PAFR and Neuropathic Pain Figure 10. Schematic representation of the proposed mechanism underlying the PAF/PAFR system-mediated neuropathic pain after peripheral nerve injury. After peripheral nerve injury, cPLA2 is activated by Ca2+ signaling evoked by P2X3 or P2X2/3 receptors (subtype of ionotropic purinergic receptors) and voltage-gated Ca2+ channels (VDCC). The Ca2+-dependent cPLA2 activation involves Ca2+/calmodulin-dependent protein kinase II (CaMKII). cPLA2 supply lyso-PAF which in turn converts into PAF by the lyso-PAF-acetyltransferase LPCAT2. PAF activates PAFR expressed in macrophages. Activation of PAFR may lead to production and release of proinflammatory cytokines, TNFa and IL-1b. These cytokines may increase the excitability of DRG neurons that link to nerve injury-induced tactile allodynia. doi:10.1371/journal.pone.0010467.g010 COX-1 antibody (1:50, Cayman chemical, Ann Arbor, MI, USA), anti-COX-2 antibody (1:50, Cayman chemical), and anti- LPCAT2 antibody (1:500, [34]). Identification of neurons was performed with a marker of neurons, microtubule associated protein 2 (1:1000, Chemicon, Temecula, CA, USA). Following incubation, the DRG sections were incubated with anti-rabbit immunoglobulin G (IgG)-conjugated Alexa Fluor 488 or anti- mouse IgG-conjugated Alexa Fluor 546 (1:1000, Molecular Probes, Eugene, OR, USA). The sections were then analyzed by a confocal microscope (LSM510, Zeiss, Oberkochen, Germany). 2 DRG neurons were characterized as small (,600 mm ), medium (600-1200 mm 2 ), and large (.1200 mm2)-sized neurons according to their cross-sectional areas [66]. Real-time reverse transcription-PCR Rats and mice were deeply anesthetized with pentobarbital, perfused transcardially with PBS, and the L5 DRG was removed immediately. Total RNA from the L5 DRG was extracted by using Trisure (Bioline, Danwon-Gu, Korea) according to manufacturer’s protocol and purified using RNeasy mini plus kit (QIAGEN, Valencia, CA, USA). The amount of total RNA was quantified by measuring OD260 using a Nanodrop spectrophotometer (Nano- drop, Wilmington, DE). For reverse transcription, 100 ng of total RNA was transferred to the reaction with Prime Script reverse transcriptase (Takara, Kyoto, Japan) and random 6-mer primers. Quantitative PCR was carried out with Premix Ex Taq (Takara) using a 7500 real-time PCR system (Applied Biosystems, Foster City, CA) according to protocol of the manufacturer, and the data were analyzed by 7500 System SDS Software 1.3.1 (Applied Biosystems) using the standard curve method. The TaqMan probe, forward primer and reverse primer used in this study were designed as follows: rat PAFR, probe, 59-FAM- ATCTCACCGTGGCG- GACCTGCTCT-TAMRA-39, forward, 59-CCCGTCCAAGAA- ACTGAATGAG-39, and reverse, 59-TCGCCCTCGTTGGAG- TAATAGA-39; mouse TNFa, probe, 59-FAM-TACGTGCTCCT- CACCCACACCGTCA-TAMRA-39, forward, 59-GTTCTCTT- CAAGGGACAAGGCTG-39, and reverse, 59-TCCTGGTATGA- GATAGCAAATCGG-39; mouse IL-1b, probe, 59-FAM-TGC- AGCTGGAGAGTGTGGATCCCAA-TAMRA-39, forward, 59- GAAAGACGGCACACCCACC-39, and reverse, 59-AGACAA- ACCGCTTTTCCATCTTC-39; rat TNFa, probe, 59-FAM-CGT- AGCCCACGTCGTA-TAMRA-39, forward, 59-GACCCTCA- CACTCAGATCATCTTCT-39, and reverse, 59- GGTACAGCC- CATCTGCTGGTA-39; rat IL-1b, probe, 59-FAM-TCTCCA- CCTCAATGGACAGAACATAAGCCA-TAMRA-39, forward, 59-AAATGCCTCGTGCTGTCTGA-39, and reverse, 59-GTCG- TTGCTTGTCTCTCCTTGTAC-39. The primers and probe for GAPDH and 18S mRNA were obtained from Applied Biosystems. In situ hybridization Digoxigenin (DIG)-labeled sense and antisense RNA probes were prepared from the sequence of rat pafr mRNA (GenBank accession number: NM_053321) positioned at 1178–1819 bases. Rats were anesthetized and perfused transcardially with Tissue Fixative (Genostaff, Tokyo, Japan) 7 day after nerve injury. DRG sections were removed and again fixed with the same fixative. Paraffin-embedded tissues (6 mm) were dewaxed with xylene and rehydrated. After proteinase K treatment (8 mg/ml, 30 min, 37uC) and acetylation by acetic anhydride (0.25%), hybridization was performed with sense and antisense probes at concentrations of 300 ng/ml at 60uC for 16 h. After hybridization, a series of washing was performed, followed by RNase treatment (50 mg/ml, 30 min, 37uC). The sections were blocked with 0.5% blocking reagent (Roche, Indianapolis, IN, USA) in Tris-buffered saline containing Tween 20 and incubated with anti-DIG alkaline phosphatase conjugate (1:1000, Roche) for 2 h at room temper- ature. Coloring reactions were performed with nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate solu- tion (NBT/BCIP) (Sigma, St. Louis, MO, USA) overnight. The sections were counterstained with Kernechtrot stain solution (Mutoh, Tokyo, Japan) and then mounted with Malinol (Mutoh). Non-neuronal and neuronal cells with a higher violet intensity PLoS ONE | www.plosone.org 9 May 2010 | Volume 5 | Issue 5 | e10467