PAFR and Neuropathic Pain Figure 6. PAFR mRNA is upregulated in the DRG following peripheral nerve injury. A. Real-time PCR analysis revealed that PAFR mRNA expression in total RNA extract from the L5 DRG was markedly increased after peripheral nerve injury. The bar graphs show the average fold increase in the level of PAFR mRNA expression in the DRG compared with the mean expression level of PAFR mRNA in na¨ıve rats. Each measurement was normalized to GAPDH content. **p,0.01, ***p,0.001 compared with na¨ıve rats. {{p,0.01, {{{p,0.001 compared with the contralateral side. All data are presented as mean 6 SEM of five individual animals. B-D. DIG-labeled antisense (B: contralateral, C: ipsilateral) and sense (D: ipsilateral) probes specific for PAFR mRNA were visualized by in situ hybridization in the rat DRG 7 days after nerve injury. Strong PAFR mRNA signals were observed in the cells surrounding DRG neurons in the ipsilateral DRG 7 days after nerve injury. Arrowheads show PAFR mRNA-positive cells. ‘N’ indicates neuronal cells. Similar results were observed in each of three experiments. Scale bar, 50 mm. E,F. In situ hybridization combined with immunohistochemistry for the macrophages/microglia marker Iba1 and the satellite glia marker GFAP was performed. PAFR mRNA signals overlapped with Iba1-IR (arrowheads, E) but not with GFAP-IR (GFAP: white arrowheads, PAFR: white arrows, F). Scale bar, 25 mm. doi:10.1371/journal.pone.0010467.g006 even though the threshold in pafr2/2 mice was decreased after injury (Fig. 7). Ablation of PAFRs did not change the paw withdrawal threshold at the side contralateral to the nerve injury (Fig. 7). Thus, PAFR might be required for tactile allodynia following peripheral nerve injury. Reduction of upregulation of TNFa and IL-1b expression in pafr2/2 mice TNFa and IL-1b are proinflammatory cytokines that have been reported to be upregulated in the DRG following peripheral nerve injury [16,17,38] and be produced via PAFRs in non-neuronal cells [39,40]. We predicted that the loss of PAFRs could affect the levels of TNFa and IL-1b in the L5 DRG. To address this, we examined the levels of mRNA of these cytokines in the DRG after nerve injury using real-time RT-PCR. The expression levels of these mRNAs at the ipsilateral DRG of wild-type mice was much higher than at the contralateral side on day 7 (TNFa: p,0.01, IL- 1b: p,0.001) (Fig. 8A, B), but pafr2/2 mice failed to show upregulation of these cytokines and there were significant differences in these expression level of ipsilateral side between wild-type and pafr2/2 mice (TNFa: p,0.05, IL-1b: p,0.01). PLoS ONE | www.plosone.org 5 May 2010 | Volume 5 | Issue 5 | e10467