(peripheral sensitization) [6] and to produce allodynic behavior [7,8]. LOX products activate capsaicin receptors in primary sensory neurons, resulting in the induction of peripheral sensitization [9,10]. Furthermore, PAF injected into the hindpaw of na¨ıve animals produces nociceptive responses and mechanical hypersensitivity [11], and recent works have also shown that intrathecal administration of LPA [12] and PAF [13,14] in na¨ıve animals induces tactile allodynia. However, the role of these lipid mediators in the pathogenesis of neuropathic pain is not fully understood. In the present study, to determine the neuropathic pain-related lipid mediators downstream of cPLA2 activation in the DRG, we investigate the involvement of enzymes and lipid mediator receptors in nerve injury-induced tactile allodynia using pharma- cological, molecular, and genetic approaches. We further investigated the role of the lipid mediator receptors in the expression of tumor necrosis factor a (TNFa) and interleukin-1b (IL-1b) in the DRG, proinflammatory cytokines that are strongly implicated in nerve injury-induced tactile allodynia [15,16,17,18]. Results Effects of COX and LOX inhibitors on the development of tactile allodynia after nerve injury To determine an involvement of a COX-dependent pathway in tactile allodynia, we first performed double-immunolabeling for phosphorylated-cPLA2 (p-cPLA2) and COX. In the both side of the L5 DRG after nerve injury, COX-1-immunoreactivity (COX-1-IR) was present mainly in small-sized neurons, whereas no COX-2-IR was observed (Fig. 1A), consistent with previous studies [19,20]. Thus, we examined the effect of the selective COX-1 inhibitor SC- 560 [21] on the development of nerve injury-induced tactile allodynia. Vehicle-treated rats with an L5 nerve injury displayed a marked decrease in paw withdrawal threshold at the ipsilateral side after nerve injury (p,0.001) (Fig. 1B). Similarly, nerve-injured rats that had been treated with SC-560 showed a decreased paw withdrawal threshold (p,0.001) and there were no significant differences in the threshold of ipsilateral side between the vehicle- and SC-560-treated group (Fig. 1B). We also found that most of neurons with the translocated p-cPLA2 were not double-labeled with COX-1-IR (169/251, 67.3% of p-cPLA2-translocated neu- rons) (Fig. 1C). In a small group of DRG neurons, especially small- sized neurons, both types of immunoreactivity were observed, but their subcellular localizations were different: COX-1-IR was observed in the perinuclear area where p-cPLA2-IR was not accumulated (Fig. 1D). Next, to investigate the involvement of LOX in nerve injury-induced tactile allodynia, we examined the effects of the 5-LOX inhibitor AA-861 [22] and the 12- and 15-LOX inhibitor baicalein [23,24] and found that AA-861- and baicalein- treated rats displayed a decrease in paw withdrawal threshold with a similar time course to that of vehicle-treated rats (Fig. 2A, B). There were no significant differences in the threshold between the vehicle- and the LOX inhibitor-treated groups. The doses of the COX and LOX inhibitors used in this study are approximately equivalent to (or even higher than) doses that are known to inhibit behavioral responses mediated by COX [25,26] and LOX [27,28], respec- tively. Thus, these results suggest that COX and LOX in the DRG may not be involved in nerve injury-induced tactile allodynia. Effects of PAFR and LPAR antagonists on nerve injury- induced tactile allodynia To determine the role of lysophospholipid-derived lipid mediators such as PAF and LPA in nerve injury-induced tactile allodynia, we administered PAFR and LPA receptor (LPAR) PAFR and Neuropathic Pain Figure 1. A COX inhibitor does not prevent the development of tactile allodynia after nerve injury. A. Immunofluorescence labeling of COX-1 and COX-2 proteins in the L5 DRG 14 days after nerve injury demonstrated that immunoreactivity of COX-1, but not COX-2, was present mainly in small-sized neurons. Scale bar, 50 mm. B. The COX-1 inhibitor SC-560 (20 nmol/10 ml) administered through a catheter whose tip was positioned near the L5 DRG once daily for 14 days did not suppress the development of nerve injury-induced a decrease in the paw withdrawal threshold of tactile stimulation using von Frey filaments. ***p,0.001 compared with the threshold of the vehicle-treated group on day 0. {{{p,0.001 compared with the threshold of the inhibitor-treated group on day 0. Data are presented as mean 6 SEM of the paw withdrawal threshold of five animals. C. Double immunofluorescence labeling of p-cPLA2 with COX-1 in L5 DRG neurons 14 days after nerve injury showed that most of neurons with the translocated p-cPLA2 were not double-labeled with COX-1-IR. Scale bar, 50 mm. D. Highly magnified pictures of large and small diameter DRG neurons demonstrated that in large neurons, p-cPLA2 was not co- expressed with COX-1-IR. In small neurons, the subcellular localizations of p-cPLA2 and COX-1-IR were different: COX-1-immunoreactivity was observed in the perinuclear area where p-cPLA2- immunoreactivity was not accumulated. Scale bars, 20 mm. doi:10.1371/journal.pone.0010467.g001 PLoS ONE | www.plosone.org 2 May 2010 | Volume 5 | Issue 5 | e10467