Peripheral inflammatory hyperalgesia depends on the COX increase in the dorsal root ganglion

Abstract

It is well established that dorsal root ganglion (DRG) cells synthesize prostaglandin. However, the role that prostaglandin plays in the inflammatory hyperalgesia of peripheral tissue has not been established. Recently, we have successfully established a technique to inject drugs (3 μL) directly into the L5-DRG of rats, allowing in vivo identification of the role that DRG cell-derived COX-1 and COX-2 play in the development of inflammatory hyperalgesia of peripheral tissue. IL-1β (0.5 pg) or carrageenan (100 ng) was administered in the L5-peripheral field of rat hindpaw and mechanical hyperalgesia was evaluated after 3 h. Administration of a nonselective COX inhibitor (indomethacin), selective COX-1 (valeryl salicylate), or selective COX-2 (SC-236) inhibitors into the L5-DRG prevented the hyperalgesia induced by IL-1β. Similarly, oligodeoxynucleotide-antisense against COX-1 or COX-2, but not oligodeoxynucleotide-mismatch, decreased their respective expressions in the L5-DRG and prevented the hyperalgesia induced by IL-1β in the hindpaw. Immunofluorescence analysis demonstrated that the amount of COX-1 and COX-2, constitutively expressed in TRPV-1(+) cells of the DRG, significantly increased after carrageenan or IL-1β administration. In addition, indomethacin administered into the L5-DRG prevented the increase of PKCε expression in DRG membrane cells induced by carrageenan. Finally, the administration of EP1/EP2 (7.5 ng) or EP4 (10 µg) receptor antagonists into L5-DRG prevented the hyperalgesia induced by IL-1β in the hindpaw. In conclusion, the results of this study suggest that the inflammatory hyperalgesia in peripheral tissue depends on activation of COX-1 and COX-2 in C-fibers, which contribute to the induction and maintenance of sensitization of primary sensory neurons.

Fig. 1.

COX-1 or COX-2 inhibitors administered into the L5-DRG prevented the hyperalgesia induced by IL-1β in the L5 peripheral field. Indomethacin (A) (Indo, 100 μg), valeryl salicylate (B) (3, 10, or 30 μg), or SC-236 (C) (30, 70, 100, or 300 μg) administered into the L5-DRG prevented the hyperalgesia induced by IL-1β (0.5 pg per paw) in the L5 peripheral field. Indomethacin, valeryl salicylate, SC-236, or their vehicles were administered 30 min before IL-1β and the hyperalgesia was evaluated 3 h after its administration. Indomethacin, valeryl salicylate (10 µg), SC-236 (100 µg), saline (3 µL), or Tris (3 µL) into the L5-DRG had no effect on the nociceptive mechanical threshold in rats treated with saline (Sal, 50 µL) in the hind-paw. Indomethacin administered in the contralateral (CL) L5-DRG did not change the hyperalgesia induced by the administration of IL-1β in the L5 peripheral field. The asterisk (*) indicates a response significantly lower (P < 0.05, one-way ANOVA followed by the Bonferroni test) than that induced by vehicle administration (C or Tris) in rats treated with IL-1β in the L5 peripheral field, and the hash-tag (^#) indicates a response significantly lower (P < 0.05, one-way ANOVA followed by the Bonferroni test) than that induced by SC-236 (70 µg). Results are expressed as the mean ± SEM of five rats per group.

Fig. 2.

COX-1 or COX-2 inhibitors administered into the L5-DRG significantly decreased hyperalgesia induced by carrageenan in the L5 peripheral field. Valeryl salicylate (VS, 10 µg) or SC-236 (SC, 100 µg) administered into the L5-DRG significantly decreased the hyperalgesia induced by carrageenan (100 µg per paw) in the L5 peripheral field. Valeryl salicylate, SC-236, or their vehicles were administered 30 min before carrageenan and the hyperalgesia was evaluated at 3 h after its administration. The asterisk (*) indicates a significant difference (P < 0.05, unpaired t test) between the groups indicated (C; 3 µL). The results are expressed as the mean ± SEM of five rats per group.

Fig. 3.

COX-1 or COX-2 knock-down in the L5-DRG significantly decreased the hyperalgesia induced by IL-1β in the L5 peripheral field. Treatment (20 μg/5 µL per day, for 4 d) with ODN-AS against COX-1 (A) or COX-2 (B), but not with their respective ODN-mismatch (Mis, 20 µg/5 µL per day, for 4 d) or saline (Sal; 5 μL/d, for 4 d) administered into the L5-DRG, significantly decreased the mechanical hyperalgesia induced by IL-1β (0.5 pg per paw) administered in the L5 peripheral field. Specific ODN-AS, but not ODN-mismatch or saline significantly decreased the expression of COX-1 (E) or COX-2 (F). C and D show, respectively, a representative image of COX-1 or COX-2 knock-down induced by ODN-AS. The asterisk (*) indicates a response significantly lower than that of other groups (A and B, P < 0.05, one-way ANOVA followed by the Bonferroni test; and E and F, P < 0.05, unpaired t test). The results are expressed as the mean ± SEM of five rats per group.

Fig. 4.

IL-1β or carrageenan in peripheral tissue significantly increased the expression of COX-1 and COX-2 in TRPV-1⁺ cells of L5-DRG. IL-1β (0.5 pg per paw) or carrageenan (Cg, 100 μg per paw), but not saline (50 µL) in L5 peripheral field significantly increased the expression of COX-1 and COX-2 in the L5-DRG (M). COX-1 and COX-2 in normal conditions (A–C) or induced by IL-1β (D–F) or carrageenan (G–I) are colocalized in L5-DRG. COX-2 is colocalized with the TRPV-1 receptor in the L5-DRG (J–L). The asterisk (*) indicates a response significantly different between the groups indicated (P < 0.001, unpaired t test). The results are expressed as the mean ± SEM of 50 cells per group. (Scale bars, 25 µm.)

Fig. 5.

EP4 or EP1/EP2 receptor antagonists administered into the L5-DRG prevented the hyperalgesia induced by IL-1β in the L5-peripheral field. The administration of AH23848 (EP4 receptor antagonist; 10 μg) or AH6809 (EP1/EP2 antagonist; 7.5 ng) into the L5-DRG prevented the mechanical hyperalgesia induced by IL-1β (0.5 pg/paw) administered in the L5-peripheral field. The asterisk (*) indicates a response significantly lower than that of rats treated with IL-1β (0.5 pg per paw) and with saline in the L5-DRG (P < 0.001, one-way ANOVA followed by the Bonferroni test). The results are expressed as mean ± SEM of five animals per group.

Fig. 6.

The administration of indomethacin into the L5-DRG blocked the PKCε translocation induced by carrageenan in the L5-peripheral field. The administration of carrageenan (Cg, 100 µg per paw) in the L5 peripheral field significantly increased PKCε expression in the membrane of L5-DRG cells. This increase was blocked by the administration of indomethacin (100 µg/3 µL) into the L5-DRG (A). (B) A representative figure of carrageenan-induced PKCε expression in the membrane of L5-DRG cells and its decrease by indomethacin. The asterisk (*) indicates a response significantly greater (P < 0.05, unpaired t test) than that of other groups. The results are expressed as the mean ± SEM of five rats per group.