Figure 7. Deletion of pafr reduces tactile allodynia. While wild-type mice with an L5 nerve injury showed a progressively decreased paw withdrawal threshold, pafr2/2 mice showed an attenuation of the decrease in paw withdrawal threshold after nerve injury. **p,0.01, ***p,0.001 compared with the threshold of wild-type mice on day 0. {{p,0.01 compared with the threshold of pafr2/2 mice on day 0. #p,0.05, ##p,0.01 compared with the threshold of the wild-type mice at each time point. All data are presented as mean 6 SEM of six to eight animals. doi:10.1371/journal.pone.0010467.g007 Ablating PAFRs did not change cytokine expression at the contralateral DRG. These results indicate that PAFRs might contribute to tactile allodynia via TNFa and IL-1b production in the DRG following nerve injury. Figure 8. Upregulation of TNFa and IL-1b gene expression in the DRG is reduced in pafr2/2 mice. A, B. Real-time PCR analysis demonstrated that following nerve injury, the expression of TNFa (A) and IL-1b (B) mRNAs in the ipsilateral DRG of wild-type mice was much higher than in the contralateral side on day 7. However, pafr2/2 mice failed to show upregulation of these cytokines and there were significant differences in these expressions of the ipsilateral side between wild-type and pafr2/2 mice. Each measurement was normalized to 18S mRNA content. **p,0.01, ***p,0.001 compared with the contralateral side of wild-type mice. #p,0.05, ##p,0.01 compared with the ipsilateral side. n.s. means ‘‘not significant’’. All data are presented as mean 6 SEM of six to eight animals. doi:10.1371/journal.pone.0010467.g008 PAFR and Neuropathic Pain Effect of PAF injection on paw withdrawal threshold and cytokine production in the DRG We determined whether injection of PAF in the absence of nerve injury is sufficient to produce tactile allodynia and to upregulate cytokine expression in the DRG. A single injection of PAF near the DRG of na¨ıve rats caused a decrease in the paw withdrawal threshold to mechanical stimulation in a dose- dependent manner (Fig. 9A). The threshold began to decrease 15 min after PAF injection and peaked at 45–60 min, then gradually recovered over 4 h (Fig. 9A). The decrease in the withdrawal threshold induced by PAF was inhibited by pretreat- ment with CV-3988 (p,0.01) (Fig. 9B). We then performed real- time RT-PCR analysis using the DRG of rats that had been injected with PAF. The expression of mRNAs for TNFa and IL- 1b was increased 45 min after PAF injection (Fig. 9C, D), when the threshold was markedly reduced. This effect was also prevented by pretreatment with CV-3988 (Fig. 9C, D). These results suggest that administering PAF near the DRG causes a decrease in the withdrawal threshold and production of TNFa and IL-1b via PAFR. Discussion In the present study, we provide the first evidence that in the DRG, PAFR is important in the pathogenesis of tactile allodynia, a major behavioral consequence of nerve injury, and are responsible for production of proinflammatory cytokines such as TNFa and IL-1b. We have previously shown that cPLA2 is translocated to the plasma membrane mainly in medium- to large-diameter DRG neurons [5], but COX-1 was present around the perinuclear region of small-sized DRG neurons. In addition, our behavioral studies demonstrated that administering the COX-1 inhibitor SC- 560 near the injured DRG showed no anti-allodynic effect at doses that are considered to inhibit this enzyme [25,26]. A previous study reported that administration of SC-560 to the lumbar enlargement of the spinal cord at a higher dose than our study prevented a decrease in the paw withdrawal threshold after nerve injury [41], which seems to be partly inconsistent with our present results. However, peripheral nerve injury causes an increase in COX-1 and COX-2 expression in the spinal cord [42,43], but not in the DRG, and a high dose of SC-560 also slightly inhibits COX-2 [21]. Thus, it is possible that the discrepancy in the effect of SC-560 on tactile allodynia might be explained by the difference in dose and region of drug administration. These results thus suggest that COX in the DRG is not primarily involved in the cPLA2-dependent tactile allodynia. These data may also provide a putative explanation for the resistance or controversial effects of COX inhibitors on allodynic behavior reported in both animals and patients with neuropathic pain [44]. Similar to SC-560, neither the 5-LOX inhibitor AA-861 nor the 12- and 15-LOX inhibitor baicalein showed the inhibitory effect on nerve injury-induced tactile allodynia. Products of lipoxygenases such as leukotrienes, hydroxyeicosatetraenoic acids (HETEs) and hydroperoxyeicosatetraenoic acids (HPETEs) have been implicated in mediating inflammatory nociception because they are produced during inflammation [45] and cause hyperalgesia when injected intradermally [46,47], but so far there have been no reports that clearly show the involvement of lipoxygenases in tactile allodynia. These findings and the data presented here suggest that in the DRG, LOX does not also play a major role in nerve injury-induced tactile allodynia as in the case of COX, while we can not completely exclude the possibility that COX and LOX in the spinal cord are involved in tactile PLoS ONE | www.plosone.org 6 May 2010 | Volume 5 | Issue 5 | e10467