PAFR and Neuropathic Pain Figure 9. Injection of PAF near the DRG induces a decrease in the paw withdrawal threshold and upregulation of TNFa and IL-1b mRNAs. A. Paw withdrawal threshold was decreased by single administration of several different doses of PAF near the DRG in normal rats. *p,0.05, **p,0.01, ***p,0.001 compared with the threshold of the vehicle-treated group. B. The PAF-induced decrease in paw withdrawal threshold was suppressed by pretreatment with CV-3988. ***p,0.001 compared with the threshold of the vehicle-treated group. ##p,0.01 compared with the threshold of the PAF-treated group. n.s. means ‘‘not significant’’. C, D. Real-time PCR analysis showed that administration of PAF near the DRG increased expression of TNFa (C) and IL-1b (D) mRNAs in the DRG 45 min after the administration. The bar graphs show the average fold increase in the level of TNFa and IL-1b mRNAs in the DRG compared with the mean expression level of these mRNAs in the vehicle-treated group. Each measurement was normalized to 18S mRNA content. *p,0.05, **p,0.01, ***p,0.001 compared with the vehicle-treated group. #p,0.05, ###p,0.001 compared with the PAF-treated group. n.s. means ‘‘not significant’’. All data are presented as mean 6 SEM of four to six animals. doi:10.1371/journal.pone.0010467.g009 allodynia. Furthermore, because COX-1 expression was observed mainly in small-diameter DRG neurons and LOX products activate capsaicin receptors in primary sensory neurons [9,10], COX and LOX in the DRG may participate in thermal hyperalgesia after nerve injury. The crucial role of PAFR in neuropathic pain was demonstrat- ed for the first time by the data obtained from the behavioral analyses using pharmacological and genetic tools. Administration of the PAFR antagonist CV-3988 near the injured DRG reduced the development and expression of nerve injury-induced tactile allodynia. By contrast, the reducing effect of the development of tactile allodynia by administration of CV-3988 to the lumbar enlargement of the spinal cord was much less than that by injection of the antagonist near the DRG. Furthermore, DRG neurons with activated cPLA2 also expressed the lyso-PAF- acetyltransferase LPCAT2, a critical enzyme that produces PAF [32,33,34]. Our data from real-time RT-PCR analyses showed that PAFR gene expression was increased in the ipsilateral DRG after nerve injury. These findings thus suggest that the PAF/PAFR system may be activated in the injured DRG and contribute to nerve injury-induced tactile allodynia, although a possible involvement of PAFR in the spinal cord can not be completely excluded [13]. Moreover, our in situ hybridization experiments revealed that following peripheral nerve injury a marked increase in PAFR mRNA expression was seen mainly in non-neuronal cells positive to Iba1. It is conceivable that peripheral nerve injury causes the transcriptional upregulation of PAFR in macrophages in the DRG. Alternatively, infiltration of macrophages expressing PAFR in the DRG after nerve injury might result in the upregulation of PAFR mRNA, because PAFR is expressed in macrophages [35] and is involved in macrophage chemotaxis [48] and the number of macrophages in the DRG is increased following peripheral nerve injury [49]. Interestingly, PAFR- deficient mice showed a marked suppression of the upregulation of TNFa and IL-1b expression in the injured DRG. This is consistent with the notion that macrophages are one of the major sources of these proinflammatory cytokines and regulate pain signaling [49,50,51,52], although it remains unknown whether PAFR activation leads to upregulation of proinflammatory cytokine expression in macrophages. PLoS ONE | www.plosone.org 7 May 2010 | Volume 5 | Issue 5 | e10467