The absence of the leukotriene B4 receptor BLT1 attenuates peripheral inflammation and spinal nociceptive processing following intraplantar formalin injury

Abstract

Background: Leukotriene B4 (LTB4) is a potent lipid mediator of inflammation, and its biological effects are mediated primarily through the high affinity LTB4 receptor BLT1. Although numerous studies have reported that LTB4-BLT1 signaling is involved in inflammatory diseases, the role of BLT1 signaling in pain remains undefined. To clarify the role of LTB4-BLT1 signaling in acute inflammatory pain induced by tissue injury, we performed pain behavioral analysis and assessment of local inflammation induced by peripheral formalin injections in BLT1 knockout mice. We examined the phosphorylation of cAMP response element-binding protein (CREB) in the spinal cord both in wild-type and BLT1 knockout mice because phosphorylation of CREB in spinal cord neurons is important for nociceptive sensitization following peripheral injury. We also examined the effect of a BLT1 antagonist on formalin-induced pain responses in mice.

Results: BLT1 knockout mice exhibited markedly attenuated nociceptive responses induced by intraplantar formalin injections. Edema formation and neutrophil infiltration in the paw were significantly decreased in BLT1 knockout mice compared with wild-type mice. Phosphorylation of CREB in the spinal cord after the intraplantar formalin injection was decreased in BLT1 knockout mice. In addition, mice pretreated with a BLT1 antagonist showed reduced nociception and attenuated CREB phosphorylation in the spinal cord after the formalin injection.

Conclusions: Our data suggest that LTB4-BLT1 axis contributes not only to the peripheral inflammation but also to the neuronal activation in the spinal cord induced by intraplantar formalin injections. Thus, LTB4-BLT1 signaling is a potential target for therapeutic intervention of acute and persistent pain induced by tissue injury.

Figure 1

Attenuation of formalin-induced pain behavior in LTB ₄ receptor type 1 knockout (BLT1KO) mice. (A) Time course of pain behaviors after formalin injection (* p < 0.05, **** p < 0.0001 vs. BLT1-wild-type (BLT1WT) mice). (B) Total duration of pain behaviors during the 1st (0–10 min) and the 2nd phases (11–40 min) (*** p < 0.001 vs. WT mice, n = 5, a two-way ANOVA with a Bonferroni post hoc test).

Figure 2

TRPV1 and CGRP expression profiles of nociceptive neurons are unaffected by BLT1 receptor knockout (BLT1KO). (A) Transient receptor potential vanilloid 1(TRPV1)- and (B) calcitonin-gene related peptide (CGRP)-positive neurons in the L4 dorsal root ganglion (DRG). (C) Representative immunofluorescence images show the localization of TRPV1 (green) and CGRP (red) in L4 DRG neurons (magnification, 200×). Scale bar represents 50 μm. (D, E) Representative immunofluorescence images showing the localization of TRPV1 (green), CGRP (green) and NeuN (red) in the lumbar spinal cord (magnification, 100×). Scale bars represent 100 μm. (F) Quantitative analysis of TRPV1-immunopositive density. β-Actin and TRPA1 mRNA levels in the DRG (G) and spinal cord (H) analyzed by quantitative RT-PCR. (n = 5–7, a Kruskal-Wallis with Dunn’s multiple comparison test ). Data was expressed as Ct value.

Figure 3

Local inflammation induced by formalin injections is attenuated in BLT1KO mice. (A) Paw edema formation 1 h after formalin injection. (* p < 0.05 vs. BLT1WT mice, n = 5, unpaired Student’s t-test with Welch’s correction). (B) Quantification of Evans blue dye extravasation 1 h after formalin or vehicle injection. (p = 0.2366 vs. BLT1WT mice, n = 5-7, * p < 0.05 vs. vehicle, n = 5–7, Kruskal-Wallis with Dunn’s multiple comparison test). (C) Myeloperoxidase (MPO) activity in the paw 1 h after formalin injection. (* p < 0.05 vs. BLT1WT mice, n = 8–9, two-way ANOVA with a Bonferroni post hoc test).

Figure 4

The expression of phosphorylated CREB (pCREB) in the dorsal horn is attenuated in BLT1KO mice. Representative diaminobenzidine-stained images showing pCREB-positive neurons in sections of the ipsilateral (A) and contralateral (B) dorsal horn of the lumbar spinal cord before or 20 min after a peripheral formalin injection (magnification, 100×). Arrowheads indicate pCREB-positive neurons. The number of pCREB-positive neurons in the ipsilateral (C) and contralateral (D) dorsal horn of the lumbar spinal cord prior to or 20 min after formalin injections. (**** p < 0.0001 vs. BLT1WT mice, n = 6, two-way ANOVA with Bonferroni post hoc test).

Figure 5

Effects of ONO-4057 on pain behavior in the formalin test. Time course of pain behavior in mice pretreated with intraperitoneal (i.p.) (A), intraplantar (ipl.) (C) or intrathecal (i.t.) (E) injections of ONO-4057 (* p < 0.05, *** p < 0.001 vs. vehicle). Total duration of pain behaviors in mice pretreated with i.p. (B), ipl. (D) and i.t. (F) injections of ONO-4057. (* p < 0.05, *** p < 0.001 vs. vehicle; n = 5–7; two-way ANOVA with Bonferroni post hoc tests).

Figure 6

ONO-4057 pretreatment affects peripheral inflammation and pCREB expression in the dorsal horn 20 min after intraplantar formalin injection. (A) Paw edema formation 1 h after formalin injection. (* p < 0.05, ONO-4057 (i.p. 0.25 mg) treated mice vs. vehicle (i.p.) treated mice, n = 5–6, unpaired Student’s t-test with Welch’s correction). (B) Quantification of Evans blue dye extravasation 1 h after formalin or vehicle injection. (** p < 0.01,*** p < 0.001, n = 6, Kruskal-Wallis with Dunn’s multiple comparison test). The number of phosphorylated CREB (pCREB)-positive neurons in the ipsilateral (C) and contralateral (D) dorsal horns of the spinal cord after intraplantar injections of formalin in mice pretreated with intraperitoneal ONO-4057 (0.25 mg, i.p.). (** p < 0.01, *** p < 0.001 vs. vehicle-pretreated mice, n = 4–5, unpaired Student’s t-test with Welch’s correction). Counts of pCREB-positive neurons in the ipsilateral (E) and contralateral (F) dorsal horn of the spinal cord after intraplantar formalin injection in mice pretreated with intraplantar ONO-4057 (2.35 μg, ipl.) injections. (* p < 0.05 vs. vehicle-pretreated mice, n = 5, unpaired Student’s t-test with Welch’s correction).