Lipoxins and aspirin-triggered lipoxin inhibit inflammatory pain processing

Abstract

Inflammatory conditions can lead to debilitating and persistent pain. This hyperalgesia reflects sensitization of peripheral terminals and facilitation of pain signaling at the spinal level. Studies of peripheral systems show that tissue injury triggers not only inflammation but also a well-orchestrated series of events that leads to reversal of the inflammatory state. In this regard, lipoxins represent a unique class of lipid mediators that promote resolution of inflammation. The antiinflammatory role of peripheral lipoxins raises the hypothesis that similar neuraxial systems may also down-regulate injury-induced spinal facilitation of pain processing. We report that the lipoxin A(4) receptor is expressed on spinal astrocytes both in vivo and in vitro and that spinal delivery of lipoxin A(4), as well as stable analogues, attenuates inflammation-induced pain. Furthermore, activation of extracellular signal-regulated kinase and c-Jun N-terminal kinase in astrocytes, which has been indicated to play an important role in spinal pain processing, was attenuated in the presence of lipoxins. This linkage opens the possibility that lipoxins regulate spinal nociceptive processing though their actions upon astrocytic activation. Targeting mechanisms that counterregulate the spinal consequences of persistent peripheral inflammation provide a novel endogenous mechanism by which chronic pain may be controlled.

Figure 1.

Intravenous administration of lipoxins reduces inflammation-evoked hyperalgesia and edema. Paw withdrawal latency (PWL) is plotted versus time for the ipsilateral (ip; injected) and contralateral (c; uninjected) hind paw showing that i.v. injection of LXA₄, ATLa (A and C), and aLXB₄ (B and C), but not 8,9-aLXB4 (B and C), before injection of carrageenan reduces thermal hyperalgesia. HI (C) is calculated for 0–4 h. Paw thickness (D) was measured by calipers at different time points after induction of inflammation. Each time point and bar represents the mean ± SEM (n = 4–6). *, P < 0.05 as compared with vehicle (ip) measurements.

Figure 2.

Intrathecal injection of lipoxins reduces inflammation-evoked hyperalgesia. Paw withdrawal latency (PWL) is plotted versus time for the left ipsilateral (ip) and contralateral (c) hind paw, showing that i.t. pretreatment (−2 min) with equimolar doses of LXA₄ (A and C), LXB₄, and ATLa and a higher dose of 8,9-aLXB₄ (B and C), as well as posttreatment with LXA₄ (injection at 120 min or at 120 min followed by a second injection at 190 min; E and F), reduces inflammation-evoked hyperalgesia. HI is calculated for 0–4 h (C) and 2–6 h (F). None of the i.t.-delivered lipoxins altered paw thickness (D). Each time point and bar represents the mean ± SEM (n = 6–8). *, P < 0.05 as compared with vehicle (ip) measurements.

Figure 3.

ALXR is expressed on astrocytes in the naive rat spinal cord. (A) Representative Western blots depicting ALXR expression in mouse and rat spinal cords 0–24 h after injection of carrageenan to the paw. Rat spleen (from rats 8 h after carrageenan injection) was used as positive control. Immunohistological processing of spinal cord sections from the naive rat show that ALXR is homogenously expressed throughout the dorsal horn of the spinal cord (B), colocalizing with the astrocyte marker GFAP (C–E, green; F, enlarged image) but not with the microglia marker OX-42 (K, green) or the neuronal marker NeuN (L, green). ALXR immunoreactivity was detected with both fluorescence (G) and avidin–biotin–3,3-diaminobenzidine (H) detection in spleens from carrageenan-injected rats (F). ALXR antibody replaced with goat serum IgG was used as a negative control (I). ALXR immunoreactivity was absent in dorsal root ganglia (DRG; J). Bars, 50 μm.

Figure 4.

ATLa prevents ATP-evoked ERK and JNK phosphorylation in primary astrocyte cultures. (A) Representative images demonstrating that ALXR colocalizes with the astrocyte marker GFAP in cultured primary astrocytes. Bar, 50 μm. (B) Western blots probed for phosphorylated ERK and JNK in samples from primary astrocytes stimulated with ATP, SP, IL-1β, and TNF-α for 15 min. Incubation with 10 nM ATLa, starting 30 min before TNF-α stimulation, had no effect on JNK phosphorylation (C), whereas ATLa prevented both ERK and JNK phosphorylation evoked by ATP (D and E). Each bar represents the mean ± SEM (n = 4–5). *, P < 0.05 as compared with control; #, P < 0.05 as compared with PBS + ATP.