Figure 4. Inhibition of PAFR induces a relieving effect on existing tactile allodynia. A. The decrease in paw withdrawal threshold was attenuated by a single administration of CV-3988 (10 nmol/10 ml) (A), but not Ki16425 (10 nmol/10 ml) (B), through a catheter whose tip was positioned near the L5 DRG on day 7 after nerve injury. ***p,0.001, {{{p,0.001 compared with pre-injury baseline (Pre). ###p,0.001 compared with the threshold on day 7. n.s. means ‘‘not significant’’. All data are presented as mean 6 SEM of the paw withdrawal threshold of five to eight animals. doi:10.1371/journal.pone.0010467.g004 by a single administration of Ki16425 (Fig. 4B), consistent with a previous study [12,31]. Alterations in motor behavior after CV- 3988 and Ki16425 treatment were not observed (data not shown). These results thus indicate that the PAFR has an important role in the development and maintenance of tactile allodynia whereas the LPAR in the DRG is important only for the development of tactile allodynia. Lyso-PAF-acetyltransferase LPCAT2 expression in the DRG Because a mechanism underlying LPAR-mediated tactile allodynia has been previously demonstrated [12,31], in the present study we investigated the role of the PAF-PAFR system in the DRG. First, we examined whether DRG neurons with activated cPLA2 express the lyso-PAF-acetyltransferase LPCAT2, a critical enzyme that produces PAF [32,33,34]. However, it is difficult to perform double-immunolabeling of DRG sections with p-cPLA2 and LPCAT2 antibodies, because they were raised in the same host species (rabbit). Thus, in this experiment, we used two adjacent DRG sections and singly immunostained one section with each antibody. We observed DRG neurons that were positive for both p-cPLA2 and LPCAT2 in the injured DRG 7 days after nerve injury (indicated by arrowheads, Fig. 5). These results suggest that LPCAT2 and activated cPLA2 are co-expressed in the injured DRG. In addition, we observed many small cells that are strongly positive to LPCAT2-IR around DRG neurons, and, surprisingly, these are not overlapped with p-cPLA2-IR (indicated by arrows, Fig. 5). It remains unclear whether the role of LPCAT2 in these small cells in neuropathic pain, and further investigations will be needed to clarify its role. Upregulation of the PAFR in the DRG after peripheral nerve injury We next examined the level of PAFR mRNA in total RNA extracts from the L5 DRG ipsilateral and contralateral to an injury PAFR and Neuropathic Pain Figure 5. LPCAT2 is expressed in DRG neurons positive to p- cPAL2 following peripheral nerve injury. Immunohistochemical analysis using two adjacent DRG sections (singly immunostained one section with p-cPLA2 or LPCAT2 antibody) revealed that there were DRG neurons positive for both p-cPLA2 and LPCAT2 in the injured DRG (arrowheads) 7 days after nerve injury. Arrows indicate unknown cells that were strongly positive to LPCAT2-IR and negative to p-cPLA2.-IR. Scale bar, 50 mm. doi:10.1371/journal.pone.0010467.g005 to the L5 spinal nerve. We found that the expression of PAFR mRNA in the ipsilateral DRG was markedly increased after nerve injury (Fig. 6A). A significant increase was observed from day 3 after injury (p,0.01 compared with the value of the contralateral hindpaw) and PAFR mRNA levels peaked on day 14 (day 7: p,0.01, day 14: p,0.001 compared with the value of contralateral hindpaw and day 7: p,0.01, day 14: p,0.001 compared with that of na¨ıve group) (Fig. 6A). The upregulation of PAFR mRNA expression on day 14 was not suppressed by CV-3988 administered for 14 days (116% of the ipsilateral DRG of vehicle-treated rats). In in situ hybridization analysis, we found that the intensity of PAFR mRNA signals and the number of cells with strong signal of PAFR mRNA were increased in the ipsilateral DRG 7 days after nerve injury (Fig. 6C) compared with the contralateral DRG (Fig. 6B). The expression of PAFR mRNA was increased in the cells (arrowheads) surrounding DRG neurons (indicated as ‘N’) in the ipsilateral DRG (Fig. 6C). Such specific signals of PAFR mRNA were not seen in sections hybridized with a corresponding sense probe (Fig. 6D). In addition, we performed in situ hybridization using another sets of probes (NM_053321 positioned at 2518-3111 bases), and similar data of PAFR mRNA were observed (data not shown). We validated the hybridization efficiency of antisense and sense probes used in this study with the spleen sections where the PAFR expression is known to be high [35] (Fig. S3). To identify the type of cells expressing PAFR, we performed in situ hybridization combined with immunohistochemistry for the macrophages/ microglia marker Iba1 (ionized calcium-binding adapter mole- cule-1) and the satellite cells marker GFAP (glial fibrillary acidic protein). We showed that the PAFR mRNA signals in the injured DRG were restricted to cells labeled with Iba1 (Fig. 6E, arrowheads), but not GFAP (Fig. 6F; GFAP, arrows; PAFR mRNA, arrowheads). These results indicate that PAFR expression in the DRG is upregulated in macrophages after nerve injury. Attenuation of nerve injury-induced tactile allodynia in pafr2/2 mice To precisely determine the functional relevance of PAFR, we used PAFR-deficient mice (pafr2/2 mice) [36]. pafr2/2 mice showed no major defects in basal mechanical sensitivity or motor coordination in the rotarod test [37]. Wild-type mice with an L5 nerve injury showed a progressively decreased paw withdrawal threshold (day 1: p,0.01, day 3, 7, 10 and 14: p,0.001) (Fig. 7). There were significant differences in the threshold between wild- type and pafr2/2 mice (day 7: p,0.01, day 10 and 14: p,0.05) PLoS ONE | www.plosone.org 4 May 2010 | Volume 5 | Issue 5 | e10467