It has been reported that TNFa [38,53,54,55,56] and IL-1b [16,17] are upregulated in the DRG following peripheral nerve injury and are implicated in tactile allodynia. Increased synthesis and release of TNFa and IL-1b can directly modulate neuronal activity and elicit spontaneous action potential discharges in the DRG [49]. TNFa enhances tetrodotoxin-resistant Na+ currents in DRG neurons through TNF receptor 1 [57]. Both injured and adjacent uninjured primary afferent neurons become more sensitive to TNFa after spinal nerve ligation [18]. Similarly, IL- 1b application has been shown to increase the excitability of sensory neurons by potentiating voltage-dependent Na+ currents [58]. Therefore, these cytokines in the injured DRG might be involved in sensitization of primary sensory neurons that link to nerve injury-induced tactile allodynia. It remains unclear whether the expression of these cytokines results from PAFR activation in non-neuronal cells in the DRG. In the future, by generating using cell type-specific PAFR-knockout mice, and by studying their phenotypes, this issue will be determined. LPA could also be a candidate signaling molecule that is responsible for nerve injury-induced tactile allodynia. Our behavioral study demonstrated that injection of the LPAR antagonist Ki16425 near the injured DRG reduced the develop- ment, but not maintenance, of tactile allodynia, which is consistent with a previous study [12]. Because among LPARs, LPA1R is mainly expressed in the DRG and LPA1R-deficient mice attenuate nerve injury-induced tactile allodynia [12]. LPA1Rs mediate demyelination and upregulate the expression of the a2d1 subunit of the voltage-gated calcium channel in the DRG [12], and these might be the mechanisms underlying LPA-dependent tactile allodynia. Furthermore, LPA increases the intracellular calcium concentration in adult DRG neurons [59] and intraplantar injection of LPA causes a nociceptive flexor response [60], suggesting that LPA could produce direct effects on primary afferent neurons and modulate nociceptive responses. Based on the results obtained from our present and previous studies, we proposed the following mechanism (Fig. 10). After peripheral nerve injury, cPLA2 is activated by Ca2+ signaling evoked by P2X3 or P2X2/3 receptors (subtype of ionotropic purinergic receptors) [5]. These receptors are activated by extracellular ATP, which presumably released from neighboring cells such as satellite glia. Activity of voltage-gated Ca2+ channels (VDCC) is also involved in cPLA2 activation [61]. The Ca2+-dependent cPLA2 activation is mediated by Ca2+/calmodulin-dependent protein kinase II (CaMKII) [61]. cPLA2 supply lyso-PAF, a precursor of PAF, which in turn converts into PAF by LPCAT2 (although the role of LPCAT2 expressed in unknown cells in the DRG remains unclear). PAF may be released from DRG neurons and then activates PAFR expressed in macrophages. Activation of PAFR may lead to production and release of proinflammatory cytokines. These cytokines increase the excitability of DRG neurons that link to nerve injury-induced tactile allodynia. In summary, the present study provided the first evidence that the activation of the PAFR may be a key event in the development and maintenance of tactile allodynia and production of proin- flammatory cytokines such as TNFa and IL-1b in the DRG. Thus, blocking the PAFR might be a new therapeutic target for treating neuropathic pain. Importantly, basal pain sensitivity was not altered in pafr2/2 mice, suggesting the possibility of a therapeutic benefit of blocking PAFRs in the treatment of tactile allodynia without affecting normal pain sensitivity. As the PAFR has been implicated in other neurodegenerative disorders such as ischemia [62] and multiple sclerosis [39], interfering with PAFRs could be a therapeutic strategy widely applicable to central nervous system diseases. PAFR and Neuropathic Pain Materials and Methods All animal experiments were conducted according to relevant national and international guidelines ‘Act on Welfare and Management of Animals’ (Ministry of Environment of Japan) and ‘Regulation of Laboratory Animals’ (Kyushu University), and under the protocols approved by the Institutional Animal Care and Use committee review panels at Kyushu University and The University of Tokyo. Animals Male Wistar rats (250–270 g, Japan SLC, Shizuoka, Japan) and wild-type and pafr2/2 mice [36] were used. Animals were housed in individual and groups of 2,3 per cage at a temperature of 2261uC with a 12-h light-dark cycle (light on 8:30 to 20:30), and fed food and water ad libitum. Neuropathic pain model We used the spinal nerve injury model [63] with some modifications [64]: in male Wistar rats a unilateral L5 spinal nerve was tightly ligated and cut just distal to the ligature. For the experiments using mice, the left L5 spinal nerve was transected [65]. The mechanical allodynia was assessed by using calibrated von Frey filaments (0.4–15.1 g for rats, 0.02–2.0 g for mice; Stoelting, Wood Dale, IL, USA) and the paw withdrawal threshold was determined as described previously [64]. Drug treatment Rats were implanted with catheters for drug injection near the L5 DRG according to the method described previously [64,65]. Under isoflurane anesthesia, a sterile 32 gauge catheter (Re- CathCo, Allison Park, PA, USA) was inserted into the intrathecal space through the atlanto-occipital membrane and to the L4 or L5 DRG and externalized through the skin [5]. After the experi- ments, we confirmed that the tip of the catheter was positioned near the L5 DRG. Rats were injected with each drug through the catheter using a 25 ml Hamilton syringe with a 30-gauge needle once a day from day 0 (just before the nerve injury) to day 13. The drugs used in this study are listed below: SC-560 (20 nmol/10 ml, Calbiochem, San Diego, CA, USA), AA-861 (20 nmol/10 ml, Sigma, St Louis, MO, USA), baicalein (20 nmol/10 ml, Calbio- chem), CV-3988 (10 nmol/10 ml, Biomol, Plymouth Meeting, PA, USA) and Ki16425 (10 nmol/10 ml, Sigma). The paw withdrawal threshold was tested 21–24 hr after the injection of each drug at 1, 3, 7 and 14 days post-injury. For the experiment in which the effect of a single administration of CV-3988 or Ki16425 on the established allodynia was examined on day 7 after nerve injury, behavioral test was performed immediately before and after the injection of CV-3988 (10 nmol/10 ml) or Ki16425 (10 nmol/ 10 ml). To examine the effect of PAF on pain behavior, the paw withdrawal threshold was assessed before and after a single injection of PAF near the DRG of na¨ıve rats. Rats were injected with CV-3988 (10 nmol/10 ml) through the catheter 10 min before the injection of PAF. Immunohistochemistry Rats were deeply anesthetized by pentobarbital (100 mg/kg, i.p.) and perfused transcardially with 4% paraformaldehyde. DRG sections were removed, postfixed with the same fixative, and placed in 30% sucrose solution for 24 hr at 4uC. The DRG sections (15 mm) were incubated in a blocking solution [3% normal goat serum/0.3% Triton X-100/phosphate-buffered saline (PBS) (2)] and then with anti-phospho-cPLA2 (anti-p- cPLA2) antibody (1:1000, Cell Signaling, Beverly, MA, USA), anti- PLoS ONE | www.plosone.org 8 May 2010 | Volume 5 | Issue 5 | e10467