Peripheral inflammation initiates changes in spinal nociceptive processing leading to hyperalgesia. Previously, we demonstrated that among 102 lipid species detected by LC-MS/MS analysis in rat spinal cord, the most notable increases that occur after intraplantar carrageenan are metabolites of 12-lipoxygenases (12-LOX), particularly hepoxilins (HXA 3 and HXB 3 ). Thus, we examined involvement of spinal LOX enzymes in inflammatory hyperalgesia. In the current work, we found that intrathecal (IT) delivery of the LOX inhibitor nordihydroguaiaretic acid prevented the carrageenan-evoked increase in spinal HXB 3 at doses that attenuated the associated hyperalgesia. Furthermore, IT delivery of inhibitors targeting 12-LOX (CDC, Baicalein), but not 5-LOX (Zileuton) dosedependently attenuated tactile allodynia. Similarly, IT delivery of 12-LOX metabolites of arachidonic acid 12(S)-HpETE, 12(S)-HETE, HXA 3 , or HXB 3 evoked profound, persistent tactile allodynia, but 12(S)-HpETE and HXA 3 produced relatively modest, transient heat hyperalgesia. The pronociceptive effect of HXA 3 correlated with enhanced release of Substance P from primary sensory afferents. Importantly, HXA 3 triggered sustained mobilization of calcium in cells stably overexpressing TRPV1 or TRPA1 receptors and in acutely dissociated rodent sensory neurons. Constitutive deletion or antagonists of TRPV1 (AMG9810) or TRPA1 (HC030031) attenuated this action. Furthermore, pretreatment with antihyperalgesic doses of AMG9810 or HC030031 reduced spinal HXA 3 -evoked allodynia. These data indicate that spinal HXA 3 is increased by peripheral inflammation and promotes initiation of facilitated nociceptive processing through direct activation of TRPV1 and TRPA1 at central terminals.eicosanoid | pain | central sensitization T issue injury and inflammation are associated with hyperalgesia mediated by facilitated spinal nociceptive processing that can be modulated by lipids derived from arachidonic acid (AA) and other polyunsaturated fatty acids (PUFA), including eicosanoids synthesized via three enzymatic pathways: (i) cyclooxygenase (COX)-prostaglandins (PG); (ii) 5-, 12-, and 12/15-lipoxygenases (LOX)-leukotrienes, hydroxyeicosatetraenoic acids (HETEs), hepoxilins (HXA 3 and HXB 3 ), lipoxins, resolvins, and protectins; and (iii) cytochrome P450-epoxyeicosatrienoic acids and HETEs (1). Substantial evidence indicates that peripheral injury or direct activation of spinal dorsal horn receptors [Neurokinin 1 (NK1), AMPA, and NMDA] increases eicosanoid formation and that spinal delivery of COX inhibitors reduces the associated hyperalgesia (2, 3). Recently, we reported that paw carrageenan increases spinal production of both COX and 12-LOX metabolites of AA, including 12(S)-HETE in cerebrospinal fluid (CSF) and hepoxilins in the lumbar spinal cord (4).Several groups point to a peripheral role for 5-and 12-LOX in nociception, as shown by antihyperalgesic actions of LOX inhibitors administered via systemic routes (5-9). It has been suggested that spinal 12-LOX may play a r...

Previously, we observed significant increases in spinal 12-lipoxygenase (LOX) metabolites, in particular, hepoxilins, which contribute to peripheral inflammation-induced tactile allodynia. However, the enzymatic sources of hepoxilin synthase (HXS) activity in rats remain elusive. Therefore, we overexpressed each of the 6 rat 12/15-LOX enzymes in HEK-293T cells and measured by LC-MS/MS the formation of HXB3, 12-HETE, 8-HETE, and 15-HETE from arachidonic acid (AA) at baseline and in the presence of LOX inhibitors (NDGA, AA-861, CDC, baicalein, and PD146176) vs. vehicle-treated and mock-transfected controls. We detected the following primary intrinsic activities: 12-LOX (Alox12, Alox15), 15-LOX (Alox15b), and HXS (Alox12, Alox15). Similar to human and mouse orthologs, proteins encoded by rat Alox12b and Alox12e possessed minimal 12-LOX activity with AA as substrate, while eLOX3 (encoded by Aloxe3) exhibited HXS without 12-LOX activity when coexpressed with Alox12b or supplemented with 12-HpETE. CDC potently inhibited HXS and 12-LOX activity in vitro (relative IC50s: CDC, ~0.5 and 0.8 μM, respectively) and carrageenan-evoked tactile allodynia in vivo. Notably, peripheral inflammation significantly increased spinal eLOX3; intrathecal pretreatment with either siRNA targeting Aloxe3 or an eLOX3-selective antibody attenuated the associated allodynia. These findings implicate spinal eLOX3-mediated hepoxilin synthesis in inflammatory hyperesthesia and underscore the importance of developing more selective 12-LOX/HXS inhibitors.

Pain is the primary motivation for seeking medical care. Although pain may subside as inflammation resolves or an injury heals, it is increasingly evident that persistency of the pain state can occur with significant regularity. Chronic pain requires aggressive management to minimize its physiological consequences and diminish its impact on quality of life. Although opioids commonly are prescribed for intractable pain, concerns regarding reduced efficacy, as well as risks of tolerance and dependence, misuse, diversion, and overdose mortality rates limit their utility. Advances in development of nonopioid interventions hinge on our appreciation of underlying mechanisms of pain hypersensitivity. For instance, the contributory role of immunity and the associated presence of autoimmune syndromes has become of particular interest. Males and females exhibit fundamental differences in innate and adaptive immune responses, some of which are present throughout life, whereas others manifest with reproductive maturation. In general, the incidence of chronic pain conditions, particularly those with likely autoimmune covariates, is significantly higher in women. Accordingly, evidence is now accruing in support of neuroimmune interactions driving sex differences in the development and maintenance of pain hypersensitivity and chronicity. This review highlights known sexual dimorphisms of neuroimmune signaling in pain states modeled in rodents, which may yield potential high-value sex-specific targets to inform future analgesic drug discovery efforts.

Previous reports utilizing pharmacological antagonists implicate kainate receptor (KAR) activation in the development of morphine tolerance, dependence, conditioned place preference (CPP), and locomotor sensitization, but the role of glutamate receptor (GluR) 5-containing KAR in these effects remains unclear because of limited selectivity of the inhibitors employed. Therefore, we examined responses to systemic morphine treatment in mice expressing a constitutive deletion of GluR5 [GluR5 knockout (KO)]. Unlike wild-type (WT) littermates, GluR5 KO mice do not develop tolerance after repeated morphine administration by subcutaneous injection or via subcutaneous pellet implantation. In contrast, GluR5 KO mice do not differ from WT with respect to thermal or mechanical nociceptive thresholds, acute morphine antinociception, morphine disposition in the central nervous system (CNS), morphine physical dependence as revealed by naloxone-precipitated withdrawal or development of place preference and locomotor hyperresponsiveness after chronic morphine administration. It is surprising that continuous subcutaneous infusion of the GluR2/GluR5-preferring antagonist LY293558 [(3S,4aR,6R,8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]decahydroisoquinoline-3-carboxylic acid] decreased the number of naloxone-precipitated jumps to a similar extent in WT and GluR5 KO mice. We observed opioidinduced hypersensitivity in both groups during morphine withdrawal as demonstrated by equivalent reductions in thermal and mechanical thresholds; however, this hypersensitivity was not evident during continuous systemic morphine infusion. These data collectively indicate that KARs containing the GluR5 subunit contribute to the development of morphine tolerance without affecting nociceptive thresholds, morphine analgesia, or disposition in CNS of morphine and its metabolite morphine-3-glucuronide. In addition, constitutive deletion of GluR5 does not alter the morphine-induced increase in locomotor activity or the acquisition of morphine reward as measured by a CPP paradigm.

Although nonsteroidal anti-inflammatory drugs are the first line of therapeutics for the treatment of mild to moderate somatic pain, they are not generally considered to be effective for neuropathic pain. In the current study, direct activation of spinal Toll-like 4 receptors (TLR4) by the intrathecal (IT) administration of KDO2 lipid A (KLA), the active component of lipopolysaccharide, elicits a robust tactile allodynia that is unresponsive to cyclooxygenase inhibition, despite elevated expression of cyclooxygenase metabolites in the spinal cord. Intrathecal KLA increases 12-lipoxygenase-mediated hepoxilin production in the lumbar spinal cord, concurrent with expression of the tactile allodynia. The TLR4-induced hepoxilin production was also observed in primary spinal microglia, but not in astrocytes, and was accompanied by increased microglial expression of the 12/15-lipoxygenase enzyme 15-LOX-1. Intrathecal KLA-induced tactile allodynia was completely prevented by spinal pretreatment with the 12/15-lipoxygenase inhibitor CDC or a selective antibody targeting rat 15-LOX-1. Similarly, pretreatment with the selective inhibitors ML127 or ML351 both reduced activity of the rat homolog of 15-LOX-1 heterologously expressed in HEK-293T cells and completely abrogated nonsteroidal anti-inflammatory drug-unresponsive allodynia in vivo after IT KLA. Finally, spinal 12/15-lipoxygenase inhibition by nordihydroguaiaretic acid (NDGA) both prevents phase II formalin flinching and reverses formalin-induced persistent tactile allodynia. Taken together, these findings suggest that spinal TLR4-mediated hyperpathic states are mediated at least in part through activation of microglial 15-LOX-1.