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...

Background We investigated the role of different voltage sensitive calcium channels expressed at presynaptic afferent terminals in substance P release and on nociceptive behavior evoked by intraplantar formalin by examining the effects of intrathecally delivered N- (ziconotide), T- (mibefradil) and L-type voltage sensitive calcium channels blockers (diltiazem and verapamil). Methods Rats received intrathecal pretreatment with saline or doses of morphine, ziconotide, mibefradil, diltiazem or verapamil. The effect of these injections upon flinching evoked by intraplantar formalin (5%, 50μl) was quantified. To assess substance P release, the incidence of neurokinin 1 receptor internalization in the ipsilateral and contralateral lamina I was determined in immunofluorescent stained tissues. Results Intrathecal morphine (20μg), ziconotide (0.3, 0.6 and 1μg), mibefradil (100μg, but not 50μg), diltiazem (500μg, but not 300μg) and verapamil (200μg, but not 50 and 100μg) reduced paw flinching in phase 2 as compared to vehicle control (P < 0.05), with no effect upon phase 1. Ziconotide (0.3, 0.6 and 1μg) and morphine (20μg) significantly inhibited neurokinin 1 receptor internalization (P < 0.05), but mibefradil, diltiazem and verapamil at the highest doses had no effect. Conclusion These results emphasize the role in vivo of N-, but not T- and L-type voltage sensitive calcium channels in mediating the stimulus evoked substance P release from small primary afferents and suggest that T- and L-type voltage sensitive calcium channels blockers exert antihyperalgesic effects by an action on other populations of afferents or mechanisms involving post synaptic excitability.

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Background Gabapentin binds at the extracellular α2δ1 subunit of voltage sensitive calcium channels. Some voltage sensitive calcium channels regulate substance P release from small primary afferents. We sought to determine in vivo if spinal and/or systemic gabapentin at antihyperalgesic doses will attenuate substance P release. Methods Rats prepared with chronic intrathecal (IT) catheters received IT vehicle or gabapentin 10 min before intraplantar formalin (5%, 50μl) injection. For systemic studies, vehicle or gabapentin was delivered intraperitoneally (IP) 15 min before formalin injection. In separate groups of rats, to assess the effect of IT or IP gabapentin upon formalin-evoked substance P release, animals received similar treatment for assessment of flinching, but underwent transcardial perfusion with 4% paraformaldehyde 10 min after the formalin injection. Substance P release was determined by the incidence of neurokinin 1 receptor (NK1r) internalization in the ipsilateral and contralateral superficial dorsal horn in immunofluorescent stained tissues. Results Unilateral intraplantar formalin evoked biphasic hindpaw flinching. IT gabapentin (100 and 200μg) and IP gabapentin (100 and 200 mg/kg) resulted in a dose-dependent reduction in phase 2, but not phase 1 flinching as compared to vehicle-treated rats. Intraplanatar formalin resulted in NK1r internalization in the ipsilateral, but not contralateral, superficial dorsal horn. IT gabapentin (200, but not 100 μg) and IP gabapentin (200, but not 100 mg/kg) significantly reduced ipsilateral NK1r internalization as compared to vehicle-treated control. Importantly, internalization evoked by IT substance P was not blocked by IT gabapentin. Conclusion Systemic and spinal gabapentin have an acute inhibitory effect on the release of substance P from small primary afferents and a concurrent effect upon the initiation of facilitated pain states.