Behavioral phenotypes of mice lacking purinergic P2X4 receptors in acute and chronic pain assays

A growing body of evidence indicates that P2X receptors (P2XRs), a family of ligand-gated cation channels activated by extracellular ATP, play an important role in pain signaling. In contrast to the role of the P2X3R subtype that has been extensively studied, the precise roles of others among the seven P2XR subtypes (P2X1R-P2X7R) remain to be determined because of a lack of sufficiently powerful tools to specifically block P2XR signaling in vivo. In the present study, we investigated the behavioral phenotypes of a line of mice in which the p2rx4 gene was disrupted in a series of acute and chronic pain assays. While p2rx4-/- mice showed no major defects in pain responses evoked by acute noxious stimuli and local tissue damage or in motor function as compared with wild-type mice, these mice displayed reduced pain responses in two models of chronic pain (inflammatory and neuropathic pain). In a model of chronic inflammatory pain developed by intraplantar injection of complete Freund's adjuvant (CFA), p2rx4-/- mice exhibited attenuations of pain hypersensitivity to innocuous mechanical stimuli (tactile allodynia) and also of the CFA-induced swelling of the hindpaw. A most striking phenotype was observed in a test of neuropathic pain: tactile allodynia caused by an injury to spinal nerve was markedly blunted in p2rx4-/- mice. By contrast, pain hypersensitivity to a cold stimulus (cold allodynia) after the injury was comparable in wild-type and p2rx4-/- mice. Together, these findings reveal a predominant contribution of P2X4R to nerve injury-induced tactile allodynia and, to the lesser extent, peripheral inflammation. Loss of P2X4R produced no defects in acute physiological pain or tissue damaged-induced pain, highlighting the possibility of a therapeutic benefit of blocking P2X4R in the treatment of chronic pain, especially tactile allodynia after nerve injury

Activation of cytosolic phospholipase A2 in dorsal root ganglion neurons by Ca2+/calmodulin-dependent protein kinase II after peripheral nerve injury

<p>Abstract</p> <p>Background</p> <p>Peripheral nerve injury leads to a persistent neuropathic pain state in which innocuous stimulation elicits pain behavior (tactile allodynia), but the underlying mechanisms have remained largely unknown. We have previously shown that spinal nerve injury induces the activation of cytosolic phospholipase A₂(cPLA₂) in injured dorsal root ganglion (DRG) neurons that contribute to tactile allodynia. However, little is known about the signaling pathway that activates cPLA₂after nerve injury. In the present study, we sought to determine the mechanisms underlying cPLA₂activation in injured DRG neurons in an animal model of neuropathic pain, focusing on mitogen-activated protein kinases (MAPKs) and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII).</p> <p>Results</p> <p>Pharmacological inhibition of either p38 or extracellular signal-regulated kinase (ERK) in the injured DRG, which led to suppression of the development of tactile allodynia, did not affect cPLA₂phosphorylation and translocation after nerve injury. By contrast, a CaMKII inhibitor prevented the development and expression of nerve injury-induced tactile allodynia and reduced both the level of cPLA₂phosphorylation and the number of DRG neurons showing translocated cPLA₂in response to nerve injury. Applying ATP to cultured DRG neurons increased the level of both phosphorylated cPLA₂and CaMKII in the vicinity of the plasma membrane and caused physical association of these two proteins. In addition, ATP-stimulated cPLA₂and CaMKII phosphorylation were inhibited by both a selective P2X₃R/P2X₂₊₃R antagonist and a nonselective voltage-dependent Ca²⁺channel (VDCC) blocker.</p> <p>Conclusion</p> <p>These results suggest that CaMKII, but not MAPKs, has an important role in cPLA₂activation following peripheral nerve injury, probably through P2X₃R/P2X₂₊₃R and VDCCs in primary afferent neurons.</p

Usefulness and Problems of Endoscopic Ultrasonography in Prediction of the Depth of Tumor Invasion in Early Gastric Cancer

The objectives of this study were to evaluate the accuracy of endoscopic ultrasonography (EUS) in local and regional staging of early gastric cancer, to analyze the factors influencing the accuracy of EUS, and to reveal the usefulness and problems of EUS in pre-treatment staging of gastric cancer. We examined 105 lesions in 104 patients with histologically confirmed gastric cancer and retrospectively evaluated them with EUS. The diagnostic accuracy, sensitivity, and specificity of EUS were determined by comparing the pre-treatment EUS with the postoperative histopathological findings. The overall diagnostic accuracy of EUS for the depth of cancer invasion was 86%. The overall sensitivity and specificity were 60% and 96%, respectively. The accuracy significantly declined in lesions located in the upper-third of the stomach (70%). Type 0-I lesions tended to be over-staged (12&), and the upper-third lesions tended to be under-staged (23%). The accuracy significantly declined in differentiated adenocarcinoma with massive submucosal invasion (56.5%). EUS is useful for evaluating the depth of gastric cancer invasion which determines the feasibility of endoscopic treatment. However, it is noteworthy that the diagnostic accuracy of the invasion depth diminished for lesions in the upper third of the stomach

Role of PAF Receptor in Proinflammatory Cytokine Expression in the Dorsal Root Ganglion and Tactile Allodynia in a Rodent Model of Neuropathic Pain

BACKGROUND: Neuropathic pain is a highly debilitating chronic pain following damage to peripheral sensory neurons and is often resistant to all treatments currently available, including opioids. We have previously shown that peripheral nerve injury induces activation of cytosolic phospholipase A(2) (cPLA(2)) in injured dorsal root ganglion (DRG) neurons that contribute to tactile allodynia, a hallmark of neuropathic pain. However, lipid mediators downstream of cPLA(2) activation to produce tactile allodynia remain to be determined. PRINCIPAL FINDINGS: Here we provide evidence that platelet-activating factor (PAF) is a potential candidate. Pharmacological blockade of PAF receptors (PAFRs) reduced the development and expression of tactile allodynia following nerve injury. The expression of PAFR mRNA was increased in the DRG ipsilateral to nerve injury, which was seen mainly in macrophages. Furthermore, mice lacking PAFRs showed a reduction of nerve injury-induced tactile allodynia and, interestingly, a marked suppression of upregulation of tumor necrosis factor alpha (TNFalpha) and interleukin-1beta (IL-1beta) expression in the injured DRG, crucial proinflammatory cytokines involved in pain hypersensitivity. Conversely, a single injection of PAF near the DRG of naïve rats caused a decrease in the paw withdrawal threshold to mechanical stimulation in a dose-dependent manner and an increase in the expression of mRNAs for TNFalpha and IL-1beta, both of which were inhibited by pretreatment with a PAFR antagonist. CONCLUSIONS: Our results indicate that the PAF/PAFR system has an important role in production of TNFalpha and IL-1beta in the DRG and tactile allodynia following peripheral nerve injury and suggest that blocking PAFRs may be a viable therapeutic strategy for treating neuropathic pain

Reduced spinal microglial activation and neuropathic pain after nerve injury in mice lacking all three nitric oxide synthases

<p>Abstract</p> <p>Background</p> <p>Several studies have investigated the involvement of nitric oxide (NO) in acute and chronic pain using mice lacking a single NO synthase (NOS) gene among the three isoforms: neuronal (nNOS), inducible (iNOS) and endothelial (eNOS). However, the precise role of NOS/NO in pain states remains to be determined owing to the substantial compensatory interactions among the NOS isoforms. Therefore, in this study, we used mice lacking all three NOS genes (<it>n/i/eNOS^-/-</it>mice) and investigated the behavioral phenotypes in a series of acute and chronic pain assays.</p> <p>Results</p> <p>In a model of tissue injury-induced pain, evoked by intraplantar injection of formalin, both <it>iNOS^-/-</it>and <it>n/i/eNOS^-/-</it>mice exhibited attenuations of pain behaviors in the second phase compared with that in wild-type mice. In a model of neuropathic pain, nerve injury-induced behavioral and cellular responses (tactile allodynia, spinal microglial activation and Src-family kinase phosphorylation) were reduced in <it>n/i/eNOS^-/-</it>but not <it>iNOS^-/-</it>mice. Tactile allodynia after nerve injury was improved by acute pharmacological inhibition of all NOSs and nNOS. Furthermore, in MG-5 cells (a microglial cell-line), interferon-γ enhanced NOSs and Mac-1 mRNA expression, and the Mac-1 mRNA increase was suppressed by L-NAME co-treatment. Conversely, the NO donor, sodium nitroprusside, markedly increased mRNA expression of Mac-1, interleukin-6, toll-like receptor 4 and P2X4 receptor.</p> <p>Conclusions</p> <p>Our results provide evidence that the NOS/NO pathway contributes to behavioral pain responses evoked by tissue injury and nerve injury. In particular, nNOS may be important for spinal microglial activation and tactile allodynia after nerve injury.</p