially peripheral heat sensitization via facilitating the activation of the transient receptor potential (TRP) channel V1 after subcutaneous PGE injection . Furthermore, experiments using local injections of presumed selective EP1 receptor blockers have suggested that EP1 receptors also are involved in mechanical sensitization at the spinal cord level. These rat studies utilized the EP1 selective antagonist ONO-8711 in the Propylthiouracil model of inflammatory pain and in a model for postoperative pain . However, the contribution of this receptor to spinal inflammatory hyperalgesia is not firmly established and our own work in genetically modified mice has assigned the EP2 receptor a major role in spinal pain sensitization during inflammatory pain states , . Here, we have used EP1 mice to investigate the peripheral versus central contribution of this receptor to mechanical- and heat sensitization during inflammation. Behavioural experiments were carried out in sex-matched groups of 7–9weeks old EP1 (, EP1) and EP2 (, EP2) receptor-deficient mice, and wild-type mice (C57BL/6). On day 1 of the experiment each mouse was tested several times to obtain baseline values for paw withdrawal latencies or mechanical response thresholds. Paw withdrawal latencies upon exposure to defined radiant heat stimuli were measured using a plantar test apparatus (Ugo Basle). Mechanical responses were obtained using an electronic von Frey anesthesiometer (IITC). Separate groups of animals were used for thermal and mechanical testing. In all behavioural experiments, the observer was blind to the genotype of the animals being tested. For intrathecal PGE injections, PGE was dissolved in 1% ethanol and 99% saline. Intrathecal injections were done using a Hamilton syringe (with spacer) into the lower lumbar spinal canal. In total 0.4nmol PGE were injected in a volume of 4μl. For subcutaneous PGE injections, PGE was dissolved in 0.1% DMSO and 99.9% saline. A total of 5nmol PGE was injected in 5μl subcutaneously into the hindpaw using a Hamilton syringe. For subcutaneous zymosan A injections, the baker's yeast extract from was suspended in saline. A total amount of 0.06mg in 20μl was subcutaneously injected using a Hamilton syringe into the hindpaw. Permission for animal experiments has been obtained from the Veterinäramt des Kantons Zürich. Sensitization to thermal or mechanical stimuli was determined as percent changes from pre-injection baselines. Reactions scores (insets in , , ) were calculated as percent change in withdrawal latencies/thresholds integrated Propylthiouracil over time for the duration of the experiment. We first addressed a potential role of EP1 or EP2 receptors in the maintenance of baseline nociceptive sensitivity. Baseline sensitivities to noxious heat and mechanical stimulation were determined in naïve wild-type mice, and compared with those of naïve animals lacking either the EP1 or the EP2 receptor. Mechanical sensitivity (A) was assessed by applying punctuate mechanical stimuli using electronic von Frey filaments. The mechanical thresholds of wild-type mice were indistinguishable from those of EP1 or EP2 mice (3.1±0.07g, 3.2±0.09g, and 3.2±0.09g, mean±SEM). Heat sensitivity was determined by measuring the withdrawal latency in response to a defined radiant heat stimulus using a plantar test apparatus (B). Again, reactions of wild-type mice were not different from those of EP1 nor EP2 mice (16.0±0.6s, 15.9±0.6s, and 16.6±1.0s, mean±SEM). These results suggest that neither EP1 nor EP2 receptors contributed to baseline nociceptive sensitivity.